ACNP 60th Annual Meeting: P551–P830 Poster Summaries (2023)

Neuropsychofarmakologia.December 2021? 46 (Appendix 1): 369–518.

PMID:34857906

University of Maryland School of Medicine, Baltimore, Maryland, Stany Zjednoczone

Bottom:Although schizophrenia is classically thought to involve impaired filtering of attention, schizophrenic individuals (PES) exhibit stronger and more exclusive attention spans than healthy control subjects (HCS), termed hyperfocus, on several tasks. A possible explanation for this contradiction lies in the distinction between control and selective attention. Control processes direct attention to relevant sources of information, while applying selective attention refers to the actual emphasis of a selected source over others. Selective attention can be impaired because processing resources are diverted to the wrong data, reflecting control deficits. Previous findings do indeed suggest deficits in attention control in the PES. However, when control processes are not a limiting factor, a stronger application of selective attention may lead to a more intense and exclusive focus on the PES. We hypothesized that the PES would show reduced selective attention when control requirements were high, but hyperfocus when control requirements were low.

Methods:Functional magnetic resonance imaging (fMRI) was used to investigate attentional enhancement in higher-order visual processing areas termed the fusiform facial area (FFA) and parahippocampal area (PPA), which are specialized for facial and spatial processing, respectively. Forty-three PES and 43 HCS underwent fMRI responding to facial and household stimuli. Stimulus-evoked activation in FFA and PPA was analyzed according to whether participants sought a predetermined target face or target house. To test the effect of attentional control requirements on attentional regulation of FFA and PPA BOLD activity, stimuli were presented singly (sequential condition) or simultaneously as translucent facial overlays that challenged attentional control (overlay condition). Activation was tested in the FFA and PPA regions of interest, defined individually by an independent functional locator.

Results:FFA ROI coordinates did not differ between PES and HCS. On average, PPA ROIs are slightly later and lower in PES than HCS. Importantly, facial ROI sensory discrimination did not differ between groups for either locational stimuli or task stimuli, arguing against group differences in the basic function of these regions.

Task responses were slower for houses than for face stimuli in sequential conditions and slower for prioritizing houses over faces in overlapping conditions, suggesting a difference in significance between stimulus dimensions. Target detection in the PES was more impaired in the Overlapping state and less (at all) impaired when responding to facial stimuli in the Sequential state following attentional control demands.

BOLD activity reflected poorer attentional selectivity in the PSZ than in the HCS when attentional control was more difficult, that is, when faces and houses were superimposed and the task required detection of the lower house target. Specifically, HCS but not PSZ showed greater PPA activation for overlapping stimuli when the house dimension was met than when the face dimension was met [interaction group x note:eat(1,81) = 4,12,PI number= 0.046]. On the other hand, selective attention was exaggerated in the PES when control was less challenged, i.e. when the stimuli were presented sequentially and the task required greater facial target detection. Specifically, PSZ but not HCS showed greater FFA activation to facial stimuli when the face dimension was met than when the house dimension was added [interaction group x note:eat(1,81) = 4,54,PI number= 0.036]. Time-course analyzes showed that these effects were not due to group differences in the timing of haemodynamic response. Thus, hyperactivation in the PES does not reflect sustained stimulus engagement.

Conclusions:The findings are consistent with two distinct attentional abnormalities in schizophrenia leading to impairment and hyperselectivity in different conditions: deficits in attentional control and stronger use of selective attention or hyperfocus when attention is directed to a stimulus.

Keywords:Schizophrenia (SCZ), selective attention, brain imaging, fMRI, fusiform facial area (FFA), parahippocampal area (PPA)

Revelation:Nothing to disclose.

University of California San Diego, La Jolla, California, United States

Bottom:Human brain functions, including perception, attention, and other higher-order cognitive functions, are supported by the neural oscillations necessary to transmit information through neural networks. Previous research has shown that the rhythmic firing of neuronal populations is not random, but is governed by interactions with other frequency bands. In particular, the amplitude of the gamma band oscillations is tied to the phase of the lower frequency oscillations to support short and long range communication between networks. This amplitude-phase coupling (PAC) is believed to reflect the temporal fine-tuning of neural communication. Although patients with schizophrenia show abnormal oscillatory responses at many resting frequencies, including oscillations in the gamma band, it is unclear whether the functional relationships between the frequency bands are intact. Our aim was to characterize the low-frequency phase (delta/theta, 1–8 Hz) and amplitude of gamma oscillations in normal subjects and patients with schizophrenia at rest.

Methods:The relationship between the low frequency angle (delta and theta bands) and the amplitude of the gamma band was evaluated in 142 patients with schizophrenia and 128 healthy controls. We characterized the resting CAP in schizophrenic patients and healthy controls using a modified method that does not use gamma amplitude as a scalar in the CAP calculation, selecting the highest 5% of the gamma power data for testing. CAP, thus avoiding confusion of group differences in output gamma.

Results:Significant delta-gamma CAPs have been detected in widespread areas of the scalp in healthy individuals and patients with schizophrenia. The delta-gamma PAC was significantly reduced in the frontal-mid, right middle temporal, and left temporo-parietal electrodes, but significantly increased in the left parietal electrode in schizophrenic patients.

Conclusions:Delta-gamma CAP may reflect a central pathophysiological abnormality in schizophrenia. Data-driven measurements of functional relationships between frequency bands could be useful in developing new therapies. Further research is needed to determine whether these changes are specific to schizophrenia or are seen in other populations of neuropsychiatric patients.

Keywords:EEG, gamma oscillations, schizophrenia (SCZ)

Revelation:Nothing to disclose.

Hiroshima University, Hiroshima, Japan

Bottom:Clinical studies have shown that microduplications in 7q36.3, which contains VIPR2, are associated with a significant risk of schizophrenia and autism spectrum disorder (ASD). The VIPR2 gene encodes the VPAC2 receptor, a seven-transmembrane heterotrimeric G protein-coupled receptor (GPCR), for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP). Lymphocytes from patients with these microduplications showed increased expression of the VIPR2 gene and a VIP response (cAMP induction). These findings provide genetic evidence for a specific receptor-mediated (and potentially drug-targetable) signaling pathway associated with schizophrenia and ASD that is distinct from the dopaminergic, glutamatergic, and serotonergic systems. In addition to VPAC2, there are other subtypes of PACAP receptors, namely VPAC1 and PAC1, and all of these receptors belong to class B GPCRs. PAC1, VPAC1, and VPAC2 receptors have moderate amino acid sequence similarities (about 50%) to each other and very three-dimensional structural homology. Therefore, these molecular features make it difficult to discover small molecule drugs selective for the VPAC2 receptor. In 2018, Sakamoto et al. discovered an artificial 16-mer cyclic peptide VIpep-3:Ac-c(CPPYLPRRLC)TLLLRS-OH that antagonizes human and rodent VPAC2 receptor signaling pathways in vitro (Biochem. Biophys. Res. Commun. 503: 1973-1979, 2). However, this peptide contains all-natural amino acids and is highly susceptible to degradation by proteases. The in vivo efficacy of this VPAC2 receptor antagonist also needs to be determined in animal models of psychiatric disorders such as schizophrenia. The aim of this study was to generate a VIPep-3 derivative for in vivo experiments.

Methods:The in vitro antagonistic activities of the candidate peptides against the VIP-VPAC1, VIP-VPAC2 and PACAP-PAC1 signaling pathways were analyzed by the calcium flux assay. VPAC2 antagonist activity in vivo was assessed by Western blot analysis. The cognitive functions of the mouse were determined using the novel object recognition test. Golgi-Cox staining was used to visualize neurons for analysis of dendritic morphology. Experimental procedures involving animals and their care were performed in accordance with the Guide for the Care of Laboratory Animals (National Research Council, 1996) and all animal experiments were approved by the Institutional Animal Care and Use Committees of Hiroshima University (#A20-115) and the University in Osaka (No. 30-3-2). Pregnant ICR mice (CD1) at 16 days of gestation and C57BL/6J male mice at 7 weeks of age were purchased from Japan SLC Inc. (Shizuoka, Japan).

Results:Using existing structural information from the extracellular domain of VPAC2 (PDB ID: 2X57), the C-terminal structure of VIP (PDB ID: 2RRI) and the VPAC1/VIP binding model (Couvineau et al., Front. Endocrinol. 3: 139, 2012), we generated molecular design concept. After amino acid substitution and structure optimization of VIPep-3, we successfully generated a new peptide KS-133 with selective and potent VPAC2 antagonistic activity and at least 24-hour stability in plasma. KS-133 had an IC50 value of 24.8 nM against VPAC2, which was a more potent antagonist than the parent VIPep-3 (40.6 nM), although the molecular weight was reduced compared to VIPep-3 (1941, 1 g/mol) in KS -133,133 (1558.8 g/mol). KS-133 did not antagonize the VIP-VPAC1 and PACAP-PAC1 signaling pathways up to 5 μM. Subcutaneous and intranasal administration of KS-133 inhibited the VPAC2 activation-induced increase in CREB phosphorylation in the prefrontal cortex of neonatal and adult mice, respectively. We also found that repeated administration of Ro 25-1553, a selective VPAC2 agonist, on postnatal days 1–14 in mice induced adult cognitive impairment, and co-treatment with KS-133 prevented this effect. The same Ro 25-1553 treatment with postnatal restriction reduced the total branching number and length of apical and basal dendrites of prefrontal cortex neurons in mice. These morphological abnormalities were treated by co-administration of KS-133 with Ro 25-1553.

Conclusions:We generated KS-133 which possessed the following drug-like properties: (a) high selectivity and strong VPAC2 antagonistic activity; (b) remarkable resistance to degradation by proteases, a common problem with peptides. (c) prevention of cognitive decline in a pharmacological model of early postnatal overactivation of VPAC2, a relevant mouse model of schizophrenia. Our study is not only the first validation of a VPAC2 antagonist in animal models of disease, but also a good example of drug discovery for class B GPCRs.

Keywords:New treatment for schizophrenia GPCR receptor antagonist VPAC2 Cyclic peptide PACAP

Revelation:Nothing to disclose.

Signant Health, McLean, Virginia, United States

Bottom:High severity of positive symptoms is often an exclusion criterion in clinical trials of negative symptoms of schizophrenia, and these studies generally do not allow randomization of patients with a positive score greater than 22. There is a general understanding from the work of Dunayevich et al., 2014 et al., 2014 et al., that more severe positive symptoms reduce the level of improvement seen in the negative unadjusted PANSS subscale (Dunayevich et al., 2014). The current retrospective analysis assessed the effect of instability on the severity of positive symptoms at the start of screening from baseline to end of treatment, negative factor change in blinded data from several clinical studies of negative symptoms.

Methods:Baseline data were pooled from six clinical trials in schizophrenia with negative symptoms. The severity of positive symptoms at baseline was determined based on the PANSS positive subscale and PANSS positive score. Change in PANSS Positive Subscale and PANSS Factor Positive Score between study and baseline was calculated. Using regression analyses, adjusting for baseline negative and test severity, we assessed the effect of severity of positive symptoms - separate analyzes for PANSS positive subscale and PANSS factor positive, and the effect of change in positive symptoms between screening and baseline - separate analyzes for PANSS positive or PANSS positive positive change in end-of-treatment factor in PANSS negative change in factor in blinded pooled data. Given the exploratory nature of the analyses, we do not adjust multiple tests.

Results:Our data included data collected from a total of 2,474 people. The mean (SD) baseline PANSS positive severity at baseline was 13.2 (3.4) points, but it varied significantly between studies (PI number<0.001). The mean (sd) PANSS factor positive was 17.3 (3.5) and varied significantly between studies (PI number<0.001). The mean change from screening to baseline in the PANSS positive subscale was -0.15 (1.6) points, and the mean change in factor positive was -0.08 (1.7), no difference between studies (PI number> 0.05). The change from baseline to last visit in negative factor decreased significantly with increasing PANSS baseline positive subscale and PANSS positive factor severity, respectively. A change in the positive symptom subscale, but not as a result of the positive factor, significantly reduced the change in the negative factor score at the last visit (allPI number< 0,05).

Conclusions:Our data support the negative effect of the severity of positive symptoms at baseline at the last visit, the change from baseline to the PANSS negative factor score in a pooled dataset of blinded data collected in a large number of patients. An increase of 1 point in the positive PANSS subscale translated into a decrease in improvement by approximately 0.08 points, and an increase of 1 point in the positive PANSS result translated into a decrease of approximately 0.15 points. A 1-point decrease in the positive PANSS subscale between screening and baseline significantly reduced the improvement in the negative factor score by 0.13 points. On the other hand, the deterioration of the positive factor by 1 point decreased the variance of the negative factor from the last visit by 0.08 points, which did not reach statistical significance. This difference is somewhat puzzling and we plan to analyze further to determine whether it is due to any of the single items that make up the positive subscale and positive factor score. Our results should be interpreted with caution. The lack of treatment allocation data did not allow unblinded analysis, therefore we cannot answer the more critical question of whether the severity of positive symptoms and their change between screening and baseline affect signal detection.

Keywords:CNS clinical trials, Negative symptoms, Positive symptoms, Schizophrenia

Revelation:Signat Health: Employee (it)

University of California, Davis, Sacramento, California, United States

Bottom:Team-based 'coordinated specialist care' (CSC) for early psychosis (PE) is highly effective in promoting clinical and functional recovery. NIH EPINET project (https://nationalepinet.org/) seeks to work with EC programs across the US to facilitate large-scale data collection and analysis to promote the rapid dissemination of best practice. As an early EPINET node, the California EPI-CAL project (https://epical.ucdavis.edu) collaborated with 6 university and 7 district EC programs to create a balanced network of health education and anonymous contributors to the NIMH EPINET database. Initial objectives focused on 1) developing a core set of evidence-based measures based on qualitative analysis of stakeholder data, 2) co-designing a data collection platform (Beehive) for EC programs and 3) implementing Beehive in 3 pilot EP Master Data Collection Programs for 70% of eligible EP customers.

Methods:Semi-structured qualitative focus groups were completed with stakeholders (e.g. clients, family members, EC program staff/management, county/state representatives) to: 1) explore stakeholder views on the output data areas and collection method within the EPICAL network? 2) get feedback on the design, flow and functionality of the Beehive app and its dashboard; and 3) determine how best to make key aspects of EPI-CAL and the Beehive platform available to users (e.g. purpose of data collection, security and data sharing) to support informed participation and data sharing decisions. The focus group data were analyzed using mixed methods, including qualitative and quantitative methods where possible. Beehive was then implemented in 3 PE pilots to determine feasibility and acceptance for employees, clients and families. Descriptive analyzes summarize the data collected so far.

Results:1) Overview of key outcome areas from the research literature and evidence-based metrics of the PhenX Toolkit (https://www.phenxtoolkit.org/) have been presented for consideration by interested parties. Stakeholders explored all potential domains and outcome measures through 23 focus groups and four interviews (N= 189) between August 10, 2019 and March 11, 2020. The results supported the inclusion of a wide range of client-centered and self-report outcomes as the primary approach to data collection to facilitate client engagement and reduce the data burden on clinicians. The results were made available to EPINET to support the development of a core assessment battery for all EC programs in the US. 2) On March 26, 2020 – May 18, 2020, 14 stakeholder focus groups (N= 82) checked the Beehive app and digital dashboard panels. Participants provided feedback on the look/feature of the app, data visualization, and the best way to structure the app for different user roles. 3) 6 stakeholder focus groups (N= 24) were conducted between 8/2020 and 1/2021 in two parts: to share experiences and preferences regarding data sharing in general and about your mental health and, based on these initial focus groups, to provide feedback about how Beehive displays your information sharing. Participants watched an informative video created by the research team, which discussed key points regarding the purpose of the Hive, data security, the importance of a limited data set, with whom data will be shared and how users can opt out. receiving or not exchanging data for research purposes. Participants appreciated the ability to opt-out of data sharing at their discretion and agreed that the video increased their understanding of the design, application and how their information is stored at each level of data sharing permission. This increased their willingness to share a limited data set for research. The feedback shaped the final content of the video, including a longer section on the cell's potential benefits to the individual, state and country to increase data sharing adoption. So far, 27 people have completed registration for the bee. Of this group, 81.48% (N= 22) agreed to share their data with the NIMH EPINET database, and 77.78% (N= 21) agreed to share their EPI-CAL data (52.38% female, 38.10% Hispanic, 80.95% minority, 61.90% first episode/4.76% high clinical risk).

Conclusions:Stakeholder input helped develop a core assessment battery that addressed customer-level performance across all stakeholder groups, which increased the motivation for the project among participating parties. Co-development of the Beehive platform ensured that the technology met all of the provider's needs. Providing users with clear information about the purpose of the project, how to maintain data security and the possibility of sharing data outside the clinic increased participants' knowledge, confidence and willingness to share data outside the clinic. Participants approved a video format with clear and simple language to present this information. Changes to this video have been made based on participant feedback to increase engagement and understanding. The pilot data collection at 3 locations demonstrated the initial feasibility of the data collection platform and identified challenges and solutions early in the implementation process, such as the need for clear workflow integration. Data collection is being extended to other EPICAL sites and results from a larger sample will be reported.

Keywords:Early psychosis, a learning healthcare system, patient outcomes, technology, relevance to outcomes

Revelation:Safari Health, Inc.: Founder (Owner)

McLean Hospital - Harvard Medical School, Belmont, Massachusetts, Estados Unidos

Bottom:About 50% of people with early psychosis have a history of substance use problems, and many report recent use of marijuana, alcohol, and nicotine. The concomitant use of the substance significantly increases the likelihood of discontinuation and discontinuation of treatment. Community Strengthening and Family Training (CRAFT) aims to increase client readiness to switch substance use, reduce family stress, and improve client-family relationships. CRAFT topics include building motivation and self-care, communication, functional analysis of substance use, positive reinforcement of health/recovery-oriented behaviors, discussing treatment commitment, ensuring negative consequences, and problem solving. The CRAFT protocol was not targeted at families of individuals with early-onset psychosis, nor was it adapted to be delivered via a telehealth platform (e.g., Zoom) prior to this study. The current project aims to develop and evaluate the feasibility and acceptability of a telehealth coaching intervention tailoring CRAFT to improve treatment commitment and reduce stress among families of individuals diagnosed with early-onset psychosis (PE) and co-occurring substance use.

Methods:The participants were family members (incl.N= 21) relatives with early psychosis (first episode in the last 6 years) and a history of substance use (marijuana, alcohol, nicotine in the last 90 days or no immediate interest in abstinence). Family members completed an intervention that consisted of six to eight approximately 60-minute sessions of Modified CRAFT for Psychosis (CRAFT-PT) coaching by a research coach. Participants completed the assessment battery four times: before treatment, during treatment, after treatment, and a three-month follow-up. Prior to treatment, participants completed a structured clinical interview for the DSM-5 (SCID) to characterize the sample. Families also completed a questionnaire after each session to rate their satisfaction with the treatment (average of how helpful and comfortable the session was, from 0 = Poor to 5 = Excellent) and whether they had technical problems (yes/no). After 10 families completed the program, participants attended focus groups, and we reviewed session feedback, engagement, and retention data to guide treatment development. The trial with the revised manual (currently eight sessions) is almost complete with 10 new family members. Session attendance rates and descriptive statistics from treatment satisfaction ratings will be used to assess the feasibility and acceptance of the intervention.

Results:From August 5, 2021, family members who started training (N= 20: 18 mothers, 1 father, 1 sister), 75% had a DSM-5 diagnosis (e.g. major depressive disorder, mild alcohol use disorder, etc.). All had 100% session attendance and rated treatment satisfaction between sessions as near excellent (mean (SD) 4.85(0.32)) with 75% of the sessions rated as excellent despite technical issues in 20% of the sessions. Of those who completed the program, participants reported a 60-65% improvement in their current problems, and 100% of participants would recommend the program to others.

Conclusions:Preliminary data suggest that the CRAFT-PT protocol is highly feasible and acceptable as an active treatment in a randomized controlled pilot study comparing usual treatment plus CRAFT-PT with usual care. Considering that 95% of the coaching participants were women and the majority supported the DSM-5 diagnosis, our results also highlight the need to target caregivers of people with comorbid disorders and to identify new ways to engage male caregivers.

Keywords:Early psychosis, substance abuse, telemedicine

Revelation:Nothing to disclose.

University of South Carolina School of Medicine, Columbia, South Carolina, United States

Bottom:Sleep spindles are neural oscillations that occur during non-rapid eye movement (NREM) sleep in the frequency range of 10 to 15 Hz in rodents and in the frequency range of 12 to 16 Hz in humans. Starting from the thalamic reticular nucleus, sleep spindles are critical for synaptic plasticity related to the integration of new information. NREM sleep spindle aberrations are often associated with psychotic disorders such as schizophrenia (SZ) and bipolar disorder (BD), and are associated with deficits in procedural memory and cognitive function. Kynurenine acid (KYNA), a tryptophan metabolite synthesized from kynurenine by kynurenine aminotransferases (KATs) and a regulator of glial glutamatergic and cholinergic transmission, is elevated in the brain of patients with psychotic disorders. To better understand the role of KYNA in the etiology of psychotic disorders, we used two experimental systems. With fetal exposure to kynurenine (EKyn), KYNA levels are elevated prenatally and brain KYNA levels increase in adult EKyn males but not in EKyn females. Following acute kynurenine challenge in adulthood, brain levels of KYNA transiently increase in both sexes. We are currently investigating whether elevated KYNA levels have a negative effect on sleep spindle dynamics.

Methods:Sleep spindle dynamics were assessed during zeitgeber time (ZT) 0 to ZT 4, the first four hours of the light cycle in which rats easily accumulate NREM sleep. Male and female Wistar rats were implanted with telemetry transmitters to obtain polysomnography recordings combining electroencephalogram (EEG) and electromyogram (EMG) signals and activity levels. In Experiment 1, pregnant mothers were fed a formula mixed with 100 mg kynurenine (EKyn) or a control (ECon) liquid formula from embryonic day (ED) 15 to ED 22. Sleep behavior was assessed in young adults the day after birth (PD) 56, (N= 7-9 per group). In Experiment 2, adult rats were acutely challenged with kynurenine (100 mg/kg, i.p.) at a ZT of 0 (N= 10-14 per group). Manual analysis of sleep spindles during NREM was performed by applying a 10–15 Hz bandpass filter to the EEG signal and identifying the spindles by correcting the bandpass filtered signal with a cubic root mean square transform over a 750 ms window.

Results:EKyn male offspring showed a 32% reduction in NREM sleep spindle density compared to ECon (ECon: 8.4±3 spindles/min, EKyn: 5.7±5 spindles/min, ***PI number<0.001). Average spindle duration (ECon: 1.2 ± 0.04 s; EKyn: 1.1 ± 0.06 s;PI number>0.05), and the maximum spindle frequency remained between 11.5 and 12 Hz for both groups. NREM sleep spindle density was unchanged in female EKyn rats (ECon: 10.1 ± 0.5 spindles/min, EKyn: 10.4 ± 0.5 spindles/min;PI number> 0.05) and mean spindle duration also remained unchanged (ECon: 1.1 ± 0.02 s, EKyn: 1.1 ± 0.04 s;PI number> 0.05). In women, the maximum spindle frequency ranged from 12 to 12.5 Hz for both groups. Initial analysis of sleep spindles (N= 3) in an acute kynurenine challenge study tends to decrease sleep spindle density (main effect of kynurenine treatment,PI number= 0,16).

Conclusions:The results of this study investigate the effects of prenatal KYNA surge and acute KYNA surge, evaluating differences between long-term and transient KYNA shifts, on sleep spindle dynamics. We identified clear gender differences where sleep spindle dynamics are different in males and females, and only male EKyn offspring have deficits in sleep spindles compared to matched controls. Future work will investigate the mechanistic efficacy of inhibiting KYNA synthesis using the compound PF-04859989, a KAT II inhibitor, to ameliorate NREM sleep spindle dysfunction caused by KYNA elevation.

Keywords:Kynyurenic acid, sleep spindles, psychotic disorders, polysomnography

Revelation:Nothing to disclose.

Johns Hopkins University School of Medicine, Baltimore, Maryland, Stany Zjednoczone

Bottom:Given the recent legalization of marijuana in the US, the harmful effects of marijuana use during adolescence, a critical period for prefrontal cortex (PFC) maturation, have gained more attention as an environmental factor that increases the risk of mental disorders such as schizophrenia. Importantly, most cannabis users do not develop psychiatric symptoms, suggesting that cannabis exposure may be an environmental risk factor in those with a genetic predisposition to mental disorders. A previous study showed that the effect of delta-9-tetrahydrocannabinol (THC), one of the main psychoactive components of cannabis, on brain function is mediated mainly by the cannabinoid receptor type 1 (CNR1), which is widely distributed in the presynaptic regions of many types of neurons and regulates retrograde synaptic inhibition, thereby controlling cognitive and emotional behavior. Interestingly, recent studies have highlighted the importance of CNR1 expressed in astrocytes in causally mediating THC-induced cognitive deficits. However, the effect of adolescent marijuana on microglia remains unknown. In this study, we investigated the effects of adolescent THC exposure on microglial function, the disruption of which may be exacerbated by genetic traumas that increase the risk of psychiatric disorders, leading to abnormal PFC maturation and a factor in adult pathophysiology.

Methods:C57BL/6 mice (both sexes) were chronically exposed to THC (8.0 mg/kg) during adolescence via once-daily subcutaneous injections, followed by immunohistochemical and biochemical analyzes to determine the effects of juvenile exposure to THC in microglia. THC doses were selected based on our pilot tests and previous research. We also investigated whether THC treatment in adolescents and 16p11.2 duplication (16p11dup), a copy number variant (CNV) associated with psychiatric disorders, synergistically affect microglial function resulting in impaired PFC neuronal function and cognitive behavior in adulthood.

The statistical significance of the behavioral, biochemical, morphological and behavioral phenotype data was assessed using a two-tailed unpaired Student's testT-test with THC and vehicle treatment and two-way ANOVA with 16p11dup and control mice and THC and vehicle treatment as independent factors. After significant effects and interactions, multiple comparisons with post hoc Bonferroni tests were performed. Sample size was based on power analyses. The honor of itPI number< 0.05 was considered statistically significant.

Results:Treatment of adolescents with THC induced specific apoptosis of the microglia of the medial prefrontal cortex (mPFC) through CNR1-mediated mechanisms (PI number= 0,0001,T= 10.97, df = 10). These microglial phenotypes were particularly exacerbated by a predisposition to 16p11dup, leading to reduced intrinsic excitability of a particular type of mPFC pyramidal neurons and abnormalities in social novelty recognition (16p11dup x THC interaction (F1.49 392PI number= 0.0147) and memory (F1.30 = 5.665,PI number= 0.0239), causally induced by microglial CNR1. Microglia-specific RNA-seq-based gene expression profiling identified a key mediator of the convergent effect of teen THC and 16p11dup treatment (N= 4-6 per group,PI number< 0.05). We are currently investigating the intracellular molecular communication between mPFC microglia and neurons that may mediate the synergistic effects of adolescent THC exposure and the 16p11dup interaction.

Conclusions:We show that THC exposure in adolescents induces mPFC-specific microglial apoptosis through CNR1-mediated mechanisms. Adolescent exposure to THC and 16p11dup synergistically causes CNR1-mediated microglial apoptosis, leading to impaired mPFC neuronal maturation and social memory deficits. Our results indicate that modeling cell-type-specific mechanisms of rare genetic risks for mental disorders and environmental factors in adolescence may contribute to a better understanding of the pathophysiology of the disease.

Keywords:Cannabis, microglia, apoptosis

Revelation:Nothing to disclose.

Addiction and Mental Health Center, Toronto, Canada

Bottom:Introduction: Second-generation antipsychotics, such as olanzapine, are associated with significant metabolic side effects that contribute to obesity and insulin resistance, which are extremely common in patients with psychotic spectrum disorders. In addition to changes in body fat, antipsychotics interfere with glucose metabolism in the body through a mechanism directly related to the central nervous system (CNS). Postprandial maintenance of glucose homeostasis requires inhibition of hepatic glucose production in the presence of elevated glucose levels. It has been found that after a meal, the hypothalamus detects elevated glucose levels, which leads to the activation of ATP-sensitive hypothalamic potassium channels (KATP), signaling the liver to suppress hepatic glucose production. Disturbances of these pathways in the CNS hypothalamus result in dysglycaemia and insulin resistance. Upstream kinases of KATP channels also regulate this central glucose sensing pathway. The aim of this study was to identify olanzapine- and haloperidol-induced changes in hypothalamic protein kinase activity, particularly with respect to established modulators of the KATP channel, while evaluating glucose kinetics in vivo.

Methods:Methods: Pancreatic gold standard euglycemic clamps were used to assess changes in glucose kinetics in response to a continuous intracerebroventricular (ICV) infusion of glucose (2 mM, similar to CNS postprandial concentrations) or vehicle solution (5 μl/h, on day 3 ). Immediately prior to the start of the clamps, male Sprague Dawley rats were given concomitant acute subcutaneous injections of olanzapine (3 mg/kg), haloperidol (0.25 mg/kg) or vehicle at a dose based on clinical class D2.N=6-9/group. After euthanasia, the hypothalamus was harvested. Array analyzes of film hypothalamic samples consisted of determining the extent to which samples phosphorylated serine/threonine residues of reporter peptides on PamChip (PamGene International B.V), followed by deconvolution based on random sampling of protein kinase peptides using kinase substrate such as GPS 3.0, Kinexus Phosphonet (Kinexus Bioinformatics), PhosphoELM and PhosphoSite Plus.

Results:Results: As expected, ICV infusion (central) of glucose inhibited hepatic glucose production (PI number< 0.05) compared to vehicle, an effect that was abolished by treatment with olanzapine but not haloperidol. In parallel, ICV glucose stimulates the activity of several hypothalamic kinases, including extracellular signal-regulated kinase (ERK), which is known to activate K ATP channels. Haloperidol also increased the activity of kinases that can activate K ATP channels, including ERK, cAMP-dependent protein kinase (PKA), and cGMP-dependent protein kinase (PKG) (Z-score > 2, beyond 95% confidence interval), while olanzapine decreased activity (Z score > 2, outside the 95% confidence interval). It seems that changes in kinase activity induced by haloperidol and olanzapine may have prepared the hypothalamus for or against the inhibition of hepatic glucose production in the presence of ICV glucose, respectively.

Conclusions:Conclusions: Olanzapine and haloperidol have a different effect on the network of protein kinases that change the activity of K ATP channels in the hypothalamus, which may explain their differential effect on hypothalamic glucose sensitization and regulation of hepatic glucose production. Whether these changes in kinase activity directly result in dysfunctional glucose metabolism associated with antipsychotics remains to be determined.

Keywords:Antipsychotic, Kinome Array, Hypothalamus

Revelation:Nothing to disclose.

Takeda Pharmaceuticals, Cambridge, Massachusetts, Stany Zjednoczone

Bottom:The lack of effective treatment options for the negative symptoms and cognitive impairment associated with schizophrenia (CIAS) represents a significant unmet medical need and increases the associated economic burden. It is believed that deficits in glutamatergic transmission play an important role in the pathophysiology of these symptoms. Luvadaxistat, a potent inhibitor of D-amino acid oxidase, is thought to increase NMDA-dependent glutamatergic signaling in the brain by increasing levels of D-serine, an absolute agonist of the NMDA receptor. Luvadaxistat has previously been shown to improve social interaction and cognition in rodent behavioral models. Here we report the efficacy and safety results from INTERACT, a phase 2 study of the adjuvant luvadaxisstat in adults with schizophrenia with persistent negative symptoms.

Methods:INTERACT was a randomized, double-blind, placebo-controlled, placebo-controlled study conducted in Europe and North America (NCT03382639). Eligible participants were 18 to 60 years of age with symptomatic stable schizophrenia, had a baseline Short Negative Symptoms Scale (BNSS) score ≥ 28 (12 items, excluding item 4), and were receiving primary antipsychotic treatment. To overcome potential confounding effects, subjects with depressive and extrapyramidal symptoms were excluded. The study included a 28-day screening period, a 14-day single-blind placebo phase-in period for prospective assessment of adherence and stability of the BNSS score, and a 12-week double-blind treatment period.

The primary endpoint was change in negative symptoms as measured by the 12-week change from baseline in the Positive and Negative Syndrome Scale - Negative Symptom Factor Scale (PANSS NSFS). For the CIAS, secondary endpoints included the 12-week change from baseline in the Brief Assessment of Cognition in Schizophrenia (BACS) composite score, a measure of cognition in all domains, and the Cognition in Schizophrenia Scale (BACS). ) Schizophrenia (SCoRS), a measure of everyday cognitive functioning that requires input from an adult informant. Secondary endpoints are not fold-corrected. Safety endpoints included an assessment of treatment-related adverse events (TEAEs). Dose selection was based on target D-serine uptake and elevation.

Results:A total of 256 subjects were randomized in a 3:2:2:2 ratio to receive placebo, luvadaxistat 50 mg, 125 mg, and 500 mg, respectively, of whom 228 (89.1%) completed the study. The mean age of participants was 40 years (range 18 to 60 years), 168 (65.6%) were male and 208 (81.3%) were Caucasian. Demographic and baseline characteristics were evenly distributed between the treatment groups.

For negative symptoms, no significant improvement in PANSS NSFS compared to placebo was observed with luvadaxistat 50 mg, 125 mg, or 500 mg at Week 12 (PI number= 0,426,PI number= 0,362 ePI number= 0.808 respectively). From baseline to week 12, LS mean changes in PANSS NSFS were -3.3 (95% confidence interval [CI], -4.3 to -2.2), -3.4 (95% CI) , -4.4 to -2.3) and -2.5 (95% CI, -3.6 to -1.5) with luvadaxistat 50 mg, 125 mg and 500 mg, respectively, and -3.1 (95% CI, -4.0 to -2.3) with placebo.

For CIAS, a nominally significant improvement was observed for luvadaxistat 50 mg compared to placebo in the BACS total score (PI number= 0.031; effect size, 0.4) and the investigator's total SCoRS score (PI number= 0.011; effect size, 0.4), but not for luvadaxist 125 mg or 500 mg. For the composite BACS result, a 12-week LS mean change from baseline of 4.6 (95% CI, 2.7 to 6.5) was observed for luvadaxistat 50 mg compared to 2.3 (95% CI, 0.7 to 3.9) for placebo. For the SCoRS investigator overall score, a 12-week LS mean change from baseline of -3.8 (95% CI, -5.3 to -2.3) was observed for luvadaxisstat 50 mg versus -1.6 (95% CI, -5.3 to -2.3 CI, -2.9 to -0.3) with placebo.

A total of 76 subjects (29.7%) had a TEAE, of which 23 (9%) were considered drug related by the investigator. TEAEs occurring in > 5 subjects (2%) included headache, insomnia, and weight gain, which were observed at similar rates in the luvadaxisstat and placebo groups. Mild, moderate, and severe TEAE occurred in 49 (19.1%), 24 (9.4%), and 3 (1.2%) participants, respectively. Serious cases of rhabdomyolysis (placebo,N= 1) and chronic cholecystitis (luvadaxistat 50 mg,N= 1) not considered drug-related. Severe schizophrenia was reported in one subject receiving luvadaxistat 125 mg, which was considered a severe drug-related ASD. Four serious TEAEs were reported in the placebo group. none were drug related. Five participants, including three who were on placebo, experienced TEAEs that led to treatment discontinuation (increased blood creatine phosphokinase, elevated liver function tests, psychiatric disorders). No deaths have been reported.

Conclusions:Luvadaxistat showed no significant efficacy in the treatment of negative symptoms of schizophrenia at the three doses tested. For CIAS, an inverted U-shaped dose response was observed for luvadaxistat at the 50 mg, 125 mg and 500 mg doses. Only luvadaxistat 50 mg showed an efficacy signal as measured by the composite BACS score and the investigator's total SCoRS score, with a clinically significant effect size of 0.4 for each. Additional clinical trials are needed to better assess this signal of efficacy. Based on previous clinical experience, luvadaxistat was generally well tolerated in the INTERACT study.

Keywords:Cognitive Disorders Associated with Schizophrenia, Negative Symptoms of Schizophrenia, D-Amino Acid Oxidase Inhibitor

Revelation:Takeda Pharmaceutical Company Ltd, Cambridge: employee (self-employed)

University of California, San Francisco, San Francisco, California, United States

Bottom:Synchronization of alpha-band neural oscillations is the dominant EEG signal when people are awake but resting. In contrast, alpha desynchronization to relevant events is thought to shift information processing resources from the internal state to relevant external stimuli. In the current study, we hypothesized that event-related alpha desynchronization (ERD) would be insufficient during reward processing in the SZ (i.e., less alpha attenuation for wins versus losses) and that reward-related ERD deficits would be related. with negative symptoms. In addition, since the ERD is thought to reflect resting-state "desynchronization" to prepare the brain for engagement with the external environment, we hypothesized that trait rumination, a reflection of excessive internal focus, would be associated with reduced alpha ERD.

Methods:EEGs were recorded when participants with schizophrenia (SZ = 54) and healthy controls (HC = 54) performed a gambling machine task. Event-related power measures from principal component analysis were extracted into delta, theta, and alpha frequency domains and then compared between groups and peer reward outcomes. Individual differences between alpha ERD and negative symptoms (SZ) and rumination traits (HC, SZ) were also examined.

Results:Significant group X reward score interaction (PI number=   0.018) was explained by HC showing significant posterior occipital alpha ERD for wins, relative to near losses (PI number< 0.001), compared to SZ not modulating alpha power for wins vs. losses (PI number> .1). Alpha power was not associated with negative symptoms (PI number> .1) among people with SZ. However, among all participants (HC + SZ), less alpha ERD to reward performance was associated with more trait chewing, for both wins (PI number= 0.005) and losses (PI number= 0.002), with no observed group differences in the slope of these relationships and persistent rumination effects after adjusting for theta and delta power.

Conclusions:These findings indicate that event-related alpha power modulation is altered in schizophrenia during reward outcome processing, even when reward achievement poses minimal demands for higher-order cognitive processes during a simple automaton task. In contrast, theta and delta power bands showed no group differences in modulation based on reward score. We did not observe associations of negative symptoms with alpha ERD, however, high trait rumination was associated with lower ERD for rewarding feedback, regardless of reward score valency and group affiliation.

Keywords:Alpha oscillations, reward processing, schizophrenia

Revelation:Nothing to disclose.

University of California, San Diego, San Diego, California, United States

Bottom:After decades of prognostic research in schizophrenia (SZ) without an FDA-approved prognostic drug, therapy development has focused on identifying biomarkers that predict prognostic sensitivity to treatment. Negative mismatch (MMN) and auditory gamma-band steady-state responses (ASSR), electroencephalogram (EEG)-based measures of early auditory information processing (EAIP) predict clinical and cognitive benefits of targeted cognitive training (TCT) to treatment - resistant SZ patients but of moderate strength. An important question is: can we enhance the ability of EAIP 'biomarkers' to predict the cognitive and functional benefits of TCT in SZ patients? For example, the MMN and ASSR measures use isolated fragments of sound (tones, clicks), presented in the artificial context of a lab environment. It is possible that EEG measurements made using naturalistic contextual sound stimuli may more accurately reflect the brain's ability to AEIP and thus be more sensitive to AEIL deficits and treatment sensitivity. However, EAIP measurements require millisecond-level stimulus control in a structured testing session, and thus cannot be easily evaluated under naturalistic conditions. Virtual reality (VR) technology provides the naturalistic context and rigorous experimental control needed to create and evaluate more environmentally relevant AEIP measurements. Here, we developed a VR-based EEG model to measure AEIP evoked by naturalistic sound stimuli (e.g., VR-based metrics for an hour-long TCT session.

Methods:Two VR-EEG paradigms have been developed to measure MMN and ASSR. Carefully screened, clinically stable, psychiatrically healthy adult (HS) and SZ patients aged 18 to 45 with an IQ over 80 as measured by the Wide Range Performance Test-3 (WRAT-3) completed the screening visit, followed by a test day. Eligible participants underwent a comprehensive neurocognitive (MATRICS Consensus Cognitive Battery (MCCB)) and functional (Brief UCSD Performance-Based Skills Assessment (UPSA-B)) assessment at the screening visit, and on the day of the study standard laboratory EEG completed and VR measurements before and after an hour-long TCT "sound search" session in a random, balanced design. The Pearson correlation was used to determine the validity, reliability and potency of the VR-based MMN biomarker and gamma-induced enhancement (γEP) in healthy subjects (HS) and SZ patients.

Results:So far, 5 people have completed the test [N= 4(HS)eN= 1(SZ)]. Participants were in their mid-20s (28 ± 10 years), in high school (14.4 ± 2.3 years), women (60%) with WRAT-3IQ 111.7 ± 11.6, MCCB cumulative score 14 (N= 1 (SZ)) and 52.2 ± 7.3 (N= 4 (HS)) and a total UPSA-B score of 9 (N= 1 (SZ)) and 18 ± 1.4 (N= 4 (HS)). Participants reported a mild level of discomfort on the Simulator Disease Questionnaire (SSQ) and high scores on the Presence Questionnaire (PQ) (80.5 (range: 58-120)) after the VR state, the latter indicating an increased sense of presence in the VR environment. Taken together, these findings indicate that VR-based EEG paradigms were immersive and well tolerated. Baseline average TCT auditory processing speed pre-training (APS)   =   65 milliseconds vs. TCT after training APS = 36 milliseconds (N= 4 (HS)), indicating a 55% increase in APS. Overall, participants averaged 15 (range 12–21) (N= 4 (HS)) e 6 (N= 1(SZ)) training block. Physical testing and VR-based analysis of neurophysiological (EEG) data are ongoing and results will be reported in full. However, a preliminary look at the EEG data (N= 1 (HS)eN= 1 (SZ)) showed that VR-based MMN and γEP are comparable to standard VR-based MMN and γEP, with VR-based measurements in this SZ patient being reduced compared to HS and showing plasticity at one hour TCT .

Conclusions:Our preliminary findings and observations of VR-based EEG data suggest that VR-based EEG paradigms are well-tolerated, riveting, and can induce strong MMNs and γEPs in HS and SZ patients. Testing is ongoing and a full comparison of VR based EEG measurements and TCT performance will be presented.

Keywords:Virtual Reality, Negative Auditory Mismatch, Targeted Cognitive Training, Steady State Auditory Response, Schizophrenia-Related Cognitive Disorder

Revelation:Nothing to disclose.

University of Texas Medical Department, Galveston, Texas, United States

Bottom:GPR52 is an orphan G protein-coupled receptor that is selectively expressed in the brain and has been identified as a promising drug target for mental disorders including schizophrenia and substance use disorders. GPR52 is mainly expressed in the ventral striatum of the human brain in dopamine D2 spiny neurons, where it can functionally modulate cAMP signaling and antagonize neuronal signaling through D2 dopamine receptors. Here, we review the signaling crosstalk between GPR52 and D2R and report drug discovery efforts to generate novel GPR52 agonists and evaluate the lead agonist for antipsychotic-like activity.

Methods:To investigate the potential signaling interaction between GPR52 and D2R, human GPR52 and D2 receptors were co-expressed in HEK293 cells, and agonists or antagonists of both receptors were tested for cAMP signaling in living cells (Glosensor assay). Medicinal chemistry and pharmacology were then used in a systematic study of the structure-activity relationship of an indoline-carboxamide-based pharmacophore to elucidate ligand characteristics critical for GPR52 activation and optimize a GPR52-like agonist drug. GPR52 selectivity and off-target profiling were achieved using competitive radioligand binding to >30 off-target CNS and drug-like pharmacokinetics and brain penetration were determined in rats. Vehicle or increasing doses of selected GPR52 agonists were tested in vivo for activity on amphetamine-induced locomotion using c57BL6J mice (N= 12 animals/treatment group). A one-way ANOVA for cross-subject design was used for horizontal activity analysis.

Results:Expression of human GPR52 in HEK293 cells significantly increased baseline cAMP levels over 100-fold, indicating high constitutive receptor activity. We hypothesize that this highly constitutive GPR52 signaling may modulate the basal cAMP tone in cells to oppose or modulate Gi/o-coupled D2R signaling. D2R expression alone in HEK293 cells resulted in low baseline cAMP levels, and treating the cells with a D2R agonist or quinpirole antagonist haloperidol produced minimal changes in cAMP levels (∼1–2 thousand light counts/s). Notably, co-expression of GPR52 with D2R significantly increased basal cAMP levels, and this elevated basal cAMP allowed for larger windows of agonism and inverse agonism by the D2R ligands quinpirole (decrease by ~500,000 light counts/s) and haloperidol (at 150,000 light counts) /s/s). When cells were co-treated with the GPR52 agonist PW0787 to further increase cAMP, the D2R efficacy of haloperidol inverse agonism increased even more significantly. Medicinal chemistry and pharmacology were then used to elucidate ligand properties that are critical for GPR52 activation. We determined that the two lower aromatic moieties of the main agonist were susceptible to additional medicinal chemistry with substituents that appear to modulate both the potency and efficacy of the agonist. Surprisingly, when the nitrogen-containing ring of the indoline system was split into more flexible variants, this further increased the potency and efficacy of the agonist (EC50: ~40 nM) while maintaining excellent target selectivity, plasma exposure and serum concentration. Dose-dependent testing of the optimized agonist PW0787 revealed that treatment of mice with 3 and 10 mg/kg significantly reduced amphetamine-induced hyperactivity, indicating antipsychotic-like activity.

Conclusions:These studies indicate that the orphan striatal receptor GPR52 can determine cellular cAMP levels and that GPR52 expression and activation profoundly regulate D2R signaling and pharmacology. Drug discovery efforts have also elucidated several agonists with optimized potency and efficacy, with PW0787 being a novel orally bioavailable brain-penetrating antipsychotic GPR52 agonist.

Support for this research work was provided by the UTMB Addiction Research Center, NIDA T32 DA07287 (DEF), NIDA 1U18DA052543-01 (JAA), and the PhRMA Foundation Starter Grant RSGPT18 (JAA).

Keywords:Drug discovery - new approaches, schizophrenia, antipsychotics, GPCR, new targets, preclinical pharmacology

Revelation:New Atlas Biotechnologies: Contributed research (own)

University of Minnesota, Minneapolis, Minnesota, United States

Bottom:Thalamocortical disconnection is consistently observed in psychosis and may represent a clinical biomarker. However, its relationship to the underlying symptomatology across the spectrum of psychosis remains unclear. Here, we directly explore the relationship between thalamocortical connectivity and clinical and cognitive variables in a diagnostic sample of patients with psychosis (including schizophrenia, schizoaffective disorder, and bipolar disorder with psychosis), their first-degree relatives, and healthy controls.

Methods:Patients with psychosis, including those diagnosed on the schizophrenia spectrum (N= 99) or bipolar diagnosis (N= 33), your first degree relatives (N= 74) and a group of healthy controls (N= 43) underwent resting fMRI in addition to clinical and cognitive evaluation by the Psychosis Human Connectome Project. For individual pre-processing and noise removal, the ART toolkit and aCompCor in the "CONN" toolkit were used. Seed-based connectivity analyzes were then based on an anatomically defined bilateral thalamic region of interest (ROI). Whole Subject Brain Effects examined the relationship between thalamocortical connectivity and clinical symptoms (as measured by the Brief Psychiatric Rating Scale, BPRS) and general cognitive function (as measured by the Schizophrenia Cognitive Brief Rating; BACS) controlled for the subject's age and sex. Two-way tests of significant clusters were based on the correction of the false discovery rate (FDR) in the whole brainPI number< 0,01.

Results:Individual differences in psychiatric symptoms between groups were negatively associated with thalamocortical connectivity (higher BPRS scores correspond to lower connectivity) in prefrontal areas including left and right middle frontal gyrus and anterior cingulate cortex (ACC). Individual differences in global cognition between groups were positively correlated (higher BACS scores correspond to greater connectivity) with thalamocortical connectivity in the ACC and right insula. Post hoc analyzes of significant ROIs showed that these effects were robust to single-subject motion (allPI number’s < .0001). Finally, we examined whether thalamo-target ROIs significantly related to symptoms or cognition also differed between groups. Controlling for BPRS and BACS scores respectively, each ROI showed a significant group effect (allPI number’s < .012), which is caused by reduced connectivity in people with schizophrenia and bipolar disorder.

Conclusions:In the diagnostic sample, thalamocortical connectivity was negatively associated with psychiatric symptoms in cognitive control-related prefrontal areas and positively associated with global cognitive scores in the anterior cingulate and insular regions according to the salience network. Susceptibility to psychiatric symptoms and cognitive deficits in patients with psychosis and those with a genetic predisposition may be due to decreased thalamo-prefrontal and thalamo-cortical connectivity, respectively.

Keywords:Psychosis, thalamocortical connectivity, cognition, symptomatology

Revelation:Nothing to disclose.

VA Boston Healthcare System, Harvard Medical School, Brockton, Massachusetts, Estados Unidos

Bottom:Changes in brain connectivity may underlie neuropsychiatric diseases such as schizophrenia. Here we assessed the geometric pattern of structural connections between the frontal (FC) and caudate (Cd) cortices in 56 healthy control subjects (HC) and 155 early psychotic patients (108 EP-non-emotional and 47 EP-emotional) from the Human Connectome project (HCP); age: from 18 to 33 years old; Mean: 23.21 years; sex: 74 women and 137 men. We used our novel whole-brain diffusion magnetic resonance tractography (dMRI) fiber cluster analysis method to assess the organization of the brain's frontotemporal wiring, which allows us to quantify the degree of deviation from the parallel topographical pattern.

Methods:The data used in this study comes from the Human Connectome Project for Early Psychosis (HCP-EP) joint research dataset (MPI: Shenton, Breier). Diffuse MRI data from 3 HCP centers (Indiana University, Massachusetts General Hospital and McLean Hospital) were harmonized using our harmonization methodology (Cetin-Karayumak S et al., 2019). From this harmonized dataset, we generated a whole-brain tractography using the odorless 2-tensor Kallman filter (VHF) tractography methodology (Malcolm JG et al., 2010). To enable the identification of the fiber plots of the frontal cortex (FC) and the caudate nucleus (Cd), we used a data-driven fiber cluster atlas (Zhang et al., 2018), which allows the plotting of the entire brain of 2,000 fibers grouped according to the white matter anatomy (WM) ( i.e. geometric arrangement of fibers). Fiber groups of interest (i.e., FC to Cd) from the WM of the whole brain were then identified for each subject in each core group according to their combined anatomical brain regions. We examined several Freesurfer FC regions, including the orbital, lateral and medial FC regions, and the caudate nucleus. We identified 17 groups of WM fibers connecting FC and Cd in the left and right hemispheres in each study group. To quantify the topographic relationship of these fiber clusters, we measured the average fiber cluster distances between the endpoints of the fiber clusters in the frontal cortex (i.e., tail-to-tail distance).

Results:We conducted the following preliminary analyses. For each group (HC, EP-Non-Affective and EP-Affective) in each hemisphere, we generated plots (not shown) from the 17 fiber groups (with 136 pairs of fiber groups, yielding 136 data points), showing the relationship between cortical and corresponding distances and caudal distances of the received pairs of fiber clusters connecting the frontal and caudal cortex. For the left hemisphere (LH), we found a non-linear relationship between cortical and caudate distances in the HC in both groups of patients (EP-non-affective and EP-affective) based on the scores of 10-pair clusters. For the right hemisphere (HR), we found a similar non-linear relationship driven by the effects of 10-pair groups in HC and a flatter non-linear relationship in the EP-Non-Emotional and EP-Affective patient groups. Notably, one cluster of fibers originating in the inferior frontal gyrus, pars triangularis, was significantly overrepresented in these 10 cluster pairs.

Conclusions:Using dMRI fiber cluster topography analysis in the HC, we show 1) the general projection pattern of the FC wiring between the FC and the caudate nucleus in the HC deviates from the parallel topographic organization, due to the bilateral convergence pattern in region-specific fiber clusters; 2) this is similar to what we found in our previous study with healthy subjects (Levitt, 2021). and 3) groups of patients (EP-non-emotional and EP-affective patients) appear to have a similar pattern of connectivity to controls in LH, but appear to have a flatter trajectory than controls in PR. The poster will provide a more detailed, site-specific analysis of the individual fiber clusters in the different thematic groups.

Keywords:Early-onset psychosis, diffusion-weighted imaging, frontal circuits, structural connectivity of the brain

Revelation:Nothing to disclose.

Baylor College of Medicine, Houston, Texas, United States

Bottom:Although genome-wide association studies have identified hundreds of common positional variants with little effect on schizophrenia risk, identification of rare high-effect variants has proven difficult compared to other neurodevelopmental disorders. In complex disorders, those with more severe or pre-existing symptoms are at higher risk of rare and damaging high-effect variants. Consequently, extreme phenotypic sampling—exclusively examining individuals at the extremes of the distribution of a phenotypic trait—can significantly reduce the sample size needed to detect rare high-effect variants. We hypothesized that severely affected and chronically hospitalized individuals with schizophrenia are at higher risk of rare and damaging variants compared to individuals with common forms of schizophrenia and controls.

Methods:We performed whole-genome sequencing of 114 subjects with severe and extremely refractory schizophrenia (SETRS), 198 subjects with typical schizophrenia, and 4146 controlled quality controls. We defined SETRS patients as those whose disease severity required continuous hospitalization for at least 5 years in a long-term institutional facility in New York State because of primary symptoms of schizophrenia.

We compared the burden of rare and deleterious loss-of-sense and loss-of-function variants among SETRS, typical schizophrenia, and controls in "intolerant" genes with reduced functional variance in the general population. Previous studies on schizophrenia have shown that the weight of control for rare loss-of-function (odds ratio, 1.26) and deleterious missense variants (odds ratio, 1.06-1.25) is concentrated only in those genes that are abolition."

For gene pool bias analysis, we recorded the total number of rare and deleterious variants in cases and controls for a given gene pool and assessed the significance of this difference between genetically defined ancestral groups by means of a two-tailed Cochran–Mantel–Haenszel test with score sizes that are represented as odds ratios. . We computed multiple comparisons using the Benjamini-Hochberg false discovery index. Specifically, we considered each assay to qualify for missense and loss-of-function variants in each of the gene sets analyzed.

Results:Patients with SETRS had a mean age of 60.9 years, were mostly male (69.3%), and had a mean hospital stay of 24.9 years. The mean age of onset was 18.4 years for men and 18.3 years for women, and only 14.9% of participants had children, consistent with a strong negative selection effect.

Those with SETRS had a high burden of rare loss of function (odds ratio (OR) 1.97; 95% confidence interval [CI], 1.42–2.75;PI number= 6.1 x 10-5) and deleterious nonsense variants in resistant genes (odds ratio, 2.59; 95% CI, 1.77-3.78;PI number= 5.4 x 10-7). 47.4% of people with SETRS carried at least one rare and deleterious missense or loss-of-function variant in resistance genes, compared with 30.3% of people with typical schizophrenia (OR, 2.06; 95% CI, 1.25 -3.43;PI number= 3 x 10-3) e 26% doz kontrole (OR, 2,55; IC 95%, 1,72-3,78;PI number= 1,5 x 10-6).

Restriction to genes previously associated with schizophrenia risk further increased the enrichment of 8.8% of SETRS individuals carrying a deleterious missense or loss-of-function variant compared with 2.5% of typical schizophrenia (OR, 4, 62, 95% CI, 1.26– 16.PI number= 0,02) e 1,5% dos kontrole (OR, 7,57; IC 95%, 3,23-16,24;PI number= 3 x 10-9).

Conclusions:In this study, we found that screening people with extremely severe forms of schizophrenia resulted in significantly better ability to detect rare variants associated with the disease. The high prevalence of rare risk factors in people with SETRS suggests future clinical opportunities for risk prediction, prognostic stratification, and genetic counseling. These findings have implications for the design of future genetic studies in schizophrenia and highlight a strategy to reduce phenotypic heterogeneity and improve gene discovery efforts in other neuropsychiatric disorders.

Keywords:Genomics, DNA, whole genome, sequencing, schizophrenia, treatment-resistant schizophrenia

Revelation:Nothing to disclose.

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Bottom:Oxidative stress and the resulting oxidative damage to key cellular components have been suggested to contribute to the complex pathophysiology underlying the clinical syndrome of schizophrenia (SZ). The main antioxidant system in the brain, glutathione (GSH), regulates the abundance of types of oxidants and, consequently, oxidative damage. It has been proposed that insufficient antioxidant defense of the brain, caused by direct GSH availability deficiencies and/or excess oxidants overwhelming the GSH system, is a pathogenic mechanism in the SZ disease process.

While some sources of oxidants, such as mitochondrial production of reactive oxygen species, are ubiquitous throughout the brain, others are brain region dependent. For example, the striatum has a particularly rich source of oxidative forms, dopamine degradation, which is largely absent in other regions such as the dorsolateral prefrontal cortex (DLPFC), which receives infrequent dopaminergic inputs. In addition, in primates, measurements reflecting presynaptic dopamine tend to be greater in the dorsal striatum (DS) than in the ventral striatum (SV). SZ appears to be associated with an exaggeration of these physiological differences in dopamine innervation in the striatum, such that excess dopamine synthesis and release is more pronounced in SD than in SV. Since dopamine degradation may be an important additional source of oxidative species, markers of oxidative stress may be more pronounced in DS than in SV and DLPFC. To explore this idea, we used quantitative mass spectrometry to quantify the markers of 1) the GSH antioxidant system and 2) oxidative lipid damage in the DLPFC, DS and SV in individuals with SZ.

Methods:Brain samples from 50 people were processed for metabolomic analyses. Each SZ item (N= 25) were matched to the intact comparator (UC) for gender and as closely as possible for age and post-mortem (PMI). Tissue samples from all subjects were processed together in one experiment. The study groups did not differ in mean age, PMI, brain pH, RNA integrity count, or tissue storage time (allPI number>0.2). DLPFC gray matter, DS and VS were used for liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the abundance of free GSH and glutathione disulfide (GSSG). GSSG is produced by the antioxidant action of GSH. LC-MS was used to quantify the abundance of malondialdehyde (MDA), a product of lipid oxidative damage. Two-way ANCOVA models were performed that included the appropriate dependent measure, the independent variables of the diagnostic group and brain region, and any relevant covariates. by CohenHellocalculated to determine the SZ disease effect size. Statistical significance was assessed atPI number< 0.05, and the statistical significance level of the trend was assessed as 0.05 ≤PI number< 0,1.

Results:No significant effect of brain region was detected for GSH abundance (F2.96 = 1.9,PI number= 0.15). However, there was a significant main effect for the SZ diagnosis trend level (F1.47 = 3.6,PI number= 0.06). In contrast to the observed effects of oxidative stress, the disease effect of SZ was associated with higher GSH in DM (Hello= +0,43) e VS (Hello= +0.51) and no effect in DLPFC (Hello= −0.04) relative to UC. There was no significant interaction of diagnosis and brain region with GSH abundance (F2.96=2.4,PI number= 0,1).

For the GSSG, there was a significant effect for the brain region (F2.96 = 12.5,PI number= 0.00002); post hoc analyzes revealed a 44% greater number of GSSGs in DLPFC compared to DS and VS (allT= 2,9,PI number< 0.005). No significant SZ main effect was detected for the GSSG abundance (F1.48 = 0.76,PI number= 0.4). No significant interaction of diagnosis and brain region on the number of GSSGs (F2.96=0.61,PI number= 0,5).

For MDA, there was a significant main effect for the brain region (F2.96 = 13.6,PI number= 0.000006); subsequent analyzes showed a 22% higher abundance of MDA in DS compared to DLPFC (T= −3,3,PI number= 0.002). No significant main effect of SZ on MDA abundance was detected (F1.48=0.6,PI number= 0.5). However, there was a significant interaction between the level of propensity to diagnose SZ and the brain region of MDA abundance (F2.96 = 3.1,PI number= 0.05). Post hoc analysis showed no significant effect of SZ on MDA abundance in DLPFC, DS or VS (allT≤ 1,6,PI number> 0.3) and the size of the SZ disease effect on DS (Hello= −0,12), DLPFC (Hello= +0,24) e VS (Hello= +0,42).

Conclusions:In contrast to the effect of oxidative stress on GSH abundance, we detected a higher concentration of free GSH in DS and VS in SZ and no effect of SZ on GSH abundance in DLPFC. These findings suggest that the GSH system may increase antioxidant capacity in areas that may have a higher oxidative load in the SZ. The lack of a significant effect of SZ on MDA, a product of lipid oxidation, further suggests that the GSH antioxidant system adequately protects against oxidative damage. Finally, the significantly greater abundance of MDA in SD patients is consistent with a greater potential oxidative load in this region of the striatum, given the greater dopamine innervation relative to the SV and DLPFC. Collectively, these data suggest that the impaired GSH antioxidant system and increased cellular oxidative damage are not found in the DLPFC, DS or SV in individuals with SZ and thus may not represent common pathogenic mechanisms in SZ.

Keywords:Glutathione, lipid oxidation, autopsy, schizophrenia, DLPFC

Revelation:Nothing to disclose.

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Bottom:Clozapine (CLZ) shows exceptional clinical efficacy compared to other antipsychotics. Previous work has linked the major metabolite of CLZ, N-desmethylclozapine (NDMC), to prognostic effects due to its presumed effects on the cholinergic system. Here, we investigate and further extend this relationship with functional neuroimaging in a homogeneous cohort of nineteen acutely ill participants with treatment-resistant schizophrenia (TRS) in the treatment of CLZ.

Methods:We followed the cohort for 12 weeks of CLZ treatment. In addition to functional neuroimaging at rest, baseline and 12-week CLZ values ​​were recorded. Cognition and changes in the functional connectivity of the forebrain to the dorsal prefrontal cortex were studied in relation to the CLZ/NDMC relationship.

Results:Consistent with previous findings, we showed a positive association between NDMC levels and measures of general cognitive ability (PI number= 0.006), working memory (PI number= 0.00025) and notes (PI number= 0.0055). In addition, we observed a significant correlation between basal forebrain-DLPFC connectivity and CLZ/NDMC ratios in the treatment of CLZ (PI number= 0.019) and negative correlation with working memory scores (PI number= 0,04).

Conclusions:These findings may reflect the effect of CLZ and NDMC on the cholinergic system by acting on muscarinic receptors. The results of this work further support the potential pro-cognitive effects of NDMC and show a longitudinal neuroimaging correlate of plasma drug ratios.

Keywords:Schizophrenia, clozapine, N-desmethylclozapine, functional connectivity, working memory

Revelation:Nothing to disclose.

Maryland Psychiatric Research Center, University of Maryland School of Medicine, Catonsville, Maryland, Stany Zjednoczone

Bottom:People with schizophrenia have been found to exhibit higher than normal levels of inflammation (1). An important example of this is that prenatal maternal infection is associated with an increased risk of schizophrenia in the offspring (2). In addition, the presence of an autoimmune disease is associated with a 45% increased risk of developing schizophrenia (3). Studies have also shown a link between immune reactions to gluten (gliadin) and the occurrence of neurological/psychiatric symptoms (4). It has recently been discovered that approximately 30% of people with schizophrenia have high levels of antigliadin antibodies (AGA IgG) (5). In preliminary studies, a gluten-free diet has been shown to reduce negative symptoms in people with schizophrenia who have high levels of AGA IgG (6). The results also showed associations between AGA IgG and several biomarkers, including TNFα and IL-1β, which were reduced among participants on a gluten-free diet. This suggests that inflammation is related to these antibody levels. These findings have sparked interest in understanding which pro-inflammatory cytokines are associated with high AGA IgG antibodies to better understand the underlying mechanisms of the nearly 30% of people with schizophrenia with high anti-gliadin antibodies.

Methods:Serum biomarkers were analyzed from an existing dataset of participants with a DSM-5 diagnosis of schizophrenia or schizoaffective disorder aged 18 to 64 years (N= 417). Serum samples were taken to determine AGA IgG levels as well as a number of pro-inflammatory markers (GM-CSF, IFNγ, IL-17A, IL-1β, IL-6 and TNFα). AGA IgG was measured and analyzed using semi-quantitative ELISAs from Inova Diagnostics. Pro-inflammatory cytokines (GM-CSF, IFNγ, IL-17A, IL-1β, IL-6 and TNFα) were measured using the EMD Millipore MAP Human Cytokine Magnetic Bead Panel (Luminex Bead-Based Immunoassays (Millipore, Billerica NY). completed using the Bioplex 200 platform (Biorad, Hercules CA) to determine the concentration of multiple target proteins in the samples Participants were divided into groups based on the level of AGA IgG in their sera (≥20=positive, <20=negative). were compared between the two groups (AGA IgG negative vs. positive) and analyzed for significant differences using the Wilcoxon statistical test Correlations between IgG AGA (positive vs. negative) and all six cytokines were also analyzed using the Wilcoxon statistical test.

Results:The mean age of the sample was 41.96 years (SD = 12.46), with 134 females and 282 males. The racial demographics of the sample were as follows: 56% were black or African American (N= 236), 34% were white (N= 145), 2% were Asian (N= 10) and 6% are other reported or unknown racial categories. The overall AGA IgG positivity rate in the sample was 38.1% (N= 159). All mean cytokine values ​​were significantly higher in those in the AGA IgG-positive group compared to those in the AGA-IgG-negative group (PI number<0.001). Positive AGA IgG (≥20 U) was moderately associated with all pro-inflammatory cytokines: GM-CSF (R= 0,36,PI number< 0,001), IFNγ (R= 0,28,PI number<0.001), IL-17A (R= 0,23,PI number< 0.05), IL-1β (R= 0,33,PI number< 0,001), IL-6 (R= 0,37,PI number< 0,001) e TNFα (R= 0,20,PI number< 0.05). However, in the AGA IgG negative group (<20 U),Rall values ​​were less than 0.16 and only two correlations reached significance: IL-1β (R= 0,16,PI number< 0.05) i IL-6 (R= 0,13,PI number< 0.05). A sample of over 400 participants provides a high statistical power of 1.00 to detect an association between antigliadin antibodies and pro-inflammatory cytokines, with associated effect sizes ranging from moderate to highR= 0,24 eR= 0.38 for all 6 cytokines.

Conclusions:All pro-inflammatory cytokines were moderately associated with IgG AGA positivity, but no association was observed in the larger IgG AGA negative group. This suggests that the association between antigliadin antibodies and inflammatory biomarkers only exists for people with elevated antigliadin antibodies (AGA IgG ≥ 20). These results reflect previous data showing associations between positive AGA IgG and TNFα and IL-1β (7). These results further support a subgroup of schizophrenic patients with high inflammation and immune activation that may differ from patients without this component. The concept of a potential subgroup of patients with schizophrenia whose symptomatology may be associated with inflammation and immune activation may help identify new therapeutic targets and better individualize treatment.

Keywords:Schizophrenia (SCZ), Gluten, Gliadin, Inflammation, Anti-gliadin antibodies (AGA)

Revelation:Nothing to disclose.

Italian Institute of Technology (IIT), Genoa, Italy

Bottom:22q11.2 deletion syndrome (22q11DS) is a genetic syndrome with high penetrance for developmental neuropsychiatric disorders (Drew et al. 2011; Gur et al. 2017). Imaging studies in humans have shown changes in structural and functional connectivity between brain regions in individuals with 22q11DS (Schreiner et al. 2017; Jalbzikowski et al. 2014). These observations promoted the conceptualization of 22q11DS as a "developmental disconnection" syndrome that may involve the formation of atypical circuits due to abnormal neurological development. However, there is a lack of multidimensional and longitudinal studies of macro-scale development trajectories in 22q11DS.

To investigate the developmental trajectory in 22q11DS, we longitudinally mapped brain morphology, resting-state fMRI connectivity (rsfMRI) and socio-cognitive behavior in LgDel/+ mice, a well-characterized murine model of 22q11DS (Merscher et al. 2010). To correlate changes in neuronal synchronization with functional uncoupling as measured by rsfMRI, we also examined the developmental rescue induced by pharmacological inhibition of the overactivity of the GSK3β pathway that characterizes these mice (Mukai et al. 2008; 2015; Moutin et al. 20106; as previous studies have shown that this procedure normalizes anterior hippocampal synchrony in LgDel/+ mice.

Methods:We mapped functional connectivity longitudinally based on rsfMRI, diffusion tensor imaging and brain morphometry in prepubertal LgDel/+ (p33-p37) and young adult (p105-p120) (LgDel/+) mice.N= 22; WTN= 22, mixed gender). We also assessed socio-communicative functions using tests of social preference and appreciation, social reward, and temporal order memory tasks. rsfMRI time series were analyzed as previously described (Cole et al. 2010) using voxel mapping. High-resolution T2-weighted morphoanatomical images were obtained ex vivo in PFA-fixed brains and analyzed as in Pagani et al. 2019 using voxel-based morphometry. The rescue experiment was performed by injecting mice every other day with p7 to p27 SB216763 or vehicle as in Tamura et al. 2016.

Results:rsfMRI mapping revealed significant changes in connectivity in both adolescence and adulthood. However, the direction and anatomical distribution of these changes was radically different during development. Specifically, we found evidence of generalized hyperconnectivity in LgDel/+ mice during adolescence, an effect that however reverted to focal anterior-hippocampal hypoconnection in young adulthood (T>2.1, fixed FWER cluster, with cluster definition thresholdT> 2,PI number< 0.05). Brain morphometry showed a slight, focal gray matter reduction in the olfactory and cerebellar areas in the prepubertal phase, with the onset of increased gray matter volume in the corticolumbar and basal ganglia, and a focal gray matter reduction in ventral hippocampal areas.T>2.1, fixed FWER cluster, with cluster definition thresholdT> 2,PI number< 0.05). Connectivity and neuroanatomical changes did not predict sociability (two-way ANOVA, genotype,PI number= 0.005), reward processing (Ttest,PI number= 0.04) and cognitive impairment (Ttest,PI number= 0.0004) were behaviorally tested in the same mice throughout development. Interestingly, developmental inhibition of Gsk3β kinase completely rescued juvenile hyperconnectivity as well as cognition (time-series memory test) in adult mice, but did not rescue adult anterior hippocampus rsfMRI disconnection, neuroanatomical changes, or any other changes. socio-behavioral factors are analyzed at both stages of development (sociability and social memory).

Conclusions:Our data document robust developmental changes in brain connectivity and morphometry in LgDel mice. Notably, connectivity changes appear to be aberrantly expressed throughout development, with generalized hyperconnectivity during the adolescent phase but no anterior hippocampal disconnection in adulthood, depending on the overactivity of the Gsk3 pathway. The lack of a strong correlation between our imaging findings and the corresponding behavioral deficits suggests that the observed imaging phenotypes, although potentially useful biomarkers of disease progression, may be mechanistically distinct from the sociocognitive dysfunctions that characterize 22q11DS.

Keywords:22q11.2 Deletion syndrome, fMRI, neuroanatomy, behavior, LgDel

Revelation:Nothing to disclose.

University of Pennsylvania, Philadelphia, Pennsylvania, United States

Bottom:The motivation to risk psychosis is overwhelming, lacks proven treatments, portends a worse prognosis, but is poorly researched. Most fMRI studies on reduced motivation have looked at responses to external reinforcements (e.g., money). However, intrinsic motivation (IM), related to intrinsic desires such as dominance or curiosity, may be even more impaired than extrinsic motivation (EM). We have previously shown that the fMRI response in the ventral striatum (SV), the central motor region of the brain, is greater for correct than incorrect responses during cognitive tasks, even in the absence of any feedback. This IM fMRI function is reduced in schizophrenia as well as in adolescents with subclinical psychotic spectrum (PS) symptoms. Here, we report preliminary results from a completed cross-sectional phase of an ongoing longitudinal study comparing MI and EM during a cognitively demanding task, with respect to MI and EM characteristics and clinical motivation. Our hypothesis is that PS status as well as clinical mobilization will be more strongly associated with impairment in MI than in EM. We expected intrinsically motivated performance to generate signals of evaluation and prediction errors in the SV when individuals internally evaluated their performance against their expectations, and that these signals would be related to MI or EM, depending on the type of job feedback.

Methods:Time1 data collection has recently been completed and participants are now returning for a 2-year longitudinal follow-up. The preliminary results used an fMRI-analyzable Time1 sample of 94 individuals with SP and 30 typically developing (TD) controls aged 16 to 26 years (50 females, balanced group). During the fMRI, participants performed a fractal visual memory task under three feedback compensation conditions: 1) none 2) information accuracy 3) money. In each trial, participants identified which of the two fractals presented was the one previously visualized during encoding prior to scanning. Trials consisted of three phases: selection, confidence assessment and feedback, separated by neural delays. fMRI analysis focused on the SV along with regional and secondary analyzes of the whole brain. Additional research measures include negative clinical symptoms (CAINS), prodromal symptoms (SIPS, PRIME), self-report trait and IM task, behavioral measure of the free-choice DY task, and behavioral effort discounting task. MRI data also included structural MPRAGE and resting state BOLD (ABCD sequences).

Results:The PS group showed greater clinical frustration (CAINS) (T= 6,2,PI number< 0.001) and impaired IM feature (T= 2,6,PI number= 0.01). However, the EM feature was intact in PS (T= 0,14,PI number= 0.89) and PS showed a relative reduction of MI compared to EM (results of MI-EM differences,T= 3,1,PI number= 0.003). Among participants, CAINS aversive motivation was most strongly associated with the self-reported IM trait (R= -0,36,PI number= 0.01) carbon EM (R= -0,08,PI number= 0.9). The average accuracy of the memory task was 63%, indicating that the task was difficult but feasible under the conditions intended to induce MI. Confidence scores across studies were on average correlated with normal/abnormalR= 0.21 across the sample, indicating that confidence scores are an (imperfect) indicator of true memory accuracy. There were no group differences in task accuracy, confidence, or confidence-accuracy correlations (PI numbers>0.5). fMRI revealed strong VS activation for fractal stimulus phase (memory selection) and for correction vs. incorrect comments (PI number< 0.01), but not for the entire phase of the effect. There was a significant relationship between SV activation and inter-sample confidence scores during the selection phase, consistent with the judgment signal and within-sample prediction errors (outcome confidence) in the outcome phase (PI number< 0.01). All of these contrasts showed stronger activation with increasing feedback (money>information>none). Group differences in SV activation were not significant, but intrinsic trait motivation was dimensionally related throughout the sample to SV activation, particularly during the choice phase (PI number< 0,01).

Conclusions:Our findings support the hypothesis that intrinsic motivation is selectively attenuated in SP and selectively associated with clinical motivation, relative to EM. The fMRI results show that the SV responses during the cognitively demanding task indeed reflect internally generated reinforcement cues related to accuracy and confidence as well as self-reported MI trait. Ongoing work will directly compare fMRI's differential response to feedback conditions of MI tasks versus EM tasks with self-reported MI and EM measurements. Longitudinal data will assess how these neurobehavioral phenotypes develop over time. Our ultimate goal is to characterize the neural mechanisms of mobilization and develop biomarkers for specific neurobehavioral dimensions or subtypes of mobilization. These biomarkers will be tested for prognostic utility and as moderators or mediators in early interventions in at-risk youth.

Keywords:Psychosis risk, intrinsic motivation, fMRI, ventral striatum

Revelation:Nothing to disclose.

University of Texas Southwestern Medical Center, Dallas, Texas, United States

Bottom:Brain concentrations of kynurenic acid (KYNA), a metabolite of the kynurenine pathway of tryptophan degradation and an antagonist of N-methyl-D-aspartic acid and nicotinic α7 acetylcholine receptors, are increased in the prefrontal cortex of individuals with schizophrenia (SZ). . This increase may be clinically significant as hypofunction of both receptors is associated with the pathophysiology and especially the cognitive deficits of this condition.

Two enzymes, kynurenine 3-monooxygenase (KMO) and kynurenine aminotransferase II (KAT II) play a key role in regulating KYNA levels in the brain. Thus, reducing KMO activity, as seen in the brains of people with SZ, increases brain KYNA, and inhibiting KAT II, ​​which produces most of KYNA in the brain, is considered a completely new approach to preventing deficiency. cognition (Schwarcz et al., 2012).

Examined post-mortem in the anterior eye field (BA6) of SZ cases, KMO SNP rs2275163 CC (but not KMO SNP rs2275163 CT/TT) is associated with reduced KMO mRNA expression enzyme activity as well as increased KYNA levels. Furthermore, unrelated to a diagnosis of SZ, this genotype reduces predictive search function and impairs visuospatial working memory (Wonodi et al., 2011).

Methods:Based on these results, we developed a "biomarker fingerprint" of KMO SNPs rs2275163 CC ('KYNA high' phenotype) and rs2275163 CT/TT in a large cohort (N= 250) Individuals with SZ belonging to biotypes 1 and 2 as defined by the Bipolar and Schizophrenia Network (Clementz et al., 2015), using together genotype and specific biomarkers to distinguish individuals with "high KYNA". To this end, we performed four separate normal discriminant analyzes across different biotypes (each primed 1000x with 95% sampling), evaluating i) cognitive function (BACS and Stop Signal task), (ii) EEG (evoked and intrinsic activity), (iii) function oculomotor (pre/anti-saccade and fluid pursuit), (iv) cortical thickness, (v) cortical surface area, and (vi) cortical and subcortical gray and white matter volumes.

Results:The results revealed a separation of 0.7 SD between probands rs2275163 CC and rs2275163 CT/TT. It is worth noting that probands with z > 0.5 had > > 95% probability of being carriers of the rs2275163 CC genotype. This created not only a genotype but also a phenotype of interest for identifying "high KYNA" individuals.

Conclusions:These findings support the use of biomarkers as secondary outcome measures, specifically the use of CC KMO SNP rs2275163 genotyping together with CC genotype biomarker fingerprinting for population screening in clinical trials designed to explore possible outcomes. cognitive effects of CAT II inhibitors in humans. This approach of enriching the future patient population through biomarker phenotyping is expected to reduce sample sizes as well as increase the likelihood of success by targeting patients with a 'high KYNA' fingerprint. The compounds are currently being prepared for human testing.

Keywords:CNS clinical trials, kynurenic acid, fingerprint biomarkers, psychosis, cognition

Disclosures:Kynexis, Karuna, Astellas, Sunovion: Advisory Board (owned) Karuna: Shares (owned)

University of California, San Diego, La Jolla, California, United States

Bottom:Negative mismatch (MMN) and P3a are critical biomarkers of early auditory information processing in schizophrenia (SZ) and are currently prime candidates for development in biomarker-based SZ clinical trials. However, the effect of drug-related factors - especially anticholinergic drug burden - on these biomarkers has not been adequately studied. Previous studies have shown that the cumulative anticholinergic drug burden of regularly prescribed psychotropic and non-psychotropic drugs negatively affects cognition in several measures of SZ. Since MMN and P3a have been mechanistically linked to cognitive impairment in SZ, our aim was to investigate: 1) how anticholinergic drug loading affects the MMN/P3a response and 2) whether anticholinergic drug loading alters previously explored mediating pathways that link MMN/P3a, cog functionality, symptoms and functional outcomes.

Methods:Anticholinergic drug burden was assessed using the modified anticholinergic cognitive load (ACB) scale for participants with SZ (N= 774) enrolled in the Consortium on the Genetics of Schizophrenia (COGS-2) multicenter case-control study, who had complete medical records available and underwent MMN/P3a measurement. ACB scores ranging from 0 to 3 were assigned to individual drugs based on anticholinergic properties and summed to create a cumulative score of anticholinergic drug burden for each subject. SZ patients were stratified based on ACB as previously described (ACB=0,N= 52; ACB = 1 or 2,N= 194; ACB = 3 or 4,N= 185; ACB = 5 or 6,N= 93; ACB > 6,N= 87). Structural equation modeling was used to test the effects of mediation.

Results:The CBA score was significantly correlated with the MMN (eat= 5,15,PI number< 0,001) e P3a (eat= 4,34,PI number< 0.01). Compared to SZ subjects with ACB 0, subjects with ACB >6 had more attenuated MMN and P3a responses (mean [st dev], ACB=0, MMN=−1.52μV [0.87], [3a1] 3a1, 1=3.ACB >6, MMN=-0.82µV [0.96], P3a=1.01µV [1.01].The effect of ACB on functional outcomes was mediated by a direct effect on MMN/P3a, negative , AC, AC and ACB, ACB and ACB, ACB and ACB) on functional outcomes was associated with attenuated MMN/P3a amplitudes, poorer cognition and severity of negative symptoms, which ultimately predicted poorer functional outcomes).Hello= -0.3 in functional scores.

Conclusions:Calculation of the burden of anticholinergic drugs may clarify the associations between electrophysiological biomarkers, cognition and the results of future studies. Such effects may be seen in extensive genetic research of SZ and cognition, biomarker-based clinical trials of SZ, and in the development of new pro-cognitive therapies in SZ.

Keywords:Cognitive, psychosis, anticholinergic, anticholinergic drug load

Disclosures:Astellas Pharma, Heptares Therapeutics, NeuroSig, Takeda Pharmaceutical Company, Ltd: Σύμβουλος (Autônomo)

Columbia University, New York, New York, United States

Bottom:Serotonin type 3 (5-HT3R) receptor antagonists have potential in the treatment of cognitive deficits in schizophrenia. CVN058, a potent and selective brain-penetrating 5-HT3R antagonist, has shown efficacy in cognitive rodent models and was well tolerated in Phase 1 studies. In one study, we assessed CVN058 binding to a target using mismatch negativity (MMN). randomized, double-blind, placebo-controlled crossover.

Methods:The subjects were stable outpatients with schizophrenia or schizoaffective disorder, treated with antipsychotics. Subjects were not allowed to use other 5-HT3R modulators or serotonin reuptake inhibitors. Each patient received oral high (150 mg) and low (15 mg or 75 mg) doses of CVN058 and placebo in randomized order on 3 daily treatment visits at least 1 week apart. The pre-recorded primary outcome was the MMN duration range. Other deviant MMN amplitudes (frequency, loudness, frequency modulation and position), P50, P300 and constant auditory response (RAEE) were exploratory parameters.

Results:19 out of 22 randomized subjects (86.4%) completed the study. Baseline PANSS scores showed moderate impairment. CVN058 150 mg resulted in a significant improvement over placebo in the primary endpoint of MMN duration (PI number= 0.02, d CohenHello= 0.48). A significant treatment effect was also observed in a pooled analysis of all deviant MMNs (PI number< 0,001,Hello= 0.57). The effect on MMN location was independently significant (PI number< 0,007,Hello= 0.46). No other significant effects were observed for other deviations, doses or EEG measurements. There were no clinically significant treatment-related adverse events.

Conclusions:These results indicate that MMN is a sensitive biomarker of target engagement for 5-HT3R and supports the potential utility of CVN058 in correcting the excitatory/inhibitory imbalance in schizophrenia.

Keywords:Discordant Negative Auditory Response, Steady State Auditory Response, NMDA Receptor, 5-HT3 Receptors, Schizophrenia-Related Cognitive Impairment

Disclosures:Glytech, NeuroRx, Promentis,: stocks/stocks (proprietary)

Biogen: Advisory Board (Auto)

SK Life Sci, Boehringer Ingelheim, Celerion: konsultant (auto)

Miller School of Medicine, University of Miami, Miami, Florida, United States

Bottom:Schizophrenia and bipolar disorder are inherited, multifactorial disorders and major causes of disability. Combined variants of polygenic risk scores (PRS) determined from genome-wide association studies to estimate disease susceptibility at the individual level and are a promising tool for risk stratification in a clinical setting. With the advent of large-scale genomic data collection, understanding diagnosis validity or EHR as phenotypic entities is critical.

Methods:Using the VA's extensive electronic health record (EHR) and a cohort of 9,378 individuals with confirmed diagnoses of schizophrenia or bipolar disorder, we validated case control assignments based on ICD codes and compared PRS performance in the 660,000 million veterans (MVP) program. We examined the relationships between PRS and 1,700 disease categories and 70 laboratory values ​​through the wide PheWAS correlation. Given the significant genetic overlap of neuropsychiatric disorders, we used genome-wide structural equation modeling (gSEM) to extract novel, common and disorder-specific underlying genetic factors.

Results:Of the 3,953 to 5,425 people with a confirmed diagnosis of schizophrenia or bipolar disorder, 95% were correctly identified by ICD codes (>1) and 25% also met the misdiagnosis criteria. Encouragingly, the PRS performed best in a comparison of confirmed cases, followed by those who received hospital treatment, those who received any treatment, and diagnoses across the spectrum. Our PheWAS confirmed that a higher neuropsychiatric PRS increases the risk of many mental and physical health problems, and many of the findings apply to African Americans. The protective effect of schizophrenic PRS on tinnitus was due to schizophrenia-specific effects. Our WAS laboratory showed positive correlations between PRS levels in schizophrenia and cholesterol and white blood cell counts, and negative correlations with glucose and electrolyte levels.

Conclusions:Using the current neuropsychiatric PRS, we demonstrated the validity of EHR-based phenotyping approaches in different US populations. PheWAS revealed new associations and replicated previously reported associations between schizophrenia, bipolar disorder and major depression of PRS and various clinical features and outcomes, highlighting the potential of PRS in unraveling biological and mediated pleiotropy.

Keywords:Polygenic Risk Assessments, DNA, Whole Genome, Sequencing, Schizophrenia, Schizophrenia, Bipolar Disorder, Major Depression, Diagnosis

Revelation:Nothing to disclose.

University of California, San Diego, La Jolla, California, United States

Bottom:Computer-directed cognitive training (CTT) improves neurocognition and functioning in patients with schizophrenia (SZ). This TCT consists of a set of exercises aimed at auditory and verbal working memory and verbal learning performance, with repetitive results that are continuously adjusted to the difficulty of the task. While many patients show sustained improvement after 20-30 hours of TCT training, some show only moderate benefits, with "no response" rates of 40-50% being reported even after 40 hours of TCT training. This heterogeneous TCT response presumably reflects heterogeneity in the degree of learning of TCT exercises. We hypothesize that the benefits of TCT will be enhanced and/or accelerated by interventions that increase engagement and learning in TCT. Since we know that attention deficits hinder engagement and learning, we hypothesize that attention-enhancing interventions in patients with attention deficits also increase the clinical benefits of TCT. Consistent with this hypothesis, we report that the attention-altering drug d-amphetamine (AMPH, 5 or 10 mg po) improves learning during TCT "Sound Sweeps" sections in treated SZ patients with antipsychotics (AP) and especially among attention-seeking patients deficit disorder. We are currently investigating the effect of AMPH (5 mg) on ​​the therapeutic effect of 30 hours of full TCT training in SZ patients receiving AP therapy. Here we describe our study design and preliminary "feasibility" data.

Methods:Carefully matched male and female patients with SZ or schizoaffective (depressive) disorder are assessed for baseline neurocognitive functioning (MATRICS Comprehensive Cognitive Battery, MCCB). Then, over two test days separated by a week, comprehensive measures of clinical symptoms and function are obtained, along with behavioral (TCT "Sound Sweeps" performance), electroencephalographic (EEG: event potentials, auditory response status) predictive potential. ) and measures of auditory fidelity (words in noise; rapid speech in noise), after placebo (Test 1) or AMPH (5 mg po, T2). Over the next 10–12 weeks, participants perform 30 training sessions of the full TCT set (1–1.5 h/session, 2–3 sessions/week), 60 min after taking placebo or AMPH (5 mg) in a double-blind, randomized design. Outcome measures (MCCB, clinical symptoms, and functional scales) and EEG and auditory fidelity measurements are obtained at 10, 20, and 30 TCT training sessions and 12 weeks post-training (endpointN= 54; blind to breakN= 10 for preliminary analyses).

Results:So far 5 people get AP (M:eat= 3:2; age (mean (range)) = 39 (33-47)) completed screening, test days 1-2, and at least 10 TCT training sessions with post-TCT testing (split into 2 drug states). 3 completed 30 hours of training, 1 also completed 12 weeks of follow-up. Data is reported to session 10 after the TCT. There were no adverse events or loss of participants despite intensive training and testing. Clinical scores for general and specific psychiatric symptoms of psychosis (PHQ-9, BPRS, PANSS, PSYRATS, YMRS) and suicidal ideation (CSSR-S) as well as neurocognitive performance (MCCB) remained unchanged or moderately better from baseline until after the TCT session 10. The size of the blinded groups is too small to allow meaningful analyzes of the effects of AMPH, but updated comparisons of larger groups will be provided.

Conclusions:Testing the effect of AMPH on the therapeutic effect of TCT in SZ patients receiving AP is feasible. To date, participants have had no evidence of "fatigue" or other adverse effects from testing, training, or exposure to AMPH. In this small trial, changes in core symptoms, neurocognition, and functioning are not clear after 10 TCT training sessions, although blinding precludes any assessment of drug effects at this point. Initial unblinded analyzes will be presented upon completionN= 10 items.

Keywords:Computerized cognitive training, schizophrenia (SCZ), amphetamine

Revelation:Nothing to disclose.

Tufts University School of Medicine, Boston, Massachusetts, Stany Zjednoczone

Bottom:Converging evidence suggests dysregulation of the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism in schizophrenia. Kyn is a substrate for the formation of 3-hydroxykynurenine (3HK), kynurenine (KYNA) and anthranilic (AA) acids. Post-mortem examination showed reduced gene expression and insufficient activity of kynurenine 3-monooxygenase (KMO), the enzyme catalyzing the conversion of Kyn to 3HK, in the brains of patients with schizophrenia [1]. KMO deficiency increases the availability of Kyn as a substrate for the formation of KYNA and AA. Elevated concentrations (at the expense of formation of Kyn 3HK) of KYNA, an NMDA receptor antagonist, have been found in the brain and cerebrospinal fluid (but not serum/plasma) of patients with schizophrenia [2,3] and are suggested to be causally related to the underlying psychopathology of schizophrenia ( the KYNA schizophrenia hypothesis)[4]. Elevated AA levels have previously been reported in the serum [5] and plasma [6] of patients with schizophrenia. However, we are not aware of any studies evaluating the effect of antipsychotic treatment on plasma AA levels in schizophrenia.

Methods:The severity of schizophrenia was assessed using the Positive and Negative Syndrome Scale (PANSS). Plasma levels of Trp and kynurenine (HPLC/MC)[7] were assessed in fasting plasma samples from patients with schizophrenia before and after treatment with risperidol (N= 21), olanzapine (N= 10), ή seroquel (N= 11). The study was approved by the Review Board of the University of Magdeburg and written informed consent was obtained. The pairwise t-test was used to assess the statistical significance of the obtained data.

Results:PANSS scores dropped from 88.3 to 56.7 (PI number< 0.0001). Plasma 3HK levels increased by 47%, from 14.64 to 21.94 μmol (PI number< 0.001), while AA levels decreased by 64%, from 16.36 to 10.56 μmol (PI number< 0.006). There was a smaller increase in Kyn from 1.32 to 1.67 nmol (PI number< 0.0001) and Trp levels from 49.86 to 55.34 nmol (PI number= 0.055). KYNA and XA plasma concentrations remained unchanged. There were no gender differences in the plasma concentrations of the tested metabolites after antipsychotic treatment.

Conclusions:This is a preliminary report of the first observation of a dramatic drop in plasma AA levels after 6 weeks of antipsychotic treatment. The simultaneous increase in the level of 3HK suggests that antipsychotic treatment alleviates KMO deficiency and restores the pattern of further Kyn metabolism to preferential conversion (under physiological conditions) of Kyn to 3HK and KYNA, and not to AA [8]. The concentrations of the metabolites in plasma probably reflect their concentrations in the brain, with the exception of KYNA, which does not cross the blood-brain barrier [9]. Assessment of plasma AA levels can be developed as an indicator of KMO activity in patients with schizophrenia. Future studies of large patient populations may allow for the analysis of gender differences and the correlation of plasma AA levels with the severity of schizophrenia symptoms after antipsychotic treatment.

Bibliographic references:

1. Wonodi I, Stine CO, Sathyasaikumar KV, Robert RC, Mitchell BDL, Elliot Hong L, et al. Decreased kynurenine 3-monooxygenase gene expression and enzyme activity in schizophrenia and genetic association with schizophrenia endophenotypes. Psychiatry Arch. Gen. 2011; 68:665-674. 10.1001/archgenpsychiatry.2011.71

2. Schwarcz R, Rassoulpour A, Wu HQ, Medoff D, Tamminga CA, Roberts R, 2001. Increased cortical kynuren content in schizophrenia. Biol. Psychiatry 50, 521–530

3. Erhardt S, Blennow K, Nordin C, Skogh E et al. (2001). Kynureninic acid concentrations are elevated in the cerebrospinal fluid of patients with schizophrenia. Neurological Letters 313, 96–98

[PMC Free Article] [PubMed] 4. Erhardt, S., L. Schwieler, L. Nilsson, K. Linderholm, G. Engberg. Physio. Συμπεριφορά. 92, 203–209.

5. Fazio F., Lionetto L., Curto M., Iacovelli L. et al., 2015. Xanthuric acid activates mGlu2/3 metabotropic glutamate receptors and is a potential marker of schizophrenia. science representative 5, 17799–17813. 10.1038/srep17799 (PMID: 26643205).

6. Oxenkrug, G., van der Hart, M., Roeser, J., Summergrad, P., 2016. Anthranilic acid: a potential biomarker and target for the treatment of schizophrenia. Ann Psychiatry Ment Health 4, 1059-1062. Fri: 1059. PMID: 27042691

7. Oxenkrug G, HG Bernstein, P. Guest, P. Summergrad, J. Steiner. (2019) Plasma anthranilic acid correlates with obesity but not markers of insulin resistance in schizophrenia. Neuropsychopharmacology, 44, 166 10.1038/s41386-019-0545-y

8. Moroni F. Tryptophan metabolism and brain function: focus on kynurenine and other indole metabolites. European Journal of Pharmacology 375 1999 87-100

9. Fukui S, Schwarcz R, Rapoport SI et al. Kynurenine transport across the blood-brain barrier: implications for brain synthesis and metabolism. J. Neurochem 1991; 56(6):2007-2017.

Keywords:Schizophrenia, Antipsychotics, Anthranilic acid, 3-hydroxykynurenine

Revelation:Nothing to disclose.

University of Alabama at Birmingham, Birmingham, Alabama, United States

Bottom:Stress in early life, such as psychosocial stress during adolescence, is known to be a dominant risk factor for the development of postpartum mental disorders. However, the biological mechanisms by which adolescent psychosocial stress influences postnatal behavior later in life have not been well characterized. Previously, we generated a new mouse model in which the mild stress of adolescent isolation leads to deficits in social behavior one week after birth. In addition, our in vivo microdialysis study revealed that our new mouse model exhibited an abnormal decrease in extracellular glutamate levels in the prefrontal cortex (PrL). Imaging studies in humans have linked functional changes in the anterior insula (AI) and dorsal anterior cingulate [dACC, PrL homolog in rodents] with postnatal behavioral changes. Based on our in vivo microdialysis data and human imaging studies by other groups, we investigated whether adolescent social isolation coupled with stressful pregnancies/births leads to deficits in postpartum social cognition behaviors through functional switches of the glutamatergic pathway from AI to PrL.

Methods:To perform neuronal manipulation optogenetics, the adeno-associated viruses (AAV) 5-Syn-FLEX-ChrimsonR or AAV5-EF1a-DIO-eNpHR3.0 were injected into the artificial intelligence of the virgin vesicular glutamate transporter 1 (Vglut1)-Cre of a 5-week-old female mouse . To perform in vivo calcium imaging using epifluorescence microscopy for activity measurements, we inject a conditional AAV expressing the calcium marker GCaMP6f (AAV1-Syn.Flex-GCaMP6f) and simultaneously implant a scalar refractive index lens over the PrL. This strategy allowed us to manipulate glutamatergic projections from AI to PrL and simultaneously monitor the calcium dynamics of glutamatergic neurons in PrL. Following surgery, the animals were exposed to mild social isolation in late adolescence (5 to 8 weeks), which did not in itself induce endocrine or behavioral changes. Each mouse was then crossed with a C57BL/6J male and released. Three-chamber social interaction tests (SIT) were performed one week after delivery. During SIT, calcium images were recorded with or without optical manipulation of the AI-PrL pathway. Neuronal signals from a single cell were extracted by region of interest (ROI) detection using independent Principal Components Analysis and analyzed using Receiver Operational Characteristics (ROC) analysis to quantify the response of each neuron during social interactions.

Results:Calcium imaging of glutamatergic neurons in PrL during SIT was used to identify subsets of neurons that are fired or suppressed when interacting with a known or novel mouse. We found that the inhibition of new mouse sniffing behaviors in stressed mothers was accompanied by a reduced fraction of excitatory glutamatergic neurons and an increased fraction of suppressed glutamatergic neurons when interacting with the new mouse. Visual activation of the AI-PrL pathway in stressed barriers increased the time spent sniffing the new mouse by normalizing the disruptions in excitable and suppressed glutamatergic neurons when interacting with the new mouse. In contrast, inhibition of the visual pathway to unstressed barriers led to behavioral deficits in recognizing social novelties. Under neuronal silencing, the fraction of excited glutamatergic neurons decreased while the fraction of depressed glutamatergic neurons increased in PrL when interacting with new mice. The fraction of glutamatergic neurons fired or suppressed in PrL when interacting with a known mouse was not affected by social isolation or visual pathway manipulation.

Conclusions:Our study showed that social isolation during adolescence, together with stressful pregnancy/birth events, results in hypofunction of the AI-PrL pathway and leads to subsequent deficits in social behavior in the postnatal period. These data provide an opportunity to understand the causal role of the AI-PrL pathway, a new corticocortical circuit, in recognizing social novelties after birth. Our new mouse model may be a promising model for studying the pathological mechanism underlying postnatal behavioral deficits and discovering new therapeutic strategies.

Keywords:Social cognition, adolescent stress, childbirth, prefrontal cortex, anterior insula

Revelation:Nothing to disclose.

Keio University, Tokyo, Japan

Bottom:Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. GABAergic dysfunction is implicated in the pathophysiology of schizophrenia. Clozapine, the only drug approved to treat treatment-resistant schizophrenia (TRS), targets the GABAergic system as one of its targets. However, no study has examined the association between brain GABA levels, as measured by proton magnetic resonance spectroscopy (1H-MRS), and the response to clozapine in STR patients.

Methods:This study included MTC patients who failed to respond to clozapine (ultra-resistant schizophrenia: URS) and responded to clozapine (non-URS), schizophrenic patients who responded to first-line antipsychotics (first-line responders: FLR), and healthy controls (HC). We measured GABA levels in the medial cortex (MCC) with 3T 1H-MRS MEGA-PRESS and compared these levels between groups. Correlations between GABA levels and symptom severity were also studied in patient groups.

Results:A total of 91 participants (URS:N= 22; non-URS:N= 21; FLR:N= 15; CH:N= 33) completed the study. We found overall group differences in GABA levels after adjustment for smoking. Specifically, URS patients had higher GABA levels compared to HC. MCC GABA levels showed no correlation with any of the symptom severity ratings in each group or the group of patients as a whole.

Conclusions:Our study is the first to show elevated MCC GABA levels in patients with clozapine-resistant schizophrenia. Longitudinal studies are needed to assess whether GABA levels are a sufficient biomarker to predict clozapine resistance.

Keywords:Clozapine, a new treatment for schizophrenia, GABA, 1H-MRS

Revelation:Nothing to disclose.

Idorsia Pharmaceuticals Ltd., Allschwil, Switzerland

Bottom:In two large phase 3 studies (study 1:NCT03545191; attempt 2?NCT03575104), we evaluated the effects of daridorexant on the macroarchitecture of nighttime sleep in adults and elderly adults with insomnia. Daridorexant improved sleep variables without changing the proportion of sleep stages at month (M) 1 and M3 of treatment. The current analysis further explores the macroarchitecture of quarter-night sleep (QoN) in insomnia patients from Study 1.

Methods:Eligible patients were ≥18 years of age, had insomnia (according to DSM-5) with an Insomnia Severity Index [ISI] ≥15, and reported sleep disturbance >3 nights per week. patients (N= 930) were randomized (1:1:1) to a double-blind study with oral daridorexant 25 mg, 50 mg or placebo at night for 3 months. Total sleep time (TST) and sleep phases (no rapid eye movements [NREM, N]1, N2, N3, REM) were measured by polysomnography (PSG) in a sleep laboratory for two consecutive nights during a single-blind placebo study. period (baseline, BL) and months 1 and 3 (M1, M3) of treatment. Change from baseline in TST and nocturnal bedroom sleep stages were exploratory parameters. Data are presented as change from baseline in absolute time (min).

Results:Among the 930 randomized patients, the median age was 55 years and 67% were women. In BL, the average post-sleep awake time ranged from 95 to 103 minutes in the three groups, the average latency to sustained sleep ranged from 64 to 67 minutes, the average total sleep time ranged from 319 to 328 minutes, and the mean ISI scores were 19 in each group. Data from 923 patients (363 aged ≥65 years) were included in the analysis.

TST spent in N1, N2, and REM all night increased from BL in each treatment group by 2–7 min, 16–38 min, and 9–16 min, respectively, with little or no change over time devoted to N3. The proportion of TST spent in each stage of night sleep was similar in each treatment group (daridorexant 25 mg, 50 mg and placebo): the proportion of time spent in N2 and REM sleep increased, the proportion of time spent in N3 decreased, and the proportion of time spent in N2 and REM decreased N3 spending increased while N1 spending remained unchanged.

The absolute increase in BL in N1 was similar in each group and of the order of 1–2 min in all quarters of the night. However, the N1 index decreased in the first quarter of the night (Q1) but remained unchanged in the subsequent quarters (Q2–4) in all treatment groups.

The absolute increase in BL in N2 was consistently greater in daridorexant than placebo recipients in Q1-4, with the largest increase (14-15 min in daridorexant recipients) observed in Q1. Despite the greater increase in N2 duration in Q1, the percentage of TST spent on Q1 N2 was similar to BL (52–57%) across all groups. In contrast, the percentage of TST spent on N2 increased in Q2-3 in all groups and in Q4 only in daridorexant recipients.

The absolute time spent in N3 increased in Q1 by approximately 5 to 7 minutes in daridorexant recipients and then decreased in Q2 to 4 by approximately 2 minutes in all groups. The percentage of TST in N3 remained unchanged from BL in Q1 (21-23%) and similarly decreased in all groups in Q2-4.

The absolute time spent in REM sleep increased in all groups in trimesters 1–4. The change in the percentage of time spent in REM sleep in Q2–4 was similar in all groups.

Conclusions:Daridorexant increased the duration of most sleep phases at night, while the percentage of each sleep phase was comparable to placebo, suggesting preservation of sleep macroarchitecture. The greatest changes were seen in the first quarter, but effects were still detected in the fourth quarter, suggesting an overall benefit of daridorexant at night and maintaining sleep cycle progression. After treatment with daridorexant, the increase in time spent in N2 along with the increase in REM sleep in the first quarter of the night suggests that these phases are involved in generating better sleep quality in insomniac patients. However, it remains to be determined how these subtle changes in sleep macroarchitecture are reflected in insomnia symptoms such as daytime functioning.

Keywords:Sleep architecture, insomnia, dual orexin receptor antagonist

Revelation:Idorsia employee (study sponsor): Employee (own)

Idorsia Pharmaceuticals Ltd., Allschwil, Switzerland

Bottom:A key symptom of insomnia disorder is the prolonged duration of episodes of wakefulness during the night. Daridorexant, a novel dual orexin receptor antagonist, reduced wakefulness after sleep onset (WASO) in patients with insomnia in a Phase 2 dose-finding study. The purpose of this post hoc analysis was to assess the duration and evolution of wakefulness episodes during treatment with daridorexant versus placebo during 8-hour overnight polysomnography (PSG) after day 1 and 29 days of treatment.

Methods:Eligible patients were 18 to 64 years of age with insomnia disorder according to DSM-5 criteria, self-reported sleep time ≥30 minutes, WASO ≥30 minutes, and total sleep time (TST)  ≤ 6.5 hours for ≥3 nights in the week. After a single-blind placebo period, patients were randomized (1:1:1:1:1:1) to receive a double-blind treatment with oral daridorexant (5, 10, 25, or 50 mg), zolpidem 10 mg, or placebo for 30 days.

The primary efficacy outcome was the change in wake time after falling asleep (WASO) from baseline to Days 1 and 2 as measured by PSG. In the current analysis, episodes of wakefulness at baseline, day 1 and day 29 were studied in patients receiving daridorexant 25 or 50 mg, zolpidem or placebo. Days 1 and 29 were chosen to assess the immediate and 1-month effect of daridorexant, respectively. Daridoxant 25 mg and 50 mg were selected for this analysis because they demonstrated efficacy on sleep variables in pivotal Phase 3 studies (NCT03545191).

Analyzes were performed using the wakefulness data for each patient, calculating the total awake time during the entire 8-hour PSG night, in half-minute increments (i.e., 1 period). Data were pooled and averaged for all patients in each treatment group.

Results:Of the 359 randomized patients, the mean age was 45 years and 64% were women. This post-hoc analysis included data from patients treated with daridorexant 25 mg (N= 60), 50 mg (N= 61), zolpidem 10 mg (N= 60) e placebo (N= 60).

At baseline, there was no difference in the average cumulative duration of wakefulness sessions between the treatment groups. The cumulative mean duration of wakefulness increased progressively more with zolpidem and placebo compared to daridorexant 25 mg and 50 mg on Day 1 and Day 29. two quarters of a night for patients in each of the four groups.

Over the last two-quarter hours of the night, the average cumulative time of awakening was similar in those receiving placebo and zolpidem on day 1 and longer in those receiving zolpidem than those receiving placebo on day 29, suggesting an increase in the duration of awakening compared with both first trimesters. night rooms. Daridorexane 50 mg resulted in a consistently shorter mean cumulative awake time than zolpidem or placebo over the last two nocturnal trimesters, while the mean cumulative awake time was similar between daridorexane 50 mg and zolpidem over the first two nocturnal trimesters. This reduction in awake time was statistically significant in the latter part of the night with daridorexant 50 mg compared with zolpidem.

All treatments increased the number of short awakening sessions and reduced the number of longer awakening sessions on days 1 and 29 compared to baseline, with the greatest effect observed with daridorexant 50 mg, thereby reducing the number of awakenings that were most contributed to the total awake time. The differences in cumulative wake time between daridorexant, zolpidem and placebo were largely due to the reduction in time spent in longer wake sessions in daridorexant recipients.

Conclusions:In this analysis, daridorexant (25 and 50 mg) consistently reduced nighttime wakefulness compared to placebo. this maintenance of night sleep with daridorexant can be explained by the optimized pharmacokinetic profile of daridorexant. Patients receiving daridorexant had less wakefulness in the second half of the night compared with zolpidem 10 mg and placebo. Daridorexant reduced wakefulness primarily by reducing the duration of prolonged periods of wakefulness, thereby targeting the main symptom of insomnia.

Keywords:Daridorexant, dual orexin receptor antagonist, insomnia disorder, sleep architecture

Revelation:Nothing to disclose.

Virginia Commonwealth University, Richmond, Virginia, United States

Bottom:There are gender differences in the risk and treatment of substance use disorder (SUD). Therefore, a deeper understanding of the role that gender, as a key biological variable, plays in SUD is necessary. One area that is receiving more attention as a goal for LDS is sleep. Sen and SUD show a bi-directional relationship, which increases the complexity of SUD trajectories. Substance use itself can negatively affect sleep quality. At the same time, sound sleep may increase or decrease the risk of developing SUD and responding to treatment. There are gender differences in sleep parameters in the general population, with sleep disturbances generally being more common in women than in men. Previous efforts to assess the relationship between sleep and gender-related SUDs have been limited and inconclusive. The aim of the study was to compare, by gender, sleep parameters between people with SUD and non-addictive controls.

Methods:This initial hypothesis-generating study included secondary analyzes of a large cross-sectional study investigating the feasibility and acceptability of a new Neurofunctional Phenotype Assessment Battery (NIDA PhAB). The PhAB was designed for possible use in clinical trials to allow people with SUD to be classified into neurofunctional domains (e.g., behavioral phenotype) and to eliminate heavy reliance on DSM-5 criteria and primary drug to determine treatment strategies. Briefly, those with SUD were between the ages of 18 and 70 and met the DSM-5 criteria for current SUD with opioids, cannabis, and/or cocaine as the primary drug diagnosis. Severe comorbid alcohol use disorders were excluded. Substance-free controls met the same criteria, except that they could not meet the DSM-5 LDS criteria. LDS and control subjects were recruited through local advertisements and an established research registry. People with LDS were also recruited through the university's LDS treatment clinic. Self-reported sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI). This 19-item self-report measure assesses overall sleep quality (range 0-21; higher scores indicate poorer overall sleep) with seven sub-scores (range 0-3; higher scores indicate poorer sleep). layered sexT-Studies compared sleep between LDS subjects and controls, while crossover analyzes looked at group differences in the proportion of subjects reporting poor sleep (defined as PSQI > 5).

Results:Data from 162 men (44 controls, 118 LDS) and 146 women (64 controls, 82 LDS) were included in this study. More people with LDS identified as black (79% men, 73% women) compared to controls (39% men, 39% women). Among men with LDS, about a third had a primary diagnosis of opioid use disorder (OUD;N= 53) followed by cocaine (N= 37) and marijuana (N= 28) use disorder. For women LDS, OUD (N= 46) was the most common primary drug diagnosis, followed by cannabis (N= 22) to cocaine (N= 14) use disorder.

In men, total PSQI scores were better in the control group (M=5.11, SD=3.08) than in LDS subjects (M=6.92, SD=3.72);T(159) = -2,88,PI number= 0.005. Control men also had significantly lower total PSQI scores than men with OUD (M=7.62, SD=3.80) and cannabis use disorder (M=7.36, SD=3.43);PI number=   0.001 and 0.005 respectively. Overall, control men reported statistically shorter sleep latency, longer sleep duration, and fewer sleep disturbances than LDS men with any underlying drug diagnosis. In women, the PSQI total score was better in the control group (M=5.66, SD=2.53) than in the LDS group (M=7.27, SD=2.99);T(143) = -3,45,PI number= 0.001. Control women also had significantly lower global PSQI scores than women with OUD (M = 7.63, SD = 2.83) and cannabis use disorder (M = 7.19, SD = 3.12);PI number<0.001 and 0.05, respectively. Unlike their male counterparts, women in the control group reported no PSQI items that were statistically superior in all primary drug diagnoses in those with SUD. Finally, the proportion of men reporting poor sleep as defined by the PSQI did not differ between controls and those with SUD, χ2(1,N= 161) = 2,05,PI number> 0.05, nor the LDS subgroup with primary drug diagnosis. However, for women, a significantly lower proportion of controls reported poor sleep as defined by the PSQI than those with SUD, χ2 (1,N= 145) = 5,64,PI number< 0.05 or persons with primary drug diagnosis of OUD, χ2(3,N= 145) = 8,63,PI number< 0,05.

Conclusions:Sleep problems and SUDs overlap substantially in neurobiology as well as in socioecological complexity. Sleep disorders and SUDs vary by gender, as gender is one of the critical variables shaping a person's overall health and day-to-day functioning. In our sample of LDS subjects, we found poorer sleep quality compared to opposite-sex controls, but clinically significant poor sleep was only more common among women with LDS compared to controls. These differences in sleep duration were most noticeable in those with opioid and cannabis use disorders. Overall, our findings showed that sleep is a promising way to address SUD in a biopsychosocial model. Future work on the intersection of SUD and sleep should prioritize research into its interaction with gender to identify targets for individualized SUD interventions. These findings have important implications for providers and addiction researchers, especially given the increased awareness of LDS during the overdose crisis.

Keywords:Sleep disorders, gender differences, opioid use disorder, cocaine use disorder, marijuana use disorder

Revelation:Nothing to disclose.

Idorsia Pharmaceuticals Ltd, Allschwil, Switzerland

Bottom:Orexin neuropeptides stabilize alertness and promote alertness. This is especially important later in the day when levels of the neuropeptide orexin increase to counteract the increased pressure during sleep. Orexins also increase levels of arousal and attention in response to internal and external environmental stimuli, are secreted during pleasurable experiences, and increase the motivation to earn rewards.

Dual orexin receptor antagonists (DORAs), including the FDA-approved drugs suvorexant and lemborexant, promote sleep and are indicated for the treatment of insomnia. Daridorexant is a new experimental drug from DORA for the treatment of insomnia in adults. Formulated with an optimized pharmacokinetic and pharmacodynamic profile to act over time including overnight. It is currently under review by the FDA.

Regulatory safety requirements for hypnotics include non-clinical testing for abuse potential. Such assays are necessary for DORA, despite the known biological role of the orexin system in the brain's reward pathway, with orexin receptor antagonism expected to have "anti-reward" properties and not induce reinforcement.

We used a well-established battery of animal experiments to assess the abuse potential of daridorexant while meeting regulatory requirements. To date, neither suvorexant nor lemboreexant have shown signs of abuse potential in non-clinical studies at clinically relevant concentrations. The actual evidence (based on reports from FEARS, NSDUH and other US federal studies) for suvorexant, the first DORA approved in 2014, has so far revealed only very rare cases of non-medical use or abuse, supported by the lack of such results in animal studies.

Methods:The potential reinforcing properties of daridorexant were tested in an intravenous self-administration model using female rats that were initially trained to self-administer cocaine. Following successful training, vehicle or daridorexant (0.1, 0.3, and 1 mg/kg/injection) was replaced with cocaine for a total of 6 daily swap sessions (N= 9/group).

The endorceptive similarity of the carrier or daridorexant (15, 30 and 60 mg/kg, orally) to standard hypnotics and zolpidem, a positive modulator of the GABA-A receptor (3 mg/kg, orally), was tested in a surgical drug discrimination study in female rats (N= 11/cross pattern).

Physical dependence on daridoxant was studied by monitoring potential withdrawal symptoms in female rats after cessation of chronic (4 weeks) administration of daridoxant by oral gavage at 20 or 200 mg/kg/day (N= 10/group). The original sleep inducer benzodiazepine and chlordiazepoxide (titrated to 200 mg/kg/day) were used as positive controls.

Results:Daridorexant did not tolerate self-administration in rats with a prior history of cocaine self-administration. Rats injected with daridorexant or vehicle showed a reduced response from the first replacement session compared to baseline cocaine (PI number<0.01; NEW). In addition, the rate of extinction of cocaine-seeking behavior during the substitution phase was similar across doses of daridorexant and vehicle (ANOVA;PI number> 0,4).

The potential inhibitory effect of daridoxant did not generalize to that of zolpidem. Rats treated with various doses of daridorexant almost exclusively selected the operant lever previously associated with the vehicle. This applied to the first food prize and the entire trial period. The response rate for daridorexant was not different from vehicle (ANOVA;PI number> 0,7).

Discontinuation of chronic treatment with daridorexant did not induce withdrawal symptoms. During chronic treatment, daridorexant caused a slight decrease in body temperature and locomotion, consistent with its pharmacological hypnotic effect. After discontinuation of treatment, body temperature and mobility returned to normal. No significant changes in physiological, neurobehavioural or motor activity indicative of deprivation were observed. In contrast, after discontinuation of chronic chlordiazepoxide treatment, the rats showed a significant decrease in body weight (ANOVA;PI number< 0.01) accompanied by a decrease in food intake (ANOVA;PI number< 0.01) and the appearance of neurobehavioral symptoms such as chewing and chewing.

Conclusions:In rats, daridoxant showed no evidence of abuse or dependence potential in three established models at exposures comparable to or greater than human exposure. similar to that seen previously with suvorexant and lemboreexant. These non-clinical data indicate no abuse potential for daridoxant in humans.

Keywords:Abuse liability, dual orexin receptor antagonist, rat models, drug discrimination, self-administration

Revelation:Nothing to disclose.

Eisai Inc., Woodcliff Lake, New Jersey, Canada

Bottom:Central respiratory depression is a safety concern with commonly prescribed sleep aids (benzodiazepines and benzodiazepine non-receptor agonists), especially in the frail elderly and/or those with underlying lung conditions (i.e. obstructive sleep apnea [OSA]). It is important to determine whether hypnotics with different mechanisms of action will be associated with similar risks. Lemborexant (LEM) is a dual orexin receptor antagonist (DORA) approved for the treatment of insomnia in several countries, including the United States, Japan, Canada, and Australia. In the previous study with the highest approved dose of LEM (10 mg, LEM10), there was no difference between LEM10 and placebo (PBO) in peripheral oxygen saturation (SpO2) and apnea-hypopnea index (AHI) in adults and elderly with mild OSA after single and multiple dose administration (NCT03471871). The current study, required post-marketing, will be the first to establish the respiratory safety of LEM and DORA in patients with moderate to severe OSA.

Methods:This study (NCT04647383) is a multi-center, multi-dose, randomized, double-blind, PBO-controlled, 2-period crossover study in adults (≥45 to <65 years) and elderly (≥65 to ≤90 years) with moderate (15 ≤ AHI < 30) to severe OSA (AHI ≥ 30). All patients were recruited and randomized to two 8-night treatment periods (separated by a ≥14-day washout period) with LEM10 or PBO. Laboratory polysomnography was performed at screening and on the first and last night of both treatment periods.

Results:Forty-eight people were selected. of these, 33 (68.8%) were randomized. Only one randomized patient did not complete treatment due to an adverse event classified as unrelated to study drug (COVID). The majority of subjects were Caucasian (24/33 [72.7%]) and male (23/33 [69.7% each]). The mean age was 60.6 years. 22/33 patients (66.7%) were ≥45 to <65 years of age and 11/33 (33.3%) were ≥65 to ≤90 years of age. The median BMI was 31.5. 8/33 subjects (24.2%) had a BMI ≥25 but <30, and 22/33 subjects (66.7%) had a BMI ≥30.93.5%, and the average AHI was 44.2.

Conclusions:This study will provide important new data for LEM, the first DORA to be evaluated in patients with severe OSA. These data, which will be available at the ACNP Annual Meeting, will complement the known respiratory safety of multiple and single doses of LEM in adults and older adults with mild to moderate or severe OSA, objectively measured by SpO2 and AHI.

Keywords:Lemborexant, Respiratory Safety, Obstructive Sleep Apnea

Revelation:Eisai Inc.: employee (self-employed)

University of Maryland School of Medicine, Baltimore, Maryland, Stany Zjednoczone

Bottom:Sleep is essential for physical and mental health and is a critical physiological function of the human nervous system. An important measure of brain health is the integrity of the brain's connectivity structures, such as the white matter pathways that connect regions of the brain. Studies have shown a correlation between sleep characteristics and white matter integrity. However, many factors affect sleep and white matter measurements. from relatively simple demographic variables to more complex factors that are more difficult to measure, such as environmental factors such as stress.

Our study used a sample of the rural Amish population, whose members are exposed to a more homogeneous environment compared to the general US population, such as education that generally ends in the eighth grade and limited access to technology that can disrupt sleep. Our goal was to test whether the association between sleep quality and white matter was present after adjusting for specific environmental factors such as stress.

Methods:The study included 240 members of an Old Order Amish/Mennonite family [137 females and 103 males, age (37.9 ± 17.9, mean ± SD)] from Pennsylvania and Maryland without any serious mental or medical disorders during their lifetime. White matter integrity of duct 42 was measured by diffusion tensor imaging to obtain fractional anisotropy values ​​using a Prism 3 Tesla scanner. Current stress was measured with the Perceived Stress Scale and life stress was measured with the Life Stress Inventory. Sleep quality was determined by the self-reported Pittsburgh Sleep Quality Index (PSQI).

Results:The integrity of several white matter tracts was significantly associated with sleep quality, all located in frontal lobe areas (allPI number< 0.05 after adjusting for multiple comparisons using the false discovery rate).

In multiple regression analyzes to account for stress factors, models showed that the PSQI remains a significant predictor of white matter integrity in these frontal tracts after adjusting for age, gender, current stress, and duration of life stress (allPI number< 0.01), while current and lifetime stress were not significant predictors in any of the four models. Meanwhile, current stress was a significant predictor of sleep quality (allPI number≤ 0.01) in a model where both white matter pathways and current stress were predictors, further suggesting that current stress was associated with poorer sleep quality as expected, which was independent and additive to the effects of these substance pathways white on the quality of sleep. Life stress was not a significant predictor of sleep quality.

Conclusions:Sleep quality was significantly associated with multiple anterior white matter segments connecting brain structures important for sleep regulation. For example, the anterior inner capsule contains fibers that connect the basal ganglia and the prefrontal cortex. Similarly, the ciliary body contains ascending fibers that connect the thalamus to the cerebral cortex and descending fibers that connect the frontal cortex to the basal nuclei, where the pons, hypothalamus, thalamus, and prefrontal cortex are important structures that regulate sleep and sleep. . Stress can affect sleep and white matter integrity, but despite the strong relationship between stress and sleep, the data showed that stress levels were not a significant confounder in this study of white matter integrity and sleep quality.

Moreover, our findings come from a population with much less environmental heterogeneity than the general population, which may indicate that environmental factors known to affect sleep and/or white matter, such as technology and education, are not confounding factors. sleep quality and white matter integrity.

Taken together, better sleep quality is associated with greater white matter integrity in frontal areas of the brain, particularly in the pathways connecting structures involved in sleep physiology.

Keywords:Sleep, perceived stress, diffusion tensor imaging (DTI), white matter integrity

Revelation:Nothing to disclose.

University of Florida, Gainesville, Florida, United States

Bottom:Based on recent national estimates, one in four adult cannabis users will develop a cannabis use disorder (CUD) in their lifetime. Many factors are associated with the onset of CUD in adolescents. However, most studies have used cross-sectional designs, and few have explored the role of decision-making (DM) as a potential risk factor. The current longitudinal study examined the role of erectile dysfunction in predicting the development of problematic cannabis use and increased polydrug use among cannabis-using adolescents.

Methods:The study involved five assessments every six months over two years among 315 adolescents aged 14 to 17 who reported using cannabis at the start of the study. Decision making (DM) was assessed at single time points using the Iowa Gambling Task (IGT). Problem cannabis use was assessed at all time points using the reported number of symptoms of abuse and dependence using the Cannabis Use Disorders Structured Clinical Interview (CUDn) and Marijuana Problem Scale (MPS) scores. Polydrug use was assessed at all time points using a drug use history questionnaire based on the number of drugs other than cannabis (DOTC). Latent growth curve models were used to investigate bidirectional influences between DM and outcomes of interest.

Results:Primary DM (b=0.06,PI number= 0.635) and the rate of change in DM (b = 0.26,PI number= 0.08) did not predict the escalation to CUDn. Similarly, baseline DM (b = 0.16,PI number= 0.189) and rate of change in DM (b = 0.14,PI number= 0.501) did not predict the escalation of MPS results. Neither baseline DM nor its rate of increase was associated with an increase in the number of DOTCs used. The results of subsequent exploratory analyzes revealed that a one-unit increase in DOTC overtime hours was associated with an increase in problematic cannabis use based on DSM-IV cannabis use disorder and MPS score.

Conclusions:The results do not support the role of DM (as assessed by the IGT) as a risk factor for problem cannabis use or polydrug use among adolescent cannabis users. Future studies examining refined aspects of DM or evaluating the studied relationship between specific subgroups of the population may help clarify our findings.

Keywords:Cannabis use, cannabis use disorder, decision making, polydrug use, youth

Revelation:Nothing to disclose.

National Institute of Mental Health, IRP, NIH, Bogota DC, Colombia

Bottom:Drug craving often leads to reuse and relapse. Desire can increase the value of certain goods, resulting in the decision to seek and consume them. Similarly, emotional states influence decision-making, and desire often precedes or co-occurs with negative emotional states. While much has been researched about the neurobiology of hunger and emotion, we still don't understand how hunger is related to emotion and how it can influence decision-making in ways that are a hindrance to recovery, even for those seeking treatment. In this study, we assessed how different levels of hunger affected emotional expression during a delay-discounting task (a measure of impulsivity). Our approach involved electromyographic (EMG) analysis of muscles specifically involved in positive valence emotional expression (zygous muscle) and negative valence emotional expression (frowning muscle). We chose facial EMG because it allows continuous measurement of emotional expression rather than discrete self-reported measurements of emotional state.

Methods:31 individuals with an opioid use disorder (OUD) who confirmed a recent craving for heroin or other opioids were recruited from a methadone treatment program to attend 2 sessions: one before the participant took methadone and one after peak methadone levels. The order of the two sessions was randomized between subjects. We used proven tools to assess craving, subjective severity of withdrawal symptoms, and current anxiety levels. Participants then performed a 12-minute delay discounting task. We calculate the discount rate parameter for each session. During the task, surface (required for frowning) and zygomatic (required for smiling) electromyography (EMG) were measured. The received EMG signal was filtered (30-400 Hz) and rectified. Muscle activity was calculated as root mean square (RMS) during the session or at specific times in each trial.

Results:Participants reported greater opioid craving during the pre-methadone session than after the session (T= 3,66,PI number= 0.001). In the post-treatment session, the discount rate among participants was negatively correlated with yoke activity (positive valence) and positively correlated with sulcus activity (negative valence). Interestingly, these relationships were reversed in the premedication session so that the negative valence was now inversely proportional to the discount rate.

Conclusions:Taken together, these preliminary results indicate that the emotional facial expression of opioid users during impulsive choice is not context-invariant. In situations of high desire, more impulsive individuals show less expression of negative valence, suggesting that desire may moderate the emotional response in decisions about immediate and delayed rewards. Further elucidation of the physiological relationship between craving, emotion, and decision making may help to understand, predict, and prevent relapse in OUD.

Keywords:Face emotion processing, opioid addiction, impulsiveness, hunger

Revelation:Nothing to disclose.

University of California, San Diego, La Jolla, California, United States

Bottom:The use of electronic nicotine delivery systems (ENDS) or e-cigarettes remains a popular drug delivery method. Recent preclinical vapor inhalation models have shown that exposure to vaporized Δ9-tetrahydrocannabinol (THC) can induce long-term behavioral and neurobiological changes. In addition, studies have confirmed that e-cigarette aerosols, with and without nicotine, pose a significant threat to the developing nervous system. Understanding the long-term effects of repeated exposure to e-cigarettes will be critical for assessing harm and identifying mechanisms for potential therapeutic targets.

Methods:Male Wistar rats were repeatedly exposed to Δ9-tetrahydrocannabinol (THC, 200 mg/ml), nicotine (30 mg/ml) or vaporized propylene glycol (PG) twice daily for up to 2 weeks. Rat brain tissues (hippocampus and frontal cortex) were collected for immunohistochemical analysis and Western blotting. Tissues were immunostained for cannabinoid receptor 1 (CB1), glial fibrous acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA1). Western blots of CB1, GFAP, IBA1, peroxisome proliferator-activated receptor alpha (PPAR-α) and complement protein C3 were performed using frontal cortex tissue. In a separate study, pregnant female Wistar rats were repeatedly exposed to either THC (100mg/ml) or PG vapor for up to 2 weeks. The offspring were tested for anxiety-like behavior using the elevated plus maze procedure.

Results:Repeated inhalation of THC vapor significantly reduced CB1 receptor expression in the hippocampus (PI number< 0.05). In addition, repeated exposure to PG carrier vapors increased the number of GFAP+ cells (PI number<0.05) and IBA1+ cells (PI number= 0.06), while repeated exposure to THC or nicotine resulted in mixed effects. Western blot analyzes confirmed some changes in GFAP and IBA1, but only a small change in PPAR-α or C3. Finally, rats born to mothers exposed to repeated THC vapors showed a shorter time spent in open arms compared to rats born to mothers exposed to the PG vehicle, confirming the effects of prenatal vapor exposure.

Conclusions:Overall, this study confirms that repeated inhalation of THC vapor via e-cigarettes reduces the density of CB1 receptors in rats, which is consistent with tolerance effects and may produce a persistent, age-dependent effect on behavior. These data also suggest that repeated exposure to e-cigarette vapor inhalation can selectively modulate microglia and astrogliosis in rat brains.

Keywords:Electronic cigarette (e-cigarette), delta9-tetrahydrocannabinol, THC, nicotine

Revelation:Nothing to disclose.

High Point University, Fred Wilson School of Pharmacy, High Point, Karolina Północna, USA

Bottom:Dopamine receptors (D1 type (D1R, D5R) and D2 type (D2R, D3R, D4R)) are G protein-coupled receptor proteins that regulate physiological functions such as movement, emotion and cognition. D4R is enhanced in the prefrontal cortex, where it plays an important role in cognition, attention, decision making, and executive functions. Studies have shown that selective D4R ligands are a promising drug for the treatment of neuropsychiatric conditions, including Alzheimer's disease, ADHD and cocaine use disorder (CUD). D4R ligands have been shown to alter cognition and behavior in animal models of drug addiction, and DRD4 gene variants have been associated with novelty seeking and risk taking as well as ADHD. A better understanding of D4R-mediated signaling is essential for understanding and treating D4R-related disorders, including substance abuse disorders. Despite its clinical importance, there are currently no FDA-approved drugs targeting D4R and CUD therapy. This study focuses on the design of D4R ligands based on the parent phenylpiperazine backbone with pharmacokinetic analysis in rat and human liver microsomes followed by preliminary in vivo behavioral analysis.

Methods:Based on the 4-phenylpiperazine scaffold, a series of selective, high-affinity D4R ligands was designed using computer modeling. The final compounds were purified and analytically characterized, followed by elemental CHN combustion analysis. Their receptor affinity was determined in vitro using HEK293 cells expressing D2-type dopamine receptors (D2R, D3R, D4R). These binding studies were combined with functional studies using β-arrestin recruitment and cAMP inhibition assays. For various selective D4R ligands, we estimated in silico brain permeation using multi-parameter optimization of the chemical properties of the central nervous system (CNS MPO) and performed Caco-2 membrane permeability assays. For selected compounds, we performed in vitro and in vivo pharmacokinetic analyzes and preliminary in vivo behavioral analyzes in rats.

Results:Selective D4R ligands predicted from the parent scaffold were synthesized and characterized. Compounds were designed using radioligand binding displacement assays, β-arrestin recruitment assays, cAMP inhibition assays, and computational modeling. We identified several compounds with high binding affinity and D4R selectivity (Ki ≤ 100 nM and > 100-fold over other D2-type receptors) with different partial agonist and antagonist profiles. Based on the binding profiles, a subset of analogs were evaluated using functional assays in CHO cells transfected with human D2, D3 and D4 receptors. Several analogs have shown strong partial agonist and antagonist profiles in functional studies, and some have been selected for in vitro metabolic stability in rat and human liver microsomes, some of which have shown acceptable stability profiles. Of these, CAB-01-019 (with a good stability profile) was selected for in vivo pharmacokinetics in rats where it showed excellent brain penetration with AUC brain/plasma >3. The complete antagonist CAB-01-019 (5, 15 and 30 mg/kg (IP)) was tested in a preliminary study of cocaine self-administration in rats using a multi-session self-administration procedure. CAB-01-019 was found to dose-dependently reduce the number of injections taken for three unit doses of cocaine in the FR3 recall regimen, suggesting that the selective D4R antagonist reduces the rewarding effects of cocaine.

Conclusions:One of our most selective, high affinity D4R antagonists, CAB-01-019, has been discovered as a prime drug candidate. Both in vitro metabolism data in rats and humans and in vivo pharmacokinetic data in rats support its development. Some partial agonist ligands, although metabolically stable in human liver microsome assays, were unstable in rat species. Structural modifications of the phenylpiperazine backbone led to the discovery of new compounds with high D4R binding affinity and subtype selectivity. In preliminary efficacy studies, we found that the selective D4R antagonist CAB-01-019 reduced cocaine self-administration in rats.

Keywords:Dopamine D4 receptor, antagonist ligands, partial agonist ligands, cocaine use disorder

Revelation:Nothing to disclose.

University of Texas at Austin, Austin, Texas, United States

Bottom:Alcohol (ethanol, EtOH) intoxication is known to trigger homeostatic mechanisms that attempt to normalize the functioning of the central nervous system, but with repeated use, it causes neuroadaptations that facilitate the transition to addiction. Under these conditions, cessation of exposure to EtOH causes a transient increase in excitatory (glutamatergic) signaling associated with varying degrees of neurotoxicity. These changes are usually quickly corrected during withdrawal, although the appearance of behavioral rigidity over a longer period of time suggests a more lasting effect on the cognitive areas of the brain. In this case, we use proteomics and neurobehavioral analyzes to investigate whether deprivation-induced hyperactivity states mobilize the overtones of glutamatergic signaling processes in the medial prefrontal cortex (mPFC) that may underlie susceptibility to alcohol-induced cognitive impairment.

Methods:Animal studies were conducted with the approval of the Animal Care and Use Committee of the University of Texas at Austin and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, male Long-Evans rats received passive administration of vaporized EtOH or uncontaminated air (N= 12 per group) in their cages (14 hours on/10 hours off) for 5-7 weeks (mean blood EtOH = 169.1 ± 13.3 mg/dL). Separate mouse cohorts (N= 9 per group) underwent similar procedures in a modified regimen of 5 days of exposure to EtOH and 2 days of abstinence in their cages (mean BEC = 158.1 ± 12.6 mg/dL). While abstinent, the rats were trained in an operating environment to press one of 2 levers to receive a palatable food reward. Following addiction induction, the rats were subjected to 10-14 days of EtOH abstinence prior to testing in a functional model of change-strategy-configuration. Briefly, performance was assessed on a "set" task where the active lever was simultaneously paired with the indicator light directly above it. After 10 consecutive correct answers were obtained, the program order was changed to the "shift" task, where the active lever was kept on one side of the chamber (left or right) regardless of the signal lights. The rats continued testing until a criterion was reached, based on which we analyzed the total number of trials and the types of errors made during testing. All mice were humanely sacrificed and the brains were extracted and sectioned into the dorsal and ventral regions of the mPFC. This two-region approach allowed us to compare the molecular processes of cognitive behaviors that are thought to be functionally distinct in these brain regions. mPFC samples were digested into peptide fragments and labeled with isobaric tandem mass tags for quantification.

Results:Analyzes identified approximately 5,000 proteins per brain region, 200–400 of which were significantly altered by chronic intermittent exposure to EtOH.N= 3 biological replicates per group). Among these changes, we identified peptides belonging to the Grm2 gene encoding metabotropic glutamate receptors (mGluRs) known to regulate glutamatergic signaling through a presynaptic mechanism. In this regard, EtOH withdrawal mice showed reduced levels of Grm2 in the ventral mPFC as well as decreases in overlapping peptides from the Grm3 gene in the dorsal mPFC (PI number< 0.05). We tested the potential of the mGluR2 positive allosteric modulator diphenyllindone A (BINA; 20 mg/kg, 5 ml/kg, IP) to modulate cognitive performance in a strategy switching task. In this regard, abstinent EtOH rats showed an increase in perseverative errors made during the set-shift task (PI number< 0.05). Alternatively, pre-treatment of EtOH rats with BINA significantly reduced the number of trials required to reach the criterion, partially reducing this tendency towards error-prone behavior.PI number< 0,05).

Conclusions:Taken together, our studies of the mPFC proteome revealed common protein signaling elements that normally attenuate glutamatergic tone but are likely to be downregulated by CIE-induced hyperglutamanergic states. Further work uses phosphoprotein and antibody capture tools to determine whether modulation of mGluR2 activity interacts with other signaling components (NMDA or calcium-calmodulin kinases) that may stabilize glutamatergic signaling during this period of increased susceptibility to alcohol-induced dysfunction.

Keywords:Glutamatergic transmission, alcohol abstinence, proteomics, cognition, behavioral flexibility

Revelation:Nothing to disclose.

Wayne State School of Medicine, Detroit, Michigan, USA

Bottom:Preclinical studies have shown that administration of opioids induces pro-inflammatory responses in the periphery and in the brain. These pro-inflammatory responses influence the appetitive and aversive addiction processes and thus may contribute to the development of opioid use disorder (OUD) and/or the perpetuation of chronic opioid use. Here, in two studies, we examined the neuroimmune effects of morphine using positron emission tomography (PET) imaging with [11C]PBR28. [11C]PBR28 is a radiolabel that binds to the 18 kDa translocation protein (TSPO), a marker sensitive to proinflammatory neuroimmune stimulation.

Methods:Study #1: The first study included 10 healthy subjects with prior exposure to medical opioids (3F; 34.7 years [range=26-49 years]; BMI=26.4 [range=20-33]). In one day, subjects took two 120-minute [11C]PBR28 PET scans: one before and one 2 hours after morphine administration (0.04 mg/kg IM or 0.07 mg/kg IM ["low dose" vs. . "high" )) . Two patients did not complete the PET scan after morphine because of nausea: both received a "high" dose of morphine. Arterial blood was drawn during each scan to measure metabolite-corrected arterial inflow function. Total volume of distribution (TV), i.e. TSPO availability, was calculated for the 12 brain regions of interest (ROI) using multilinear analysis-1 (MA-1;T*=30). The effect of morphine on peripheral VT of [11C]PBR28 was evaluated using linear mixed-effects models with rs6971 genotype (high vs moderate affinity ligands), morphine dose (low vs high) and time (pre-) vs. "meta"-morphine) as constant factors and peripheral VT as a recurring intra-individual factor. Subjects also completed a visual analog scale (0–100 mm) to measure the subjective effects of morphine. The regional biomarker findings were not available at the time of abstract submission but will appear on the poster. Study #2: In non-human primates, we investigated the putative neurobiological mechanism underlying the neuroimmune effects of morphine: the Toll-like-4 receptor (TLR4). Two adult male rhesus monkeys (9 years and 10 kg, 11 years and 15 kg) completed [11C]PBR28 PET imaging procedures identical to study #1 before/after morphine (1 mg/kg EM). One monkey received (+)-naloxone (1 mg/kg i.v.) 10 minutes before morphine, while the other monkey received no pre-treatment. (+)-naloxone is a TLR4 antagonist with negligible affinity for the μ, Δ or κ opioid receptors. These studies have been validated by the IRB and IACUC of Yale University, respectively.

Results:Study #1: A significant main effect of time showed that morphine increased the availability of TSPO in the ROI,eat(1,203) = 282,2,PI number< 0.001. Re-analysis, after excluding the non-responder (statistical outlier), improved the fit of the model and showed a significant effect of time,eat(1,180) = 361,7,PI number< 0.001 and dose interaction after one year,eat(1,180) = 13,3,PI number< 0.001, indicating that the regional availability of TSPO increased significantly more after the "high" dose of morphine compared to the "low" dose (38–52% vs. 19–30%, respectively). In addition, a significant time effect indicated that morphine induced a subjective "high"eat(4,24) = 3,06,PI number= 0.036. Controlling for morphine dose (mg), subjective "high" post-scan was positively correlated with percentage increase in caudate nucleus TSPO availability (R2 = 0.57,PI number= 0.03). Study #2: Morphine increased the regional availability of TSPO by 24-54% in the first monkey, consistent with our human data. For the first monkey, pre-treatment with (+)-naloxone reduced the effect of morphine on TSPO by an average of 43% across all ROIs.

Conclusions:First, our findings provide the first evidence that morphine induces a neuroimmune response in humans. Second, the magnitude of the neuroimmune response in the caudate nucleus was linearly related to the magnitude of the morphine-induced "high" and accounted for 57% of the variance. Given the established role of the caudate nucleus in mesolimbic reward circuitry, our data suggest that neuroimmune signaling may modulate opioid-induced euphoria. Third, pretreatment with (+)-naloxone, a TLR4 antagonist, reduced the neuroimmune effects of morphine by almost half, suggesting that the TLR4 system is a target for drug development. Taken together, our findings highlight the role of neuroimmune signaling in opioid-induced euphoria, a process central to the development of OUD, and suggest that the TLR4 system is a drug target. Future studies are needed to investigate opioid-neuroimmune relationships in patients with OUD.

Keywords:Neuroimmune activation, Opioid addiction, Morphine, Toll-like receptors (TLRs), Mesolimbic reward system

Revelation:Nothing to disclose.

Yale University School of Medicine, New Haven, Connecticut, Stany Zjednoczone

Bottom:Across species, chronic alcohol consumption is associated with extensive brain and body changes. These changes are particularly pronounced in stress-related processes: in humans, alcohol use disorder (AUD) is associated with peripheral changes in the stress hormone cortisol, as well as central changes in the structure and function of stress-related brain networks. These stress-related changes may play a key role in enhancing the development and relapse of problem drinking. However, although stress-related regional changes have also been observed in risk-taking drinkers who do not meet criteria for addiction (e.g., Blaine and Sinha 2017), it is unknown whether adaptations in anticipatory brain circuits are also evident at this early stage. Here we assessed whether regular drinkers, an increasingly common high-risk group for developing AUD, exhibit distinct patterns of stress-related functional connectivity.

Methods:We recruited 104 male and female participants (N= 53 heavy drinkers/51 light drinkers/abstinents per NIAAA criteria) to participate in a validated functional MRI stress induction procedure (Sinha et al. 2016). Specifically, these groups were matched for age, gender, and measures of stress, including current stress, anxiety and depression, as well as a history of traumatic childhood events. We used connectome-based predictive modeling (CPM; Shen et al. 2017) to determine whether whole-brain functional connectivity patterns during a continuous stressful task could successfully predict subjective stress ratings in both cohorts and developed further analyzes to identify non-parametric differences in: (1) which brain regions contributed to successful predictions; and (2) how strongly different brain regions were associated with subjective stress. To determine the prospective validity of the identified predictive brain networks for light and heavy drinkers, we collected data from our participants' diaries for 30 days after scanning to assess how the involvement of these networks related to their subsequent experiences in the real world. stress... and controlling drinking.

Results:With different models, we were able to (allPI number< 0.001) and comparatively predict subjective stress using whole-brain connectivity in light and heavy drinkers. Although there were no significant differences in subjective anxiety ratings between the groups (PI number>.25), we identified generalized group differences in the neural correlations of this stress response. All significant effects were non-parametrically thresholdedPI number< 0,001.

Heavy drinkers have more visual and motor connections in their stress prediction networks, with visual and salient connections contributing significantly more to the successful prediction of stress in heavy drinkers than in light drinkers. At the network node level, stress predictions for heavy drinkers had significantly more connections to the visual and primary motor cortices, anterior cingulate, insula and ventral gyrus, and much stronger brain/stress associations in the inferior temporal gyrus and cerebellum. In contrast, the stress prediction networks for light drinkers had significantly more connections to the ventral prefrontal cortex (vmPFC), angular gyrus, hippocampus, amygdala and posterior cingulate gyrus, with the vmPFC and angular gyrus also showing stronger brain/stress correlations. Comparison of stress representation patterns in the brain (after Kriegeskorte et al. 2008) additionally revealed group differences in the cerebellar, ventral, and subcortical networks.

Finally, prospective projections revealed that the brain prediction networks identified for heavy drinkers (BDN) were more specialized than those for light drinkers (LDN). BDN involvement during the fMRI scan predicted later levels of binge eating stress (b=0.025 [0.008],PI number= .0013), but not light drinkers (PI number>0.27; paroxysmal vs mild: b=0.02 [0.008];PI number= .0041); LDN did not show this discrimination (PI number> .2). In addition, the share of BDN predicted less control over future alcohol consumption, while LDN did not (F1.82=4.78,PI number= 0.032), suggesting that the neural correlates of stress in heavy drinkers may be more specific to stress-induced alcohol motivation.

Conclusions:These results suggest alcohol-induced adaptation in stress-related brain circuits, with a shift towards over-reliance on primary sensory and motor networks to predict subjective stress responses in binge drinkers and stronger brain/stress correlates in frontolateral networks. and standard function (including vmPFC, hippocampus, and posterior cingulate) in light drinkers. These neural differences were evident even when the subjective responses themselves (as well as participants' stress histories) were similar, demonstrating the utility of these analyzes in revealing group differences that are not apparent when examining the brain or behavior alone. Interestingly, this light drinking circuit has previously been identified as contributing to adaptive stress responses (Sinha et al. 2016; Goldfarb et al. 2020), and the differences between heavy and light drinkers overlap with circuits known to that they are altered in the clinical AUD (e.g. Seo et al. 2013). Together, these findings indicate potential targets for early therapeutic intervention to attenuate the development of AUD and reveal the plasticity of the neural processes underlying the stress response.

Keywords:Acute stress, alcohol consumption, functional fMRI connectivity, instantaneous ecological assessment, brain connectivity

Revelation:Nothing to disclose.

University of Kansas, Lawrence, New York, United States

Bottom:Opioid use disorder is a chronic, relapsing mental disorder with an enormous socioeconomic burden. Relapse is one of the most difficult challenges in recovery. None of the current treatments are effective in preventing recurrence. Stress is one of the most obvious causes of relapse. Most research focuses on withdrawal stress that causes relapse. Little is known about how stress in early life increases addiction risk and vulnerability to relapse.

Adverse psychosocial factors in early childhood or adolescence impair neuronal structure and brain function, predisposing to susceptibility to substance use disorders. However, the mechanisms underlying vulnerability to stress-induced addiction in early life remain unclear, especially in the case of opioids.

Anxiety and substance abuse are associated with neuroplasticity in the mesocorticolimbic pathway. For example, maladaptive glutamatergic projection neurons of the prefrontal cortex (PFC) and their subcortical projection regions (such as the nucleus accumbens [NAc] and ventral tegmental area [VTA]) are implicated in both stress and addiction. We therefore hypothesize that ESI stress may increase vulnerability to addiction by exerting pathological damage to these key areas of the brain. To test our hypothesis, we used a mouse model of heroin self-administration to investigate how the chronic stress of early social isolation affects behavioral and neural responses to heroin in adulthood.

Methods:Male and female C57BL/6J mice were used in the current study. Based on previous publications, stress related to early social isolation (ESI) was performed after weaning from PND 21 (P21) to P60 (approximately 5 weeks). Control mice are housed in groups (GH, 4-5 mice per cage). All procedures were approved by the Institutional Animal Care and Use Committee. All animals were maintained in accordance with the National Institutes of Health guidelines of the Association for the Evaluation and Accreditation of Laboratory Animal Care. At P60, GH and ESI mice were first trained to self-administer sucrose in fixed and progressive ratio schedules. After the sucrose progressive index test, the animals underwent cervical surgery. After recovery, mice were subjected to SA heroin (50 μg/kg/injection, 3 h/session) for 10 days, followed by progressive ratio and dose-response tests (6.25, 12.5, 25 and 50 μg/kg/ shot ). Then, 14 days after the last heroin session, the animals were subjected to a 1-hour stimulus-evoked heroin-seeking test. Another set of animals were sacrificed without a re-exposure stimulus to study c-Fos expression in subregions of the mesocorticolimbic system during abstinence. Circuit-specific chemogenetic tools (AAV[AAVrg]-Cre back and Flex-hM3D-Gq) were used to identify key brain pathways that regulate heroin seeking under ESI stress.

Results:ESI stress did not alter the acquisition of sucrose or SA heroin, nor did it alter sucrose motivation in a progressive intercourse pattern. However, ESI stress induced an upward shift of the heroin dose-response curve in female mice (N= 11-14/group, F1, 45 (stress) = 10864,PI number= 0.002, F1, 45 (sex) = 8.285,PI number= 0.006), increased total responses during the heroin progressive index test (N= 11-14/group F1, 45 (stress) = 15.61,PI number= 0.0003) and during the heroin-seeking test (N= 7-9/group, F1, 29 (stress) = 38.6,PI number< 0.0001) in men and women. In addition, ESI stress decreased c-Fos expression in the prefrontal cortex (PrL,N=4/group F1.12 (stress)=62.88,PI number< 0.0001), sub-Mediterranean bark (IL,N= 4/group 62.82,PI number< 0.0001) e VTA (N= 4/group F1, 12 (stress) = 4.996,PI number= 0.045) after 14 days of forced abstinence. To investigate whether hypoprojection of the PFC into the VTA contributes to ESI-enhanced heroin seeking, we injected Cre-AAVrg into the VTA and Cre-dependent hM3D-Gq into the PrL of ESI mice during heroin withdrawal. After viral expression, we injected compound 21 to repeatedly activate the PFC-VTA circuit for 3 days (3 mg/kg, ip once daily). 24 hours after the last vehicle or C21 injection, the mice were subjected to a heroin-seeking test. We found that C21 treatment significantly reduced heroin seeking in ESI mice (N=7-11/group, t13.47=2.5,PI number= 0,02).

Conclusions:These data indicate that ESI stress leads to increased heroin seeking and motivation, which may be related to marked alterations in neural activity in the mesocorticolimbic system, where underactive PFC-VTA projection may contribute to ESI-enhanced heroin seeking. .

Keywords:Opioid addiction, prefrontal cortex, ventral tegmental area, social isolation stress, chemogenesis

Revelation:Nothing to disclose.

University of Minnesota, Minneapolis, Minnesota, United States

Bottom:We used data-driven methods to discover subtypes in people with substance use disorder (SUD) from a large community sample, the NKI-Rockland Enhanced Sample (NKI-RS). The NKI-RS study used a broad phenotyping approach to assess impairments in three general functional domains that have been associated with addiction: 1) approach behavior, 2) negative affectivity, and 3) executive function. To our knowledge, this is the first study of lesion heterogeneity in these three domains in a sample with mixed DUB diagnoses (with different primary drugs of choice). We hypothesized that deficits in these three functional domains represent three independent neurobehavioral mechanisms associated with addiction, which would warrant a subtyping and individualized medical approach to addiction treatment. Thus, we expected to show the existence of at least three distinct addiction subtypes, regardless of the main drug chosen: "reward users" for deviant behavior, "relief users" with high negative affectivity, and "poor functioning". users with reduced executive function.

Methods:Data were collected and ethical approval was obtained from the Nathan-Kline Institute in New York. In the sample with complete data (N= 644. 66% women; 55% of women in LDS. 74 results per subject), we initially arrived at 12 hidden phenotypic factors using parallel analysis to determine the number of factors. Subtypes were then identified by clustering using latent profile analysis of these latent factors within those with prior SUD [N= 172.55% women;N=74 Alcohol use disorder, 67% women;N= 30 Cannabis use disorder, 49% women.N= 68 polydrug users, most commonly cocaine use disorder, 44% women]. The optimal model was selected using the Bayesian information criterion. Subtypes were behaviorally characterized by profiling deficits in these latent factors, controlling for significant differences for healthy controls. Resting-state connectivity analyzes based on graph theory (Brain Connectivity Toolbox) were performed for each subtype in a subsample with full fMRI and NIDA Quick Screen (formerly SUD) dataN= 104, 58.6% women; controlsN= 302, 73.5% women). Differences in global nodal efficacy, local efficacy, and mediation were measured for each subtype compared with healthy controls by regression of graph theory measures of lifetime drug use, while tobacco use covariates were for past 6 years, months, and comorbidities.

Results:Latent factor analysis revealed three hypothetical phenotypic domains. Individuals with a history of SUD showed mild impairments in all three domains (PI number< 0.05, uncorrected. in all subtypes). Latent profile analysis revealed three subtypes among people with a history of SUD, each showing a distinct disability profile (allPI number< 0.05, corrected by Holm-Bonferroni): (1) Reward users showed high sensation seeking, social risk taking, rule breaking and openness, and reduced risk perception. (2) "Aid users" showed a high degree of internalization, general psychiatric symptoms and negative affect, and a lack of persistence. and (3) "Low Users" had low openness/sensitivity, crystalline/fluid IQ, and below average sensation seeking. Importantly, these three subtypes were evenly distributed among subjects with different primary SUD diagnoses (PI number= 0.51), demonstrating the existence of addiction subtypes independent of the main drug of choice. Resting-state analysis revealed reduced connectivity (between) to the accessory motor network in reward users compared to healthy controls when predicting the number of drugs used over a lifetime (pFDR < 0.05). On the other hand, support users showed greater connectivity (between) to the care network (7P, VIP, MIP, 6a) (pFDR < 0.05).

Conclusions:These empirical results provide evidence for a general three-domain model of addiction, extending previous empirical work on the three-domain model in alcohol use disorder for individuals with cannabis and cocaine use disorders. Importantly, our results also reveal the existence of at least three distinct 'addiction subtypes', as hypothesized, demonstrating their existence regardless of the main drug of choice of the individual. The results of the brain analysis replicate previous findings on cocaine users (unpublished data). Taken together, these results warrant future research into the importance of addiction subtypes in predicting clinical outcomes and their utility in developing personalized medical approaches. These precision medicine approaches can help differentiated treatment of patients based on their individualized impaired functional domains and subsequently improve treatment outcomes.

Keywords:Addiction Phenotypes, Alcohol and Substance Use Disorders, Subtypes of Substance Use Disorders, Search Domain Criteria (RDoC), Personalized Medicine

Revelation:Nothing to disclose.

Duke University, Durham, North Carolina, United States

Bottom:Neurotensin (NTS) is a 13 amino acid peptide that acts as an endogenous regulator of brain dopamine (DA) signaling. Neurotensin regulates DA neurotransmission by acting on the G protein-coupled receptor, neurotensin receptor 1 (NTSR1). NTSR1 is expressed in DA cell bodies in the ventral tegmental area and substantia nigra, as well as in DA axon terminals in the striatum. NTSR1 signaling can be targeted pharmacologically to alter DA neurotransmission and psychostimulant-related behaviors (e.g., cocaine, methamphetamine). Systemic administration of a selective agonist NTSR1 is reported to transiently reduce methamphetamine self-administration in mice, and we recently showed that a β-arrestin-polarized NTSR1 agonist can reduce cocaine self-administration. NTSR1 knockout (KO) mice are resistant to methamphetamine-induced changes in AD cell firing and exhibit reduced methamphetamine self-administration and methamphetamine-seeking behaviors. However, the effect of NTSR1 deletion on cocaine self-administration and cocaine-seeking behavior remains to be determined. To this end, we used global NTSR1 KO mice to analyze the contribution of NTSR1 to cocaine-induced hyperactivity, intravenous (iv) cocaine self-administration, and cocaine seeking during signal-induced extinction and reinstatement using functional paradigms.

Methods:NTSR1 tm1Dgen mice (catalogue number 005826; Jackson Labs, Bar Harbor, ME) were purchased and crossed with C57BL/6J mice. Cocaine-induced novelty and hyperactivity and longitudinal intravenous self-administration of cocaine were evaluated in adult male and female NTSR1-null (KO) mice and their wild-type (WT) littermates. Mice with internal jugular catheters were evaluated for sensitization to novelty and cocaine-induced locomotor activity (20 mg/kg, i.p.)N(WT) = 25,N(NTSR1 KO)==27). The mice were then trained to self-administer cocaine (0.5 mg/kg/injection) in conjunction with a lever response signal in the operant chambers. During 1-hour daily sessions, mice were trained in 2-lever and active vs. lever. inactive under FR1, FR2 and FR4 booster programs per prospective study protocol. After obtaining a sustained lever response, FR4 was assessed at 5 doses of cocaine: 0.5, 0.1, 0.3, 1.0 and 3.0 mg/kg/injection (N(WT) = 15,N(NTSR1 KO)=20). Mice completed a single Progressive Ratio (PR) session with a cocaine dose of 0.5 mg/kg/injection. Dose-response and PR trials were followed by 1-hour once-daily extinction sessions in which clues were hidden and lever responses had no planned consequences. After 20 sessions of extinction, mice underwent a single stimulus-induced recall session in which cocaine-related stimuli were presented in the absence of drug reinforcement.N(WT) = 10,N(NTSR1 KO)=16). Mice without proprietary catheters and those meeting certain health parameters were removed from the study. Two-way repeated measures or mixed-model ANOVA models were used to analyze the effects of genotype on behaviors over time, drug doses, sessions, and session types.

Results:Compared to WT controls, NTSR1 KO mice showed reduced novelty-induced locomotor activity (PI number= 0.0017), but unaffected cocaine-induced hyperactivity. Time to self-administration of cocaine was comparable for both genotypes (mean ± SEM, KO: 18.2 ± 1.3; WT: 16.1 ±   1.1 sessions). Notably, the active lever response was FR and drug dose dependent for WT and NTSR1 KO mice. Cocaine self-administration of NTSR1 KO and WT mice could not be separated by cocaine intake, active lever response, inactive lever response, latency of onset of lever response, fraction of responses occurring after reinforcement, or accuracy of the lever, at any of the five doses assessed for cocaine. In the PR session, WT and NTSR1 KO mice reached comparable breakpoints. After evaluating cocaine use behaviors, cocaine seeking was assessed during extinction and recovery. The overall leverage response during the 20-session extinction period was reduced in NTSR1 KO mice compared to WT controls (PI number= 0.0178). When cocaine-related states were reinstated, both genotypes showed some degree of reinstatement of cocaine seeking, but an effect of genotype on the lever response was detected (PI number= 0.0040). In the cue-triggered recovery session, post hoc pairwise comparisons identified an active downtrend (PI number= 0.0868), but not inactive (PI number= 0.7218) lever response in NTSR1 KO mice. No differences were detected between the sexes, but there was a trend towards reduced cocaine consumption by females compared to males at doses of 0.1 and 0.3 mg/kg/injection in WT mice (PI number= 0,0669).

Conclusions:NTSR1 KO mice show intact locomotor responses in cocaine-seeking and WT-like behaviors, but have reduced novelty-induced locomotion and impaired cocaine-seeking during extinction and reintroduction. In the context of the methamphetamine literature, these findings suggest that some mechanisms leading to self-administration of cocaine and methamphetamine are different. Our work provides further evidence that the mechanisms mediating cocaine seeking and consumption behavior differ and suggest that NTSR1 deletion selectively affects cocaine seeking. These results can be used in the development of mechanism-based treatments for cocaine use disorders that reduce cravings and prevent relapse.

Keywords:Cocaine, self-administration, mouse models, neurotensin, extinction and recovery

Revelation:Nothing to disclose.

Columbia University Medical Center, New York, Minnesota, USA

Bottom:Dopamine is an essential neuromodulator involved in physiological processes such as learning and memory, motor control and reward, as well as in pathological conditions such as Parkinson's disease, schizophrenia and substance use disorders. Compared to extensive work investigating neurons, the role of astrocytes in dopaminergic signaling has not yet been fully elucidated. In addition, little is known about the role of astrocytes in the plasticity effects of psychostimulants such as amphetamine. The current work will provide new evidence that astrocytes play a key role in dopaminergic signaling and psychostimulant-induced plasticity and may serve as therapeutic targets for diseases with disrupted dopamine signaling.

Methods:Ethical Statement: All animal care procedures have been approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. To study the astrocyte response to dopamine in awake animals, we used fiber photometry. Adult male and female DAT-IRES-Cre mice were used. Two viral vectors were transfected to target dopaminergic neurons in the ventral tegmental area with a light-activated channel (AAV5-hSyn-FLEX-ChrimsonR-tdT) and astrocytes in the nucleus accumbens were targeted with the calcium marker, AAV5-GfaABC1D-cyto-SV40 . The optical fiber was placed 0.02 mm dorsal to the NAc viral core injection for calcium imaging experiments. Locomotor tests with amphetamine were performed ≥ 3 weeks after surgery. To test the effect of amphetamine on the astrocyte response to dopamine, amphetamine was administered at a dose of 2.5 mg/kg (ip) and the increase in astrocyte calcium in response to dopamine was assessed before and after amphetamine exposure. Statistics: Data are expressed as mean ± standard error of the mean (SEM). Data normality was tested using the Kolmogorov-Smirnov test. The results were compared using a two-tailed Student's t-test.testor ANOVA (α = 0.05). Fisher LSD one-way post hoc ANOVA was used for normally distributed data, and Kruskal-Wallis post hoc Dunn one-way ANOVA was used for non-normally distributed data. Non-standard MATLAB code was used to analyze the data.

Results:In awake animals, optogenetic stimulation of dopaminergic terminals (5 ms pulses for 5 s) resulted in an increase in astrocyte calcium that was attenuated in the presence of the global dopamine receptor antagonist flupentixol.N= 10,PI number< 0.001), suggesting that astrocytes respond to dopamine by activating the dopamine receptor in vivo. The psychostimulant amphetamine significantly increased the astrocyte response to dopamine in relation to the area under the curve (N= 15,PI number= 0.01). The astrocyte response to dopamine was modulated by repeated amphetamine exposure and reinstatement to amphetamine after a period of abstinence (N= 6,PI number= 0,01).

Conclusions:Taken together, the present results demonstrate that astrocytes respond to dopaminergic signaling by increasing cytoplasmic calcium levels and participate in psychostimulant-induced brain plasticity. Astrocyte calcium signaling in awake animals is modulated by repeated exposure to amphetamine and re-exposure to amphetamine after a period of abstinence, suggesting that astrocytes may serve as a novel cellular therapeutic target for targeted therapies for substance use disorders.

Keywords:Astrocyte-neuron interaction, dopamine, amphetamines, in vivo calcium imaging, reward system

Revelation:Nothing to disclose.

Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, USA

Bottom:Opioid abuse has increased dramatically over the past decade, with potent synthetic opioids such as fentanyl responsible for nearly half of opioid-related deaths. Opioid withdrawal or withdrawal creates a negative emotional state that is believed to promote relapse. Opioid withdrawal induces dendritic atrophy in the medium spiny neurons (MSNs) of the nucleus accumbens (NAc), but the molecular mechanisms remain unknown. Our previous work showed that dendritic atrophy of MSNs expressing the D1 dopamine receptor leads to anxiety-like behaviors. Therefore, we hypothesize that the atrophy induced by fentanyl withdrawal is specific to the MSN subtype, and that molecular mediators of the blockade may reverse the behavioral changes induced by opioid withdrawal.

Methods:Male and female mice were administered fentanyl in a cage for 5 days (10 μg/ml in drinking water) followed by 10 days of abstinence. We assessed anxiety-like behaviors with social interactions and the elevated maze plus test in both sexes, and we assessed increased stress sensitivity in men with an acute social stressor (N= 9-12 mice/sex/condition). To characterize structural plasticity in NAc MSNs, we used the Cre-dependent eYFP virus to sparsely label D1- and D2-MSN in D1- and A2A-Cre mice, respectively (N= 11-13 cells from 4-5 mice/sex/condition). We tagged MSN subtypes for patch electrophysiology with Cre-Off tdTomato Cre-On eGFP in D1-Cre mice (N= 3-7 cells/mouse, 4 mice/gender/condition). To profile the molecular changes and determine the molecular mechanisms of cell type-specific dendritic remodeling, we used D1- or A2A-Cre mice crossed with RiboTag mice to isolate ribosome-associated mRNA in specific cell types after discontinuation of fentanyl (N= 4 mice per sample, 6 samples/gender/cell type/state). We performed D1- and D2-MSN transcript RNA sequencing followed by weighted correlation network analysis (WGCNA). We then further validated the genes identified by RNAseq using Nanostring and RNAscope (N= 4-6 mice/gender/cell type/state). We evaluated the effects of E2F1 expression on behavior, morphology and electrophysiology using Cre-dependent E2F1 overexpression virus in D1-Cre mice (N= 4-8 mice/sex/condition).

Results:Both male and female mice show increased social withdrawal and anxiety-like behavior after fentanyl withdrawal. (e.g. reduced open hand EPM time, fentanyl vs. water:eat(1,33) = 6,102,PI number= 0.018). Anxiety-like behaviors after withdrawal were associated with reduced NAc D1- but not D2-MSN dendritic complexity (D1 Sholl Radius x Drug:eat(15,688) = 2,006,PI number= 0.0129, cortical radius D2 x Lek:eat(15,688) = 0,4386,PI number= 0.97). Using WGCNA, we identified 11 networks of MSN subtype-specific genes altered by fentanyl withdrawal. We found a cluster of genes with reduced dendritic morphology exclusively in D1-MSN that are transcriptionally regulated by E2F1. E2F1 overexpression in D1-MSN protected mice from withdrawal-induced dendritic atrophy of D1-MSN and anxiety-like behaviors (E2F1-fentanyl vs E2F1-waterPI number> 0,05).

Conclusions:Our findings indicate that fentanyl withdrawal induces unique structural, functional and molecular changes in NAc D1-MSN. Moreover, these molecular changes may be targeted to attenuate dendritic atrophy induced by withdrawal and anxiety-like behaviors.

Keywords:Fentanyl, RNAseq, Nucleus Accumbens, Dendritic Remodeling, Recall

Revelation:Nothing to disclose.

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Bottom:Unfortunately, the limited utility of available treatment options remains a major obstacle to alleviating the daunting burden of alcohol use disorder (AUD). The rational development of new pharmacological or brain stimulation treatment options will be based on a better understanding of the molecular and neuro-circuit mechanisms by which ethanol exposure alters brain function.

Methods:We used ethanol conditioning to assess learning aversion and reward. Fluorescent tomato PV-td control mice received nonconventional injections of vehicle or ethanol (2 g/kg i.p.) before or after site conditioning to induce conditioned place preference or aversion, respectively. We assessed PV-IN physiology by ex vivo whole-cell clamp electrophysiology between 3 and 7 days after ethanol exposure.

Results:We found that female and male C57BL/6J mice showed a preference or aversion to ethanol poisoning with no apparent differences between the sexes. Ethanol preference, but not aversion, was associated with reduced PV-IN excitability. In addition, ethanol preference was specifically associated with increased excitatory synaptic potency in PV-IN, and the phenotype emerged during ethanol withdrawal.

Conclusions:These findings indicate that ethanol-induced neural adaptations may vary with behavioral experience. PV-IN PFC adjustments are not made after the ethanol experiment. Rather, it appears that reduced excitability and increased synaptic strength are specifically related to learning to reward with ethanol. These findings suggest that PV-IN PFCs represent an interesting target for the regulation of enduring memories associated with ethanol reward.

Keywords:Ethanol, parvalbumin, GABA interneurons, medial prefrontal cortex

Revelation:Nothing to disclose.

University of Washington - Seattle, Seattle, Washington, United States

Bottom:Opioid overdose deaths have skyrocketed nationwide over the last 20 years. The main difficulty in tackling this epidemic is that both the therapeutic (i.e. analgesic) and addictive properties of opioids work through mu-opioid receptors (MOPRs). However, increasing evidence suggests that these different properties may be mediated by different intracellular signaling mechanisms (G-protein signaling versus beta-arrestin signaling). We recently showed that MOPRs exert their rewarding but not analgesic effects via a dorsolateral spike in the nucleus accumbens periphery (LDRN-mNAcSh). Here, we seek to determine how G protein or arrestin signaling controls MOPR reward modulation. By isolating these MOPR mechanisms, we can design more effective opioid drugs that reduce or even prevent their addiction.

Methods:Adult male and female mice (8-16 weeks) were used in all studies. Behavioral mice (7–13 per group) were tested in a food intake task in which they were allowed to freely consume sucrose pellets for one hour. Mice were tested ad libitum or after acute 24-hour food deprivation. The mice were also tested in a limeria task in which they were allowed intermittent access to a sucrose solution. Mice were tested ad libitum or after 18 hours of acute water deprivation. Statistically, we used parametric ANOVA/T- attempt. Effect sizes and confidence intervals were also calculated to complement the results. Multiple mice (3-6/group) were also used for anatomical validation.

Results:MOPR knockout mice (OPRM1 KO) ate and licked less after food or water deprivation compared to wild-type mice. In contrast, beta-arrestin 2 (Arrb2 KO) mice showed no general deficits in food intake or lycometric tests, indicating that arrestin does not primarily mediate the MOPR modulation of rewarding behavior. Additional analyzes of the lick microstructure showed that the reduction in licking in OPRM1 KO mice was due to fewer initiations of licking attacks (PI number= 0.018), while the duration of licking was similar between KO mice and wild-type mice (PI number= 0.231). Arrb2 KO mice also performed fewer sessions (PI number= 0.014), but the licking time was almost twice as long as in wild-type mice (PI number= 0.089). Perhaps indicating that arrestin may play a role in expressing rewarding behaviors. To further isolate the involvement of arrestin signaling in MOPR-mediated behaviors, we selectively expressed a G protein-sensitive MOPR mutant in OPRM1 KO mice in the LDRN-mNAcSh loop. We found that this selective rescue partially restored food intake (PI number= 0.11) and licking compared to wild-type mice. Furthermore, we found that this rescue elicited a microstructural licking phenotype similar to that of Arrb2 KO mice, where they exhibited a longer duration of licking but did not initiate licking (PI number<0.001). Ongoing research includes the development of G protein-specific CRISPR/Cas9 viral vectors to selectively cleave various G protein subunits, as well as the selective restoration of arrestin signaling in Arrb2 KO mice. Future experiments will also test how the G protein or stop signal affects exogenous opioid rewards (e.g., morphine).

Conclusions:These results indicate that G-protein and arrestin signaling contribute to MOPR-mediated reward behavior and do so in a complementary manner. In particular, G protein signaling appears to be required to initiate reward consumption, while arrestin is required to terminate reward consumption. These results have important implications for the development of therapeutic opioid drugs.

Keywords:Mu opioid receptors, bias signalling, food intake

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:The United States is in the midst of an opioid abuse and overdose epidemic that has been declared a public health emergency. Oxycodone is one of the most commonly prescribed painkillers, the first opioid many people try, and has physicochemical properties that allow it to accumulate in the brain at a higher rate than other opioids, perhaps explaining its significant potential for abuse. Over the past two decades, much research using animal models of drug addiction has focused on a small number of neurobiological systems, in particular the mesocorticolimbic dopamine system, the cortical glutamate system, and the extended amygdala. Recent advances in tissue cleaning and light microscopy technologies now allow unbiased, high-throughput study of protein expression or neural circuitry throughout the brain. In addition, the development of deep learning-based motion capture models such as DeepLabCut and behavioral segmentation/clustering enables high-throughput, unbiased behavioral analysis. By combining these techniques and taking a holistic approach to the study of addiction-like behavior, we are able to thoroughly and unbiasedly study brain region, cell type, and specific behavioral control projection mechanisms. My hypothesis is that these holistic methods could be the key to unlocking new relapse prevention methods.

Methods:We used the iDISCO+ tissue debridement method and light microscopy to examine whole-brain c-Fos expression after experimenter administration of oxycodone and after signal-induced oxycodone search. Using male C57Bl6/J mice, we first quantified c-Fos expression after acute injection of either saline or 5 mg/kg oxycodone (a dose considered appropriate in CPP paradigms.N= 6-8 per group). We then trained Fos2A-iCreER (i.e. TRAP2) x tdTomato mice to self-administer intravenous oxycodone, with saline and sucrose self-administered control groups (N=  9–14 per group) and used iDISCO+ to identify neural assemblies activated by signal-induced reinstatement. We identified the dorsal pedicle cortex (DPC) as a poorly studied structure that showed significant increases in c-Fos+ and tdTomato+ cells after acute oxycodone injection and reinstatement of oxycodone seeking, respectively. We then used single-core sequencing, RNAscope, and patch-clamp electrophysiology to fully characterize the cell types in CPD and their responses to opioids. To establish the functional role of CPD in opioid responses, we used C57, TRAP2 and MOR-Flox mice, including optogenetic and chemogenetic studies of hedonic responses to oxycodone exposure and addiction. We used DeepLabCut, a Python-based deep learning library to track individual body parts, and Variable Auto Encoder (VAME) to segment behaviors and quantify the time spent on each detected behavior. Finally, we performed whole-brain mapping of CPD outputs in TRAP2 mice using en face markers in conjunction with iDISCO +  and identified a projection to the nucleus accumbens that regulates opioid withdrawal.

Results:We found that the DPC is an aversion signaling hub in the ventral prefrontal cortex and contains a highly unique population of neurons that co-express vGluT2 and the μ-opioid receptor (MOR). Single-core sequencing, RNAscope and qPCR show that the DPC is highly enriched for MOR expression relative to the nearby hypomellite cortex. Electrophysiological characterization of MOR(+) neurons showed that they had a relatively depolarized resting membrane potential compared to neighboring MOR(-) neurons, showed increased Ih currents, and were hyperpolarized by the use of DAMGO, confirming MOR functionality in these glutamatergic neurons. Optogenetic stimulation of ChR2 in the DPC of C57 and vGluT2-Cre mice induced real-time place aversion, and this aversion was blocked by pre-administration of oxycodone. This result was recapitulated by selectively stimulating opioid-responsive neurons in TRAP2 mice, and we showed that MOR (GPCR inhibitor) binding directly stimulated c-Fos production via ß-Arrestin2 signaling. When MOR is selectively knocked out by injection of AAV-Cre into the DPC of MOR-Floxed mice, the hedonic valence of oxycodone is reversed and the rewarding dose in AAV-GFP-treated control mice produces conditioned place aversion. In opioid-dependent mice, artificial rescue of Gi-GPCR signaling in CPD by hM4D(Gi)-DREADD inhibits naloxone-induced motor withdrawal symptoms. On the other hand, optogenetic stimulation of opioid-responsive neurons in the CPD of TRAP2 mice enhances naloxone-induced withdrawal symptoms. Analysis of DPC outputs from TRAP2 mice shows dense projections to hindbrain regions known to regulate pain, stress, autonomic function and aversion, including the brachial nucleus (PBN) and the medial gyrus. Optogenetic stimulation of DPC terminals in the PBN of TRAP2 mice recapitulated the aversive effects of somatic DPC stimulation.

Conclusions:These data identify the DPC as a highly novel cortical regulator of opioid reward and aversion. Furthermore, we identified a unique cell population that co-expresses MOR and vGluT2, contradicting the canonical notion that the effects of opioids in the cortex occur through inhibition of GABAergic interneurons.

Keywords:Opioid abuse, opioid addiction, addiction circuits

Revelation:Nothing to disclose.

University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States

Bottom:We recently developed a rodent model of opioid addiction that is able to identify subpopulations of heroin-preferring and food-preferring rats under conditions of mutually exclusive choice between the two rewards. The general population makes, on average, about 50% of the choices between heroin and food, under conditions where reward price (FR3) and latency (0) are held constant. We applied the principles of behavioral economics to determine individual rats' motivation for heroin versus food, and found that the demand for heroin is much more inelastic than the demand for food. Correlation analyzes revealed no significant variables able to predict heroin choice, although predictors of relapse were identified. In addition, there was no correlation between choice and relapse, indicating that these are separate measures of opioid dependence. The above-mentioned findings are currently under publication (Nature Communications). To further validate this new model of opioid selection and relapse, we generated a reverse translation of the well-known therapeutic opioid use disorder naltrexone.

Methods:Our experimental procedures followed the guidelines outlined in the Laboratory Animal Care Guide and were approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee (IACUC). Male and female Wistar rats (N= 14), an intravenous jugular catheter was surgically implanted. After recovery, the rats were trained to self-administer heroin and food simultaneously, on opposite levers with opposite directions (tone or light, signals and lever position were balanced). Training started with the FR1 program (7 days) and then progressed to FR3 (3 days). When self-feeding, both levers were retracted while delivering the bounty + clue (5s). The selection phase then began, during which an additional 10-minute period was imposed (lever retracted, house light turned off) to limit the availability of rewards during the session, forcing the rats to exclusively choose each other's heroin over food. in every effort. After three initial screening sessions, repeated treatment with vehicle or naltrexone was initiated. Naltrexone (3 mg/kg, SQ) was administered 10 minutes before the start of the 150-minute choice session and again midway through the session to ensure that its effects were maintained throughout each session. Treatment was for seven selected sessions and a relapse test was performed the next day (~24 hours after the last naltrexone injection). During the cue relapse test, both levers were available simultaneously and delivered the appropriate heroin or food cue (FR3), but the rewards were withheld.

Results:Treatment with naltrexone reversed the choice of heroin to eat, with effects occurring on the fourth day of treatment. Selection data were analyzed using a 2-way MRI ANOVA with session selection as a repeated measure within subject and treatment group (naltrexone or vehicle) as an inter-subject variable. Session Main Score [eat(9,108) = 10,11,PI number< 0.0001] and session x treatment interaction [eat(9,108) = 4,048,PI number= 0.0002] were significant. Individual 1-way MR ANOVAs were performed for each treatment group to determine whether selection changed over time. Selection shifted from heroin (to food) only in the naltrexone group [eat(9,60) = 1,147,PI number<0.0001]. Dunnett's post hoc multiple comparisons test for the first baseline selection session (prior to treatment) showed that heroin choice decreased significantly over the last four days of treatment (PI numbers < 0.05). In addition, heroin (but not food) relapse was reduced with pretreatment with naltrexone. Relapse data were analyzed using a leveraged two-way RM ANOVA as a repeated within-subject measure and treatment group as a between-subject variable. The main treatment effect [eat(1,12) = 11,76,PI number= 0.0050] and leverage x treatment interaction [eat(1,12) = 6522,PI number= 0.0253] were significant. Multiple post hoc comparisons by Sidak showed that naltrexone reduced response compared to heroin-leverage vehicle alone (PI number= 0,0005).

Conclusions:Although opioid choice and opioid relapse measure different aspects of opioid dependence, repeated treatment with naltrexone was effective in reducing both behaviors. Moreover, the effect of naltrexone on relapse was specific to heroin and did not interfere with responding to food cues. Future research will increase the sample size of this initial experiment to determine whether naltrexone treatment is more effective in heroin-preferring rats and whether the effects of naltrexone differ by sex.

Keywords:Choice, relapse, addiction to opioids

Revelation:Kaleidescapes LLC: Founder (Auto)

Indiana University, Indianapolis, Indianapolis, Indiana, United States

Bottom:Binge drinking is a central aspect of alcohol use disorder in which alcohol use persists despite adverse consequences and is a major clinical barrier to effective treatment. Recent work has shed light on the underlying circuits and neurochemical mechanisms in resistant drinking aversion, but it still remains largely elusive. We have previously described that the anterior insular cortex, a critical mediator of emotion, motivation and internalization, plays an important role in promoting aversion-resistant alcohol consumption. Indeed, optogenetic inhibition of the striatum significantly reduced compulsive alcohol drinking in rats, as observed when the aversive stimulus was bitter-tasting quinine or intermittent shock. However, the same inhibitions were not affected when drinking alcohol alone, suggesting that this insula-related circuit is critical especially in resistant alcohol use. In addition, heavy drinkers show similar insular circuit recruitment when responding to alcohol under threat of shock. Together, these studies show that the insula plays an important role in both rodents and humans in overcoming the conflict (compulsion) of drinking alcohol. Here we hypothesize that alcohol consumption combined with aversive provocation will increase firing levels in the anterior insula described by 32-lead multiwire electrophysiology implants.

Methods:Rats were first trained to consume 20% alcohol via a periodic drinking paradigm for 3 months. Quinine, a bitter additive, was administered at two concentrations (10 mg/L and 60 mg/L) to assess compulsive and aversion-resistant motivation to drink alcohol. Animals that exhibited binge drinking were implanted with multiwire recording probes, allowing insula neuronal activity to be recorded only during the consumption of alcohol and two levels of aversion-resistant alcohol. In parallel with leukometry, licking patterns were assessed.

Results:As with previous findings, the rats maintained longer periods of licking during sessions with alcohol alone, with shorter periods when consuming quinine plus alcohol. Island electrophysiology records revealed firing data from more than 1,200 neurons in 13 rats during quinine challenge with alcohol alone and moderate (10 mg/L) and high (60 mg/L) alcohol.

Conclusions:Current analyzes focus on comparing the activation characteristics of the anterior insula neuron depending on the drinking conditions, with the original hypothesis of a greater magnitude of insular activation for the level of challenge and the amount of alcohol consumed. Preliminary analysis suggests that insula firing is greater, especially at the onset of alcohol consumption when subjected to a negative challenge, supporting our behavioral findings (Darevsky et al. 2019, 2020) that quinine-resistant alcohol consumption is associated with earlier onset and higher consumption compared to alcohol alone. Supported by R01AA024109.

Keywords:Overeating, alcohol, front of the island

Revelation:Nothing to disclose.

University of Minnesota School of Medicine, Minneapolis, Minnesota, United States

Bottom:Maladaptive behaviors that lead to alcohol use disorder (AUD) relapse include binge eating, failure to regulate negative emotions and stress, and failure to stop drinking. According to Koob and Volkow's theoretical model of addiction, these maladaptive behaviors can be divided into the following domains of addiction: motivational meaning, negative affectivity, and executive functioning. Behind these dependency domains are specific neural networks. The fractional kurtosis anisotropy (KFA) is a metric derived from an extension of the more common diffusion tensor model. KFA is responsible for the dynamics of non-Gaussian diffusion and is closely related to the microstructure of white matter. The current study directly investigated whether white matter integrity, as measured by KFA in addiction networks, is related to treatment outcome in AUD.

Methods:Multi-envelope diffusion-weighted MRI data were collected from 49 AUD subjects (Age: M=42.4, SD=9.4, 19 females) in approximately 2 weeks of abstinence. White matter tracts were identified in each subject individually by probabilistic tractography. KFA values ​​were extracted from the following pathways: fornix, hamate fascicle, and anterior thalamus radiation. These pathways were chosen because they connect regions known to mediate areas of addiction, including motivation (vault), negative affectivity (uncinate bundle), and executive function (anterior thalamic radiation). Dichotomous (abstinence vs. relapse) and continuous (number of abstinence days during follow-up) measures were collected over 4- and 8-month follow-up periods.

Results:KFA within the immobilized fascia measured during early abstinence was shown to be significantly lower (i) in subjects who subsequently relapsed during the follow-up period compared to those who remained abstinent, (ii) intoxicated periods related to the number of days of abstinence and length of observation, and (iii) ) is related to a person's confidence in being able to resist the urge to get drunk while experiencing negative emotions (as measured by the Situational Confidence Questionnaire). Fornix KFA and anterior chamber radiation measured during early abstinence did not correlate with treatment outcome measures.

Conclusions:This is the first study to directly investigate whether KFA within the white matter pathways of theoretically defined addiction neural networks is related to treatment outcome in AUD. Importantly, significant associations were specific to the uncinate fasciculus, a conduit connecting regions known to mediate negative emotions in addiction. These findings highlight the promising targets of neuromodulatory interventions to prolong abstinence.

Keywords:Alcohol Use Disorder, Diffusion Weighted Imaging, Relapse and Treatment Outcome, Negative Affectivity

Revelation:Nothing to disclose.

Harvard Medical School, Boston, Massachusetts, Stany Zjednoczone

Bottom:Despite the widespread legalization of medical marijuana in the United States, the risks and benefits of obtaining a Medical Marijuana Card (MMC) for treating pain, anxiety, depression, and/or insomnia have not been thoroughly assessed. Healthcare professionals are increasingly called upon to advise patients interested in using cannabis to treat a variety of disorders and need evidence-based research on the risks and benefits of its use, including the risk of developing cannabis use disorder (CUD), basic behaviors associated with the use and side effect profiles of MM. Unlike drugs that undergo FDA review, patients and caregivers lack basic information about the safety and effectiveness of marijuana. Here we assess the risks and benefits of cannabis for adults who wish to use cannabis for pain, anxiety, depression or insomnia. Our hypothesis was that we would investigate both the risks and benefits of MMC. That is, we hypothesized that we would see a slight improvement in pain and insomnia in the MMC group, along with a worsening of CUD and depressive symptoms over 12 weeks.

Methods:We conducted a 12-week randomized clinical trial in the Boston metropolitan area with 269 adults aged 18 to 65 who were not occasional (non-daily) users or were not using marijuana at the start of the study, but wanted to use it. the patient has symptoms of pain, anxiety, depression and/or insomnia. Participants with a diagnosis of CUD, assessed using the DSM-5 CUD checklist, were excluded from the study at the start of the study. Participants were enrolled between August 2017 and July 2020 and were randomized in a 2:1 ratio to receive MMC without delay (MMC;N= 105) or wait 12 weeks to get MMC, Waitlist Control Condition (WLC;N= 81). Participants were divided into 3 main complaints. pain (N= 37 em MMC,N= 24 in WLC); insomnia (N= 22 em MMC,N= 20 in WLC); and anxiety/depression (N= 46 em MMC,N= 37 in WLC). This was a pragmatic trial where participants in the MMC group selected their cannabis products, doses and frequency of use, allowing them to test the safety and effectiveness of cannabis use through the current system of prescribing, regulating and distributing cannabis. Main outcomes include symptom scores for CUD and change in self-reported pain (NRS, scale, 0-10), anxiety (HADS, anxiety subscale, 0-21), depression (HADS, depression subscale, 0-21), and sleep quality ( AIS, scale 0-24) during the 12-week randomization period.

Results:269 ​​participants, mean age 37.4 years (SD = 14.5; range = 18–65) and 65.1% women, were enrolled and randomized, and 186 (69.1%) completed the 12-week randomization period . Controlling for baseline cannabis use, significantly more participants switched to heavy use (≤3 days per week) at week 12 in the MMC than in the WLC (48% in MMC vs. 8.8% in WLC,PI number<0.001). MMC had a significantly greater increase in CUD symptoms compared to WLC, controlling for baseline CUD symptoms (PI number<0.001). The incidence of CUD at 12 weeks was 10% in the MMC group versus 5.4% in the WLC group (OR=2.88; 95% CI, 1.17 - 7.07;PI number= 0.02). Specifically, 19.5% of the depressive/anxiety symptom group assigned to the MMC developed CUD compared to 6.1% in the WLC group. MMC group had greater improvement in AIS scores compared to WLC (mean difference=-2.9; 95% CI, -4.3--1.5;PI number<0.001). There was no significant difference between the groups in self-reported pain, depression or anxiety scores over the 12-week period.

Conclusions:In this randomized, pragmatic study, taking MMC was associated with a significant risk of developing CUD symptoms during a short 12-week intervention period. There was no significant benefit for symptoms of pain, depression or anxiety. There was a small but significant improvement in self-reported insomnia. The risks of MMC for the development of CUD, as well as the potential benefits of MMC for insomnia, warrant further research. Contrary to our hypothesis, this study does not support the use of MMC in the treatment of pain. These results also indicate that MMC is not indicated for the treatment of anxiety and depression.

Keywords:Marijuana, medical marijuana, depression, pain, insomnia

Revelation:Nothing to disclose.

Rowan University School of Osteopathic Medicine, Stratford, New Jersey, USA

Bottom:Cocaine use disorder remains a major public health concern in the United States, due in part to the increase in cocaine overdoses in recent years and the lack of FDA-approved drugs for treatment. A particularly challenging feature of cocaine addiction is the high risk of relapse, even after long-term abstinence. Among the factors known to trigger cocaine craving and relapse is exposure to stress, and many experts argue that ending perceived distress is the primary motivation for continued cocaine use. However, the exact neurobiological mechanisms by which stressor exposure triggers drug-seeking responses have not been fully resolved. Parietal gray (PAG) has been established to play an important role in threat response and execution of various stress coping strategies, with functionally distinct subdomains mediating the production of active/proactive and passive/reactive behaviors. A possible role for PAGs in mediating some aspects of alcohol consumption has recently been reported, but no study to date has investigated whether PAGs may be similarly involved in cocaine-seeking behavior. The overall objective of this study was to assess PAG neural activity during stress-induced cocaine-seeking episodes in rats. We also examined neuronal activity in other brain regions selected on the basis of providing afferent inputs to the PAG and/or playing a known role in drug seeking or behavioral stress responses. Finally, because different neural circuits are involved by social and nonsocial stressors, we investigated whether the observed patterns of neural activity differed when cocaine seeking was triggered by psychosocial and nonsocial stressors.

Methods:Adult male and female Long-Evans rats were trained to self-administer cocaine (0.5 mg/kg/inf, intravenously) during daily 2-hour behavioral sessions for 20 days. On days 11, 14, 17, and 20, a discreet tactile cue was presented in the operating room, and these self-administration sessions were immediately followed by exposure to psychosocial stress (social failure,N= 16), extrasocial stress (shock of leg,N= 12) or stress-free control condition (N= 12). From day 21, the animals underwent extinction training in which the bar press was not reinforced. After the response expired, the rats were again exposed to a tactile cue that signaled their imminent stress/no-stress state, and cocaine seeking was measured for 2 h under extinction conditions. Immediately after this restoration assay, the animals were sacrificed and the brains were harvested and processed for expression of c-Fos as a marker of neuronal activation.

Results:When all three treatment groups were combined into one experimental cohort, the amount of cocaine seeking was found to be positively correlated with neural activity in several brain regions, including the rostral periapical gray matter (rPAG). Interestingly, the activity in the different rPAG columns during cocaine seeking varied according to the stressor used. Specifically, dorsal rPAG (rPAGdm) activation was associated with paw-related cocaine-seeking behavior, while dorsal and lateral rPAG (rPAGdl/l) activation was associated with anxiety-related cocaine-seeking behavior. In addition, activity in these distinct rPAG subdomains was differentially correlated with activation in other brain regions during cocaine seeking, as shock-related activity in rPAGdm was positively correlated with neuronal activation in the bed nucleus of rPAG terminals. Stress in rPAGdl/l was positively associated with activation of neurons in the prefrontal cortex (plPFC). Finally, an ethmographic analysis of social failure episodes showed that plPFC activity at the time of the cocaine-seeking test was positively associated with the earlier occurrence of 'active defense' coping behaviors during social emergency.

Conclusions:Our findings reveal for the first time that neural activity in the rostral PAG, a brain region well characterized for its role in behavioral responses to stress, correlates with stress-induced cocaine-seeking behavior. In addition, our results suggest that psychosocial stressors can trigger cocaine-seeking through a neural network involving the dorsal and lateral rPAG and plPFC columns. Research is underway to investigate the functional role of rPAG subregions in cocaine-seeking behavior induced by social and non-social stress.

Keywords:Cocaine self-administration and recall, psychosocial stress, gray aqueduct (PAG), c-Fos, social failure

Revelation:Nothing to disclose.

University of California, Los Angeles, Los Angeles, California, United States

Bottom:Chronic pain affects a large proportion of the population, and effective pain management has proven challenging. Although therapeutic opioids are potent analgesics, their use is limited by a number of undesirable side effects, including a high risk of abuse. Currently, preclinical and clinical data present conflicting views on whether chronic pain can confer resistance or susceptibility to addiction. Our current study aims to investigate the effects of chronic pain on various aspects of the self-administration (SA) profile of remifentanil in mice.

Methods:Remifentanil, a potent, fast-acting, short-acting μ-agonist that is as effective as fentanyl in humans and mice, is an ideal opioid for intravenous AS in rodents. Using the chronic contracture injury (CCI) model, a small cuff was placed over the left sciatic nerve. Seven days after surgery, male and female mice (N=   ~ 5-10 mice/gender/group) started with remifentanil SA for 2 hours according to the FR1 schedule. After a stable response (∼5–7 days), mice were transferred to a higher demand operant schedule (FR3, ~4–6 days), followed by a progressive intercourse (PR) day. The mice were put back on the FR1 schedule for 3 days and then put on a reverse task where active and inactive attraction were reversed to control learning. A second group of mice repeated the same program but with the "Ensure" dietary supplement as a reward.

Results:We found no differences in the total number of remifentanil reinforcements obtained between CCI and naive mice during FR1 SA, although CCI mice gained reinforcements faster in each session. However, in higher demand operant schedules (FR3 and PR), CCI mice showed a significant decrease in gains in reinforcement and a decrease in total effort compared to naïve mice. This effect was not due to changes in learning as CCI mice performed similarly to naive mice on the inverse task. In contrast, CCI mice showed no difference in workload during Assure SA.

Conclusions:Chronic pain significantly altered the motivation of CCI mice to work in exchange for remifentanil, but only during periods of high surgical demand. This effect was specific to opioids and did not affect their willingness to work for the reward of drug-free food (insurance). While it is currently unclear what causes this cross-regimen SA selective change in CCI mice to remifentanil, it is likely due to a change in the balance between the analgesic properties and the hedonic value of remifentanil. Our ongoing research will aim to explore the potential impact of time and negative emotional state on this AS profile.

Keywords:Chronic pain, opioid abuse, remifentanil, intravenous self-administration, surgical behavior

Revelation:Nothing to disclose.

Duke University, Durham, North Carolina, United States

Bottom:Addictive behaviors result in part from drug-dependent changes in cellular function and nucleus accumbens (NAc) circuitry. NAc output is strongly regulated by local interneurons and we show that interneurons expressing fast-peak parvalbumin (PV + ) are required for amphetamine locomotor sensitization and conditioned place preference (CPP). Others have shown that repeated exposure to cocaine affects the excitability and synaptic signals of these cells. During development, synaptic inputs to PV+ neurons are modulated by perineural networks (PNNs), extracellular matrix structures surrounding the soma, and proximal dendrites. In addition, the PNN Brevican (Bcan) protein has been shown to be activity-regulated, and the short Bcan2 isoform has been shown to autonomously control synaptic inputs from PV+ cells in the hippocampus, presenting an attractive molecular target for drug-induced functional changes in NAc PV+ neurons. . However, it is not known whether Bcan or other PNN components contribute to addiction-related psychostimulant-induced changes in NAc PV+ neuronal synapses.

Methods:Heterozygous adult male and female Pvalb-2A-Cre mice were used in all studies. Gene expression was measured by fluorescence in situ hybridization (ACD) qRT-PCR or RNAscope. PNNs were labeled with FITC-conjugated WFA followed by anti-FITC primary antibodies and Alexa 488 secondary antibodies to amplify the signal. To manipulate Bcan expression in PV+ NAc cells, we used Cre-dependent (shLacZ), knock-down (shBcan) or overexpression (Bcan2-HA) AAV1 control viruses. For mEPSC electrophysiology recordings in untreated mice, these viruses were mixed with AAV1-FLEX-eGFP to increase the visibility of the labeling.

The viral constructs were validated using Neuro2A transfected cells and NAc-injected mice that were injected with the virus. For analysis of cocaine-regulated gene expression, mice were subjected to the 3-pair CPP paradigm (i.p. saline or cocaine, 15 mg/kg) and brains were harvested 3 h after the first drug dose or 24 h after the first cocaine dose. trial session. To assess the effect of Bcan manipulation on CPP cocaine, virus-infected mice were used in a modified CPP paradigm (i.p. saline or cocaine, 10 mg/kg) with short, interspersed testing sessions to measure preference development. Control saline mice were used to immunostain VGlut1/2+/PSD95+/PSD95+ synapses by contacting particles from infected PV+NAc cells.

Cell culture:N= 3 holes/group

RNAscope:N= 41-95 cells from 3-5 mice/group

PCP:N= 4-11 mice/group

Synapse Color:N= 44-62 cells from 4 mice/group

mEPSCs:N= 8-13 cells from 4-6 mice/group

Data collection was performed blinded to treatment or virus group and analyzed by rmANOVA, ANOVA, Kruskal-Wallis test, Student's t-testT-test or Mann-Whitney U test with post-hoc Dunn or Bonferroni test.

Results:Bcan expression in NAc cells is regulated by acute cocaine exposure. 3 hours after an acute dose of cocaine (15 mg/kg), expression of Pvalb, Bcan2 and Bcan1 is induced in Pvalb+ cells (mean transcripts/cell, saline vs. cocaine: Pvalb 173.6 vs. 271.7, Bcan2 259.6 vs. .31915, 31915.21.5;PI number< 0.05) and Bcan1 expression is upregulated in Pvalb-/Bcan1+ cells. 24 hours after the CPP test session, Pvalb expression remains high, while Bcan1/2 expression returns to control levels in saline. We are currently analyzing PNN staining over time after acute cocaine (15 mg/kg) to map Bcan transcriptional regulation to PNN structural remodeling.

Viruses for manipulating Bcan expression have been proven in vitro and in vivo. Viral reporters correlate with PV immunoreactivity in vivo. Transfection of shBcan into Neuro2A cells significantly reduced the expression of Bcan1 and Bcan2 compared to shLacZ, while transfection of Bcan2-HA resulted in a protein of sufficient molecular weight (~80kD) that was immunoreactive against antibodies against Bcan and HA. In vivo knockdown was confirmed by quantification of total Bcan in mCherry+ cells by RNAscope.

Bcan depletion in NAc PV+ cells accelerates the development of CPP cocaine. In the 10 mg/kg CPP cocaine paradigm, shBcan mice showed a significant preference for the drug-paired chamber after single pairing. shLacZ and Bcan2-HA mice required 3 pairs (eat(15,111) = 2,806,PI number< 0.05 for shBcan 1/2/3 vs acclimation test, shLacZ and Bcan2-HA 3 vs acclimatization test).

Bcan knockdown in adult mice reduces structural and functional excitatory synapses in NAc PV+ interneurons. shBcan cells had fewer VGlut1+ and VGlut2+/µm synapses than shLacZ cells (median shLacZ vs shBcan: VGlut1 0.149 vs 0.088, VGlut2 0.124 vs 0.071,PI number<0.001). Electrophysiology yielded similar results, with shBcan mice showing a marked decrease in mEPSC frequency (mean shLac = 12.72Hz, shBcan = 3.02Hz,PI number< 0,02).

Conclusions:Bcan expression in NAc PV+ cells increases after acute cocaine exposure and returns to control levels after the full CPP paradigm. Consistent with the idea that Bcan in NAc PV+ cells plays an important role in the early stages of cocaine memory, Bcan reduction in these cells before CPP accelerates preference development, leading to a significant preference for the drug-paired chamber after the first mating. Bcan knockdown in adult NAc PV+ cells also reduces structural and functional excitatory synaptic inputs, indicating that cell type-specific changes in Bcan expression in adulthood may modulate its inputs even after circuit maturation. These data are consistent with a model where Bcan stabilizes synaptic inputs to NAc PV+ cells, limiting the development of addictive behavior.

Keywords:Cocaine addiction, parvalbumin interneurons with fast GABAergic spike, perineural networks, synaptic plasticity

Revelation:Nothing to disclose.

University of Florida, UF Center for Addiction Research and Education, Gainesville, Floryda, Stany Zjednoczone

Bottom:There has been renewed interest in the potential benefits of treatment-based cognitive training in terms of long-term (i.e. post-treatment) outcomes. We recently completed an initial clinical trial in which treatment-seeking men and women who met the criteria for an alcohol use disorder (AUD) completed a treatment as usual (TAU) or a training protocol that induces neurobehavioral processes that rely heavily on integrity. prefrontal cortex (PFC) and their connections, i.e. executive functions. In addition to the benefits of training and the transfer of training to cognitive tests, we were interested in intermediate outcomes such as alcohol consumption after treatment. Participants were contacted 30 days after leaving treatment and provided information on a range of post-treatment variables, including craving and alcohol consumption in the previous month. We predicted that alcohol consumption would decrease significantly over the follow-up period from pre-treatment levels. We asked whether cognitive training was associated with different reductions in alcohol consumption relative to TAU. Additionally, given the role of hunger in relapse, we investigate the associations between drinking, hunger, and training status. Recent data suggest that even when abstinence is not maintained, significant reductions may bode well for long-term outcomes. Therefore, in a secondary analysis, we examined intake levels at baseline and post-treatment using World Health Organization (WHO) intake levels.

Methods:The methods have been approved by UF IRB-01 and registered at ClinicalTrials.gov (NCT031376554). Participants provided written informed consent prior to the study. Men and women seeking medical attention for a substance use disorder (SUD) who met the DSM 5 criteria for moderate to severe alcohol use disorder (AUD) and reported 21 to 90 days of sobriety were eligible for consideration. Those with significant medical or psychiatric impairments were ineligible. After screening, Ss were assigned to usual care (TAU) or cognitive training groups. Subjects in the training group were included in this analysis if they completed at least six (30-minute) sessions over a period of two to three weeks. The conclusion of the trial coincided closely with their acquittal. Both groups were interviewed for 30 days after discharge from the hospital. Using time-tracking methods, respondents provided detailed information on their drinking patterns to determine average alcohol consumption (quantity/frequency ratios) and other outcomes. Craving after treatment was assessed using the Mini Alcohol Craving Experience (MACE) questionnaire.

Results:The follow-up interviews were significantly disrupted by COVID-19. Data were collected for 40% (17/42, 4 women) of subjects who completed the study. Given the sample size and nature of the WHO-related data, we relied on non-parametric and descriptive statistics. Gender differences could not be analysed. As expected, alcohol consumption decreased for all respondents. Pre-treatment alcohol consumption averaged 15.2 (± 14.2) typical (standardized) drinks per day for all participants. Seven subjects (41% of the sample) reported alcohol consumption during the follow-up period with similar rates in the training and TAU groups. The days to the first sip ranged from 2 to 20. The mean number of days to the first sip was 3 for TAU, 11 for the TAU trained group.Ntoo small to be analyzed. Among drinkers of any type of alcohol (trainers), the mean number of drinks/day after treatment was 0.42 (+0.29), while the pre-treatment estimate was 10.72 for these drinkers (Wilcoxon pairwise test, V=24 ,PI number= 0.03). Across the sample, the average MACE score was 11.4 (+9.8: range 0-26/50)). MACE scores were not associated with pre-treatment alcohol consumption. Moreover, relapsers and abstainers did not differ in scores on any of the MACE subscales (Ts < 1.12). However, for retrievers, total MACE and aggression subscale scores were associated with the maximal pattern. drinks per day in a 30-day observation period (R= ,67(PI number= .10);R= ,86 (PI number= 0.01), respectively). The correlation between MACE intensity and the average number of drinks per day was noticeable but not significant (R= ,58,PI number= ,17).

At screening, 59% of the sample met the WHO criteria for very high risk (>7.1/drinks/day for men, >4.3 for women), 24% fell to high or moderate risk (7.1, 2.9 drinks/day for men; 4.3, 1.4 drinks for women, respectively), with 8% at low risk (1-<2.9, 1.4 for men, women, respectively). This distribution reflects the observed heterogeneity in consumption among those meeting the AUD criteria. This is also illustrated by the unusually high levels of alcohol consumption reported by those seeking treatment. During follow-up, all retrievers were in the low-risk group.

Conclusions:Although ingestion after treatment was common, intake levels did not exceed the WHO low-risk category. Considering the percentage of participants at high risk at screening, this result reflects a significant benefit. though smallNDespite interpretative limitations, the training-related difference in days to first drink is difficult and deserves further investigation. The equivalence of desire in conquerors vs. Abstinence, as opposed to the association between craving and peak intake, suggests the importance of developing multimodal neurobehavioral training of cognitive control and response selection in future protocols.

Keywords:Cognitive education, WHO risk levels, alcohol use disorder, substance use disorder, craving

Revelation:Nothing to disclose.

University of Texas at Austin, Austin, Texas, United States

Bottom:Alcohol use disorder (AUD) affected 8.6% of men and 1.7% of women worldwide and accounted for 5.3% of all deaths worldwide in 2016. We have previously shown that immune genes in brain glia are important moderators of drinking behavior. TLR7, a key player in neuroimmune activation, is differentially expressed in mice in accordance with the Chronic Intermittent Exposure (CIE) treatment paradigm, a model of alcohol dependence that induces escalating alcohol consumption in mice, and neurobiological and behavioral adaptations in mice that mimic those in those with AUD. Chronic treatment with R848, an activator of TLR7, increases alcohol consumption in mice. Our goal is to identify new molecular mechanisms involved in the escalation of alcohol consumption, which will provide the opportunity to develop therapies that prevent or reverse this escalation. In this work, we identify spatially separated overlapping mechanisms specific to prefrontal cortex cells between alcohol dependence and mouse models of immune activation.

Methods:We sequenced single-stranded RNA (snRNA-seq) in microarrays obtained from the prefrontal cortex (PFC) of CIE and control mice to determine cell type-specific alcohol-induced changes in gene expression. To understand the spatial context of the overlapping mechanisms between CIE and the immunoactivation model, we generated 10-fold Genomics Visium Spatial transcriptomic data from PFC coronal sections (10 μm) from C57BL/6J mice treated with a single injection of R848 (50 μg, i.p.). ) or regular saline. We use the "anchor" based integration workflow introduced in Seurat on snRNA-seq data obtained from PFC micro punctures (reference) and spatial RNA-seq data (query), this pipeline spatially creates for each capture point a prediction score for each of the originating cell types from snRNA-seq.

Results:The snRNA-seq analysis pipelines identified 24 clusters, the expression levels of the canonical marker of each cell type were used to determine the cellular identity of the clusters. We identified differentially expressed genes (DE) and their enriched pathways in each group. Astrocyte and microglial ED genes were enriched in inflammation-related pathways (IL-2, CXCR4, IL-17 and chemokine signaling). Integration of snRNA-seq and spatial transcriptomics data showed enhanced microglial prediction scores in layers 2/3 and astrocyte prediction scores in layers 5/6, indicating a cell type-specific response to R848 treatment. In addition, we identified 19 DE genes in R848 brain slices overlapping CIE astrocytes and microglial DE genes. Gene ontology analysis shows the relationship of these genes with stress/stimulus response.

Conclusions:The integration of snRNA-seq datasets and spatial transcriptomics identifies spatially separated transcriptional changes induced by immune activation in the PFC that mediate the escalation of alcohol consumption.

Keywords:Alcohol and substance use disorders, neuroimmune activation, bioinformatics, spatial transcription, monocyte resistance

Revelation:Nothing to disclose.

State University of New York at Buffalo, Buffalo, New York, United States

Bottom:Chronic pain is a debilitating condition. Heavy reliance on opioids such as oxycodone for pain management causes dependence and addiction due to their ability to promote long-term neuroadaptations in the mesolimbic dopamine system. Research exploring the neurobiology of pain and addiction is of great importance as the two disorders overlap in their diminished hedonic responses, compulsive drug cravings and stress. The neurobiological crosstalk by which opioids relieve pain and promote the addictive phenotype is poorly understood, and the cellular mediators that regulate these overlapping neuroadaptations are unknown.

Methods:RNA sequencing was performed on mPFC tissues obtained from mice administered oxycodone or saline (ip) in the presence or absence of CFA-induced inflammatory pain. Mechanical and emotional pain were assessed by VonFrey and PEAP (escape-avoidance paradigm), respectively, following virus overexpression or pharmacological inhibition of Egr3 in the mPFC. Motivation for self-administration of oxycodone was assessed using the progressive ratio test in the oxycodone self-administration model after viral Egr3 or GFP overexpression. Quantitative polymerase chain reaction and western blot were used to determine Egr3 levels and their altered targets under various treatment conditions.

Results:Pain in combination with oxycodone induces unique transcriptional signatures compared to pain or oxycodone alone in the mPFC. Among the various altered genes, we focused on Egr3, an immediately early gene, as its expression was upregulated in the oxycodone and no-pain groups, implicating it as a potential regulator that is exclusively dysregulated by opioid-induced neuroadaptations. We observed that in the presence of oxycodone, viral overexpression of Egr3 in the mPFC is enhanced, while pharmacological inhibition of Egr3 by cyclosporine A attenuates mechanical pain relief. Egr3 overexpression in the mPFC increases motivation to receive oxycodone infusion in a progressive ratio test without impairing acquisition or maintenance of oxycodone self-administration.

Conclusions:Taken together, this demonstrates a role for Egr3 as a mediator of neuroadaptation at the intersection of recreational and prescription opioid use, influencing pain and increased motivation.

Keywords:Opioid abuse, pain, immediate early gene, medial prefrontal cortex

Revelation:Nothing to disclose.

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Bottom:Sleep disturbances often persist long after stopping chronic drug and alcohol use, which can negatively affect withdrawal symptoms and accelerate relapse. However, it is not clear which sleep component(s) may be particularly important for this regulation and why. Here, we use a cocaine self-administration (SA) model in young adult male rats that recapitulates persistent sleep disturbances after long-term abstinence. We found a set of sleep/wake and EEG characteristics after prolonged withdrawal that correlate with future relapse-like behaviors, in particular rapid eye movement (REM) sleep characteristics in individual rats. We then tested potential causality by selectively targeting REM sleep, integrating behavioral, chemogenetic and optogenetic manipulations. They then analyzed the circuit mechanism linking REM sleep and the reward network.

Methods:Young adult male Sprague-Dawley rats were trained to self-administer cocaine using the overnight paradigm + 2 hours/day x 5 days (0.75 mg/kg/injection). EEG polysomnographic characteristics after 45 days of cocaine abstinence (WD) (N= 16) were associated with cocaine-induced behaviors including SA training in active nose picking# (ANP), average cocaine injection in the last 3 days#, WD d1 ANP, WD d45 ANP and rate of incubation. Having identified REMS-cocaine behavioral associations, we used three different approaches to selectively enhance REMS to test for potential causality: ambient heating, chemotherapy (using AAV5-MCHp-DREADD(Gi)) or optogenetic stimulation (using AAV5-MCHp-ChR2) of neurons melanin concentration hormone (MCH) promoting REM in the lateral hypothalamus. We measured MCH neural activity during REM sleep under ambient heating conditions using fiber optic photometry and differentiated its activity during long and short REM sleep. We also measured cue-induced cocaine seeking after these manipulations and performed correlation analyses. To investigate the circuitry mechanisms, we used slice electrophysiology to measure the synaptic accumulation of calcium-permeable AMPA receptors in major nucleus accumbens (NAc) neurons after all three manipulations. NAc CP-AMPARs are induced by long-term abstinence from cocaine self-administration, which is a key factor contributing to the gradual increase in cocaine-seeking cue-induced after long-term abstinence (i.e., incubation of cocaine craving). We then compared the behavioral and electrophysiological effects of REMS manipulation with intra-NAc injections of the MCH peptide (1 μg/μl, 1 μl per side).N= 8-20 mice for sleep recordings, behavioral tests and courtship recordings. Two-way or paired or unpaired ANOVAT-tests were used appropriately.

Results:We observed associations between rapid eye movement (REM) sleep characteristics and cue-induced cocaine seeking in individual rats after long-term withdrawal from cocaine self-administration. Selective extension of REM sleep with ambient warming (t17 = 5.824,PI number< 0.001) decreased cue-induced cocaine seeking after abstinence (ANP control, t14 = 3.261,PI number<0.01; hot ANP, t15=0.7947,PI number= 0.44; incubation rate, t29=2.162,PI number< 0.05), with the amount of long and short REM sleep being inversely related to cocaine-seeking behavior. Warming induced a selective increase in melanin-concentrating hormone (MCH) lateral hypothalamic neuron activity during long REM episodes (t5 = 4.406,PI number< 0.01). Mimicking this effect by chemogenetic or optogenetic stimulation of MCH neurons during sleep similarly reduced cue-induced cocaine seeking (Gq: saline ANP, t18=3.437,PI number< 0,01; CNO ANP, t13 = 0,422,PI number= 0.68; incubation rate: t29=1.914,PI number= 0,066. ChR2: control ANP, t26 = 5,249,PI number<0.001; ANP stimulation, t24=1.119,PI number= 0.27; incubation rate, t50 = 2.870,PI number< 0.01). All but optogenetic stimuli also antagonized the cocaine-induced accumulation of calcium-permeable AMPA receptors at NAc synapses (warming, t26 = 5.496,PI number< 0,001; Gq, t10 = 3854,PI number< 0.01). Finally, intra NAc infusions of MCH peptide reduced cocaine craving incubation (aCSF ANP, t15 = 3.626,PI number< 0,01; MCH ANP, t25 = 0,753,PI number= 0.46; Incubation rate t14 = 2.521,PI number< 0.05) and NAc CP-AMPAR reduction (cocaine x MCH interaction, F1, 29 = 7.116,PI number< 0,05; cocaine SA/MCH vs. cocaine SA/aCSF,PI number<0.01; Tukey's two-way post hoc ANOVA with no change in sleep/wake time, suggesting that MCH receptor signaling in the nucleus accumbens is sufficient to reduce signal-induced cocaine seeking and to antagonize cocaine-induced accumulation of permeable AMPA receptors in calcium

Conclusions:REM sleep can explore itself and produce relapse-preventing effects by engaging MCH neuronal activity and signaling in part through the NAc.

Keywords:REM sleep, cocaine addiction, melanin-concentrating hormone, relapse

Revelation:Nothing to disclose.

South Carolina Medical University, Charleston, South Carolina, United States

Bottom:The current opioid epidemic highlights the need for alternative pain management strategies, and cannabis is widely legalized for medical use, including pain management. Cannabis may serve as a partial therapeutic alternative to opioids, but it is unclear whether a chronic history of THC increases the risk of developing opioid use disorder (OUD). Preclinical studies using unconditioned THC injections have shown that a chronic history of THC increases heroin self-administration (SA). However, it has been argued that because unconditioned THC is anxiogenic and can induce aversive responses, the reinforcement of SA by heroin may be due to the chronic stress of unconditioned THC injections rather than cross-sensitization between THC and THC. . Various epidemiological studies show that while the legalization of medical marijuana reduces opioid overdose mortality, marijuana use also increases the risk of developing OUD.

The neurobiological basis of the increased response to heroin after prior exposure to THC is also unknown. Morphine sensitization has been shown to depend on MOR signaling in the pallidum (VP), however, the lack of a preclinical model of chronic dual self-administration of THC and heroin (SA) precludes a detailed study of morphine-induced neuroadaptations. reward pathway that can lead to cross-sensitization to opioids.

Here we present a rat model of dual self-administration of THC and heroin (SA) to test i) whether a history of THC self-administration modulates the various stages of heroin uptake, withdrawal and craving and ii) whether these effects of prior THC exposure are gender specific and iii) explore neuroadaptations in areas of the brain involved in OUD.

Methods:The four treatment groups underwent two 10-day THC or Vehicle SA followed by a 10-day blank followed by either heroin or sucrose SA. Different tips and active leverages were used in the first and second sessions of the SA. After the last day of SA, a progressive index test was performed, with the need for response increasing exponentially with each injection of heroin. The second SA session was followed by 10 days of extinction. 24 hours after the last extinction session, the rats underwent heroin or sucrose stimuli-induced reinstatement. In addition, we performed the elevated maze plus (EPM) test the next day and the heroin recall test the day after EPM. A subgroup of animals were taken from the initial heroin trial and sacrificed to harvest brain tissue for whole cell patch-clamp recordings.

Results:Our data show that the history of THC SA significantly increased heroin consumption in both men and women. Chronic exposure to THC appears to affect cue- and cue-induced reinstatement differently in males and females. We found a strong trend towards increased stimulus-induced reinstatement in men, but not in women. Overall, women show higher onset-induced recovery than men and tend to have increased recovery after chronic THC. Furthermore, we found that bar pressure in the progressive intercourse test is reduced in men with a history of cannabis use, while it is elevated in women.

Conclusions:Our results indicate that chronic THC modulates motivation and heroin craving in a sex-specific manner.

We are currently using whole-cell patch-clamp recordings in VP slices to study neuroadaptations in CB1R and MOR signaling after withdrawal of chronic THC and heroin exposure and will be compared to adaptations induced by chronic THC +  or heroin alone in male and female rats.

Keywords:Heroin self-administration, gender differences, marijuana use, opioid withdrawal, abdominal pallor

Revelation:Nothing to disclose.

University of Colorado, Boulder, Boulder, Colorado, United States

Bottom:Despite the widespread availability of state legal cannabis markets, research on the combined and differentiated effects of the concentrated forms of the two most important cannabinoids, THC and CBD, is lacking. This study examines blood cannabinoid levels and subjective mood and drug effects after using ad libitum over-the-counter concentrates.

Methods:In this cross-group cohort study, conducted in a community and university setting, users of cannabis concentrate were randomly assigned to a THC- or CBD-dominant concentrate. The THC-dominant concentrate contained 84.99% THC and <1% CBD. The main CBD concentrate contained 79% CBD and 4.5% THC. Participants completed a baseline session followed by an experimental mobile lab session consisting of 3 time points - before, immediately after, and one hour after using the ad libitum concentrate. Of the 81 people enrolled and assessed, 54 regular concentrated marijuana users followed the study's marijuana use guidelines and had full baseline outcome data. 28 participants (51.9%) were assigned to a THC-dominant state (mean [SD] age 29.86 [8.7] years; 42.9% female) and 26 were assigned to a CBD-dominant state (mean [SD] SD] 29.88 [10.5] years); 53.8% of women).

Results:As expected, the THC-dominant concentrate was associated with higher subjective intoxication effects as well as higher drug effects and reward scores than the CBD-dominant concentrate. When it comes to positive mood, both product formulations produced an immediate feeling of euphoria, which subsided over the next hour. Negative mood varied between products, with the CBD-dominant group reporting an immediate reduction in tension and anxiety, while the THC-dominant group saw a significant reduction in anxiety after just one hour. Paranoia increased immediately after use in the THC-dominant group, which dropped back to baseline within an hour.

Conclusions:The overall positive effect of the CBD-dominant concentrate on mood was accompanied by lower levels of intoxication and no side effects such as paranoia that were experienced with the THC-dominant concentrate. The results presented here confirm the need for further research into the harm-reducing potential of concentrated CBD, both alone and in combination with THC.

Keywords:Marijuana, high potency THC, harm reduction, cannabidiol

Revelation:Nothing to disclose.

Janssen R&D Department, San Diego, CA, USA

Bottom:Nicotinic acetylcholine receptors play an essential role in various cognitive and reward processes and are involved in many mental illnesses such as depression, schizophrenia and addiction. as well as neurodegenerative disorders such as Parkinson's disease. Despite the widespread importance of these receptors in a wide variety of disorders, drug discovery efforts to identify targeted therapies have been hampered because most neuronal nicotinic receptor subtypes are not functionally expressed in recombinant systems. We previously identified NACHO, a neuronal endoplasmic reticulum protein, as a key nicotinic receptor chaperone enabling recombinant functional expression. NACHO protein-deficient mice lack expression of alpha7 complex nicotinic receptors and show reduced expression of several other subtypes of neuronal nicotinic receptors throughout the brain. In addition, NACHO knockout (KO) mice exhibit abnormalities in motor and cognitive behaviors consistent with the absence or reduced expression of several subtypes of nicotinic receptors in the brain. The high prevalence of nicotine among patients with mental illness has been attributed to self-medication attempts by patients, as nicotine can have stimulant and anticipatory effects. Here, we investigate abnormalities in nicotine-induced behavioral and physiological responses in vivo and ex vivo in NACHO KO mice compared to wild-type (WT) mice to better understand how NACHO mediates nicotinic receptor function in vivo.

Methods:Nicotine-induced dopamine release was studied in vivo (0.6 mg/kg, subcutaneous) by nucleus accumbens microdialysis (WTN= 19; IN= 21) and ex vivo from striatal synaptosomes loaded with tritiated dopamine from WT (N= 10) e NACHO KO (N= 10) mice. Nicotine-induced changes in locomotor activity were measured in an infrared chamber by quantifying the X-Y of walking 10 minutes after administration of nicotine (0.5 mg/kg, subcutaneous) or vehicle (VEH) WT (VEHN= 15; nicotineN= 15) and NACHO KO mice (VEHN= 14, nicotineN= 15). Electroencephalographic (EEG) sleep and body temperature were assessed at baseline and in response to nicotine (0.15, 0.3, and 0.6 mg/kg, subcutaneous) in WT (N= 8) e NACHO KO (N= 8) mice with an implanted telemetry device.

Results:Consistent with NACHO's role as a key regulator of expression of functional neuronal nicotinic receptors, NACHO KO mice exhibited abnormalities in several nicotine-induced behavioral and physiological responses. NACHO KO mice showed reduced nicotine-induced dopamine release in vivo (20%) and ex vivo (23%) compared to WT mice (ANOVA,PI number< 0.05). Nicotine-induced immobility in WT mice, while NACHO KO mice showed hyperlocomotor activity in response to nicotine (ANOVA,PI number< 0.05). Under basal conditions, NACHO KO mice showed a specific reduction in REM sleep time (6.26 min) during the 12-h light/rest phase and increased sleep fragmentation (increased micro-arousal) throughout the light-dark sleep cycle compared to WT mice (Plus 10 in dark cycle, Plus 16 in light cycle, bi-directionalT-test,PI number< 0.05). The wake-promoting and sleep-suppressing NREM and REM effects of nicotine observed in WT mice were essentially absent in NACHO KO mice (ANOVA,PI number< 0.05). Nicotine induced dose-dependent hypothermia in WT mice, while this response was reduced in NACHO KO mice (ANOVA,PI number< 0,05).

Conclusions:NACHO KO mice exhibit a variety of abnormalities in nicotine-induced behavioral and physiological responses. These results are consistent with the role of NACHO in mediating the functional expression of nicotinic receptors throughout the brain. So far, NACHO has not been involved in the biogenesis of other neurotransmitter receptors, making NACHO a client-specific custodian for neuronal nicotinic receptors. As NACHO depletion mediates the behavioral effects of nicotine on nicotinic receptors, this provides strong evidence that NACHO is part of the endogenous cellular pathway leading to functional expression of the nicotinic receptor. Therefore, the use of NACHO as a tool to express previously unavailable nicotinic receptors in recombinant systems could enable high-throughput drug screening and discovery of target compounds for various neuropsychiatric and neurodegenerative conditions.

Keywords:Cognition, nicotine addiction, striatum, rodents, nicotinic acetylcholine receptors, mesolimbic reward circuitry, NACHO, alpha7 nicotinic acetylcholine receptor

Revelation:Johnson and Johnson: employee (self-employed)

South Carolina Medical University, Charleston, South Carolina, United States

Bottom:Alcohol use disorder (AUD) is a chronic, relapsing condition that is difficult to treat. One of the reasons for the prevalence of AUD is that excessive alcohol consumption disrupts the body's stress systems. This increased stress can lead to increased alcohol consumption, which can perpetuate this cycle and lead to alcohol dependence. Understanding the mechanisms underlying stress-alcohol interactions is critical to the development of more effective pharmacotherapies that can address stress-related alcohol dependence and/or binge drinking, and reduce the ability of stress to trigger relapse in abstinent AUD patients. One target that regulates stress-induced increased alcohol consumption is the epigenetic modifier, G9a, which is a histone methyltransferase that regulates several genes important in AUD and substance use disorders. G9a levels are reduced in the nucleus accumbens (NAc) after exposure to cocaine, morphine or alcohol. Previous studies show that mimicking this reduction in NAc G9a levels reduces the representation of stress-induced cocaine craving and reduces stress-induced increases in alcohol consumption. However, the role of G9a in addiction-induced alcohol consumption has not been fully characterized. We hypothesized that reducing G9a levels in the NAc or systemically reducing G9a activity with a pharmacological inhibitor would reduce alcohol consumption in a mouse model combining stress and chronic alcohol exposure.

Methods:All experimental protocols in these animal studies were approved by the MUSC Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

To test the role of the G9a NAc in alcohol-related behavior, adeno-associated virus (AAV) expressing G9a shRNA or scrambled control shRNA was injected bilaterally into the NAc of 8- to 10-week-old adult male mice. Three weeks later, basal ethanol intake (15% v/v) was established under restricted access conditions (1 h/day) for 5 weeks. The mice were then treated in the CIE-FSS alcohol consumption model, where brief and repeated forced swimming (FSS) stress increases alcohol consumption in mice with a history of chronic intermittent ethanol exposure (CIE). Mice were exposed to CIE vapors in inhalation chambers (16 hours per day x 4 days) and after a 72-hour abstinence period for 5 test drinking days (as per baseline). Weekly cycles of CIE exposure were interspersed with 5-day drinking test periods for 3 cycles. FSS (10 min) was performed in half of the G9a shRNA and control shRNA groups 4 hours prior to the alcohol intake testing sessions.

To test the effect of a pharmacological G9a inhibitor on increased alcohol consumption, CIE+FSS was separated into groups of mice and then divided into 4 groups in a 2x2 pattern (CIE vs AIR, FSS vs no stress). The AIR, CIE, AIR+FSS, and CIE+FSS groups alone received (i.p.) injections of vehicle or UNC0642 (4 mg/kg) for 15 days. Injections were initiated during beverage test period 4 and continued during CIE/air exposure cycle 5 and subsequent beverage test period 5.

Results:In mice injected with the control virus into the NAc, FSS+CIE significantly increased alcohol consumption compared to the CIE condition alone. In contrast, FSS did not increase alcohol consumption in CIE-exposed mice injected with the shG9a virus inside the NAc. We also found that repeated injections of the G9a enzyme inhibitor, UNC0642, significantly reduced CIE-enhanced alcohol consumption during the fifth week of the restricted drinking test. Indeed, mice exposed to CIE consumed ~3 g/kg of alcohol per day before UNC0642 treatment, but consumed only ~1.5 g/kg of alcohol per day after treatment. Animals treated with UNC0642 also showed a significant reduction in increasing alcohol consumption in the CIE+FSS group, but no effect on body weight.

Conclusions:Specific knockdown of G9a NAc reduced the FSS-induced increase in alcohol consumption but had no significant effect on CIE-induced alcohol intake. In contrast, chronic systemic G9a inhibitor administration reduced the increase in alcohol consumption by CIE alone and CIE+FSS. Indeed, we observed an approximately 50% reduction in alcohol consumption on the CIE scale, suggesting that G9a inhibition may reduce the high levels of alcohol consumption associated with addiction to more moderate levels experienced by non-addicted mice. Since NAc-specific manipulation of G9a did not reduce increased CIE intake, but systemic UNC0642 suppressed CIE intake, these findings suggest that G9a may act in multiple brain areas to regulate alcohol intake. Together with our published findings, these new data provide further evidence that G9a inhibition can reduce alcohol dependence and stress-related alcohol consumption, and support the exploration of G9a inhibitors as a new therapeutic agent for the treatment of AUD as well as comorbidity of stress-related disorders and AUD in humans.

Keywords:ehmt2, G9a, Alcohol use disorder, new therapies, alcohol dependence

Revelation:NeuroEpigenix, LLC, founder, self-employed

NIMH, NIH, Bethesda, Maryland, USA

Bottom:Amphetamine (AMPH) and methamphetamine (METH) are closely related psychostimulants that differ only in one methyl group. However, their behavioral effects, therapeutic utility, and addiction potential vary widely. It is believed that the mechanisms of action of psychostimulants rely primarily on the modulation of dopaminergic neurotransmission. One mechanism by which AMPH and METH regulate extracellular dopamine concentrations is their ability to induce internalization of the dopamine transporter, DAT. We also found that AMPH modulates glutamatergic signaling through endocytosis of the neuronal glutamate transporter, EAAT3, resulting in enhanced excitatory neurotransmission in dopaminergic neurons. The sequence of events leading to the internalization of DAT and EAAT3 begins when AMPH enters the cytoplasm of the cell via DAT. Inside the cell, AMPH activates the intracellular GPCR for trace amines, TAAR1, which signals through the small RhoA GTPase and activates DAT and EAAT3 internalization. Internalization of these transporters contributes to the increase of extracellular concentrations of dopamine and glutamate, as well as the increase of neurotransmission. However, it remains unresolved whether METH activates DAT and EAAT3 internalization through the same RhoA-dependent mechanism as AMPH.

Methods:Cultured dopamine (DA) neurons were derived from the midbrain of E15 Swiss-Webster mice. DAT activity was measured in these cultures by measuring the 3H-DA uptake to which it was sensitiveGBR12909, a specific DAT inhibitor. EAAT3 activity was determined by measuring the sodium-dependent uptake of 3H-glutamate, which was insensitive to the EAAT2 inhibitor dihydrocainate. Total internal reflection fluorescence microscopy (TIRF) was used to study the membrane localization of fluorophore-tagged transporters in HEK293 cells in response to AMPH and METH. Forster resonant energy transfer (FRET) sensors were used to measure the time course of RhoA activation. The dependence of various effects on TAAR1 was verified in parallel experiments on HEK293 cells lacking the TAAR1 receptor. Cell surface expression of DAT and EAAT3 in acute midbrain sections was determined by a cell surface biotinylation assay.

Results:In primary cultures, DAT activity was reduced in response to thirty minutes of AMPH (10 µM) or METH (10 µM) pretreatment.N= 6,PI number< 0.05). However, AMPH loss of DAT activity could be blocked by co-administration of the DAT blocker cocaine while the effects of METH were unaffected, indicating that METH's actions are independent of DAT. Using TIRF microscopy of DAT-expressing HEK293 cells, we observed internalization of mCherry-DAT and GFP-EAAT3 in response to AMPH and METH. However, in HEK293 cells transfected with GFP-tagged EAAT3 alone, only METH stimulated transporter internalization, while AMPH had no measurable effect (N= 7;PI number= 0.0001). METH had no effect on Rho recruitment or activation in HEK2093 cells lacking the TAAR1 receptor, indicating that TAAR1 is an obligatory target for METH actions, as we have previously shown for AMPH. METH-induced internalization of DAT and EAAT3 was blocked by the dynamin inhibitor, dynasore, and the RhoA inhibitor, C3 (N= 12, 9;PI number< 0.05 and 0.001). The internalization of DAT and EAAT3 by AMPH was sodium-dependent, while the action of METH was sodium-independent (N= 9;PI number< 0.0001). The transport of radiolabeled AMPH in cultured neurons was temperature dependent, while the experiments examining the transport of METH had no significant effect on temperature.N= 11,PI number< 0.01). AMPH or METH application resulted in RhoA activation, however, the profile of RhoA activation by METH was faster and biphasic (N= 13). With METH, initial activation of RhoA was observed even in DAT-deficient cells (N= 20). In acute midbrain sections exposed to AMPH or METH, we observed a decrease in cell surface expression of EAAT3. However, when examining cell surface expression in the DAT-deficient forebrain region, only METH induced EAAT3 internalization.

Conclusions:We found that the effect of METH on transporter transport is independent of DAT expression and, unlike AMPH, METH can activate RhoA signaling in non-DA neurons as well as in DA neurons. EAAT3 is found in many neurons and thus the action of METH to promote endocytosis of EAAT3 may enhance the action of glutamate more widely throughout the brain. These differences in the delivery mechanisms of the two drugs likely determine the different pharmacological profiles of the two drugs.

Keywords:DAT/NET inhibition, glutamate transporter (EAAT3), methamphetamine

Revelation:Nothing to disclose.

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Bottom:The acute and long-term effects of exposure to the main psychoactive component of cannabis, delta-9-tetrahydrocannabinol (THC), are of interest due to the drug's potential therapeutic uses and potential for abuse. Depending on the dose administered, exposure to THC can produce both harmful and beneficial behavioral effects in humans and animals. Performance on working memory tasks requires activation of the prefrontal cortex, and THC acts on cannabinoid receptors in this area of ​​the brain. We sought to investigate the acute effects of relatively low doses of THC on working memory task performance and related neural activity in the prefrontal cortex. We hypothesized that activation of cannabinoid receptors at these doses would affect the activity patterns of excitatory cortical neurons and the accuracy of task performance.

Methods:Adult male and female rats were trained in a delayed-sampling working memory task. The rats were first trained to fill in responses to nose poking into one of 5 illuminated sample ports to receive a sucrose reward. Mice were then trained to respond to a specific lit sample port, wait for a variable latency phase, and then correctly select the initially sampled port when choosing between the 3 lit ports. Delays between the sampling phase and the selection phase ranged from 4 to 20 seconds. Prior to training, mice were injected with the genetically encoded calcium marker AAV1.CamKII.GCaMP6f, and a lenticular probe was permanently implanted in the prefrontal cortex for single-photon calcium imaging in vivo. Rats were trained on the task until they achieved at least 80% correct attempts in the shortest time before starting the imaging session. During test sessions, rats were injected (i.p.) with 0.5 mg/kg THC, 0.75 mg/kg THC, 1.0 mg/kg rimonabant or vehicle 30 minutes prior to the test session; working memory test. The data was analyzed byT-test or one-way ANOVA (α = 0.05 for all analyses). All experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Results:The rats successfully learned to perform the sample-lag-matching task, making more correct guesses at shorter lags with less accuracy at longer lags. Injecting 0.75 mg/kg of THC 30 minutes before the testing session reduced the number of trials initiated during the session, but did not affect the accuracy of the task. Injecting 0.5mg/kg THC, 1.0mg/kg rimonabant or vehicle prior to the test sessions did not significantly reduce performance. Rate of calcium events sensed by individual cells (N= 2, 220-250 cells in total) were compared within each testing session and around behavioral events. Preliminary analysis of the activity of the population of all neurons recorded during the control sessions showed a significant increase in activity in the lag phase preceding the incorrect response in relation to the activity preceding the correct response. Rates of calcium-related events were also significantly increased immediately prior to the abnormal trial after vehicle injection, but this effect was no longer seen after injection of THC or rimonabant. Neither 0.5 mg/kg nor 0.75 mg/kg THC affected the rate of calcium-related events during the sessions compared to the control days, but there was a significant increase in the event rate, especially during training. delay before correct and incorrect answers. Rimonabant reduced the mean rate of calcium-related events measured during the session compared with vehicle, but did not affect activity during the lag phase.

Conclusions:We found that THC, at doses low enough to produce minimal behavioral effects, increased the activity of key neurons in the prefrontal cortex specific to task performance. Further study of this effect can examine the activity of individual neurons during and between sessions and in response to each pharmacological challenge. These results can be used to study the acute and long-term effects of different levels of THC exposure on cognition.

Keywords:Cannabinoid, THC, working memory

Revelation:Nothing to disclose.

Harvard Medical School McLean Hospital, Belmont, Massachusetts, Stany Zjednoczone

Bottom:Prescription opioid abuse is a nationwide epidemic, exacerbated by the stress and isolation of the Covid-19 pandemic. Unlike most drugs, where the prevalence of abuse is much higher among men, women abuse prescription opioids to a similar or greater extent than men. Therefore, biological sex is a risk factor for misuse and the development of opioid use disorder (OUD), but the neurobiological mechanisms are unknown. The high rate of relapse during abstinence (Abs) is one of the major barriers to OUD treatment. Cravings and relapses are often triggered by exposure to the drug itself or drug-related stimuli. Glutamatergic synaptic transmission in the nucleus accumbens (NAc) underlies cue-based reward-seeking behaviors, and recent evidence suggests that synaptic plasticity in projections from the ventral thalamic nucleus (PVT) to the NAc cortex (NAcSh) is required for expression of morphine deprivation (WD) ) in male mice. However, it is unknown whether opioid (SA) self-administration leads to similar plasticity of PVT-NAcSh and whether there is a gender difference in synaptic mechanisms in rats. Here we determined the effects of short and long intraventricular oxycodone (oxy) in male and female rats. We hypothesized that glutamatergic transmission from PVT to NAcSh would be increased in male and female rats after prolonged Abs oxy SA.

Methods:We used slice electrophysiology with optogenetics to determine the short (24 hours) and long (14 days) effects of oxySA Abs on PVT-NAcSh glutamatergic transmission in men and women. We also determined the effect of short abs on somatic AV and long abs on stimulus reinstatement (a surrogate for drug seeking). Prior to implantation of chronic intravenous jugular catheters for self-administration, rats received a unilateral injection of AAV5-CamKIIα-ChR2-EYFP into the PVT. After recovery from catheterization, rats underwent an 8-day short-access (ShA) training regimen of oxySA (0.06 mg/kg/injection; 1 h/day) followed by a 14-day long-access (LgA) regimen (0 .06 mg/kg/injection, 6-h/day). Control rats self-administered saline (saline). All self-administration took place during the light phase in operator chambers equipped with two response levers (active and inactive), with a light signal above each lever and a traffic light. A constant-ratio 1 amplification schedule was used such that active lever pressing resulted in a 4-second oxygen infusion followed by a 6-second rest period. After the final 14d LgA SA session, rats were subjected to 24-hour or 14-day Abs periods (total animals: malesN= 14 sal,N= 14 oxy; womenN= 15 sal,N= 13 oxy). For the short Abs group, somatic symptoms of WD were recorded after 24 hours, and then the mice were sacrificed to prepare brain sections. The extended Abs group underwent reinstatement (2 hours, no pumps) on Abs day 14 and were sacrificed for brain slice preparation. Vaginal lavage was performed for all females on the day of sacrifice. Standard procedures were used to prepare and save sections. Briefly, we optogenetically stimulated PVT projections to NAcSh and recorded from medium spiny neurons (MSNs) after 24 hours or 14 days of Abs. Whole-cell patch-clamp recordings were made for synaptic plasticity with MSNs using an EPSC-10 amplifier and Pulse v8.8 software (Heka Elektronik).

Results:We compared oxy SA between men and women and found no significant differences between the sexes in the number of drug infusions per session. For the short squat group (men:N= 5 sal,N= 10 oxy; women:N= 7 sal,N= 7 oxy), rats that self-administered oxy showed physical signs of WD compared to controls (Two-way ANOVA: main drug effecteat(1,25) = 17,28,PI number= 0.003), with no gender differences. In addition, there was no significant effect of gender or drug on NAcSh or PVT excitability for NAcSh glutamatergic synaptic plasticity after a short oxy SA Abs. This included measurements of input-output curves for synaptic strength, PPR for glutamate release probability, AMPA/NMDA ratios, input resistance and EPSC rectification index. Preliminary data from the Extended Abs group show that women on oxygen have significantly greater reengagement compared to men (Two Way RM ANOVA of Active Lever Presses: main effect of gendereat(1,13) = 9,18,PI number= 0.01; women:N= 5 sal,N= 3 oxy; macho:N= 4 sal,N= 5 oxy). In addition, self-oxygenated rats show an increase in synaptic strength after prolonged bouts compared to control females (Two-way RM ANOVA: main drug effecteat(1,164) = 6,05,PI number= 0.01), while men showed no differences. Preliminary data also shows that women have increased synaptic strength compared to men after extended oxy Abs (Two Way RM ANOVA: Sidak posthoc analysisPI number= 0,001).

Conclusions:Our preliminary findings suggest that the effect of LgA oxy SA Abs on PVT-NAcSh synaptic transmission is time and sex dependent. Collectively, these data suggest that extended Ab LgA oxy SA increases PVT-NAcSh synaptic plasticity, but only in female mice. In ongoing studies, we plan to determine whether NAcSh GABAergic transmission is also affected by prolonged oxyAbs and the role of the oestrous cycle in women. We are also interested in examining the effects of 24-hour and 14-day abdominal muscle training on MSN excitability in the NAcSh of men and women. To our knowledge, this is the first and only study to investigate sex differences in the PVT-NAcSh pathway and the effect of different times of opioid withdrawal, craving incubation and ovarian hormones on this.

Keywords:Gender differences, thalamic paraventricular nucleus, nucleus accumbens, opioid use disorder, synaptic plasticity

Revelation:Nothing to disclose.

Emory University, Yerkes National Primate Center, Atlanta, Georgia, United States

Bottom:Many addictive drugs, including cocaine, increase circulating corticosterone (CORT). In addition, addictive drugs and CORT impair the ability of organisms to select actions in a goal-directed manner (causing reliance on habitual behavior), and loss of dendritic spine density on excitatory neurons in the ventromedial orbitofrontal cortex (VLO) is common. We hypothesize that cocaine induces decision errors by upregulating circulating stress hormone, activation of low-affinity glucocorticoid receptors (GR), leading to loss of dendritic spines in the VLO.

Methods:Here, we use selective and pharmacological gene silencing strategies to downregulate Nr3c1, the GR gene, in male and female mice. To quantify action-selection strategies, mice were trained to make two separate responses to food and then asked to update their response strategies when the behavior was no longer being reinforced. The use of Thy1-driven YFP enabled the imaging of VLO excitatory neurons.eatThe -actin cycle has been manipulated in vivo to reveal effects on action selection strategies.

Results:Cocaine increased circulating CORT, and exogenous CORT exposure was sufficient to disrupt goal-directed action strategies. Cocaine had the same effects, while inhibition of CORT synthesis blocked cocaine-induced response biases. Preliminary evidence indicates that knocking down Nr3c1 in the VLO also protects against cocaine-induced response bias accompanied by dendritic spine modifications in deep-layer excitatory neurons. Finally, induction of actin polymerization in the VLO enhanced the mice's ability to update action selection strategies. future experiments will use the same strategy to try to restore the plasticity of responses after taking cocaine.

Conclusions:Our results suggest that cocaine-induced decision errors are driven by repeated activation of the GR in the VLO. Future experiments will determine whether cocaine-induced CORT release interferes with reward decision making by destabilizing dendritic spines on excitatory neurons in the VLO.

Keywords:Cocaine, stress hormones, decision making, orbitofrontal cortex (OFC), dendritic spines

Revelation:Nothing to disclose.

University of Iowa, Iowa City, Iowa, United States

Bottom:Nicotine addiction is the largest preventable cause of disease and death worldwide, and current treatments are only moderately effective. Nicotine acts on nicotinic acetylcholine receptors (nAChRs) in the brain. In particular, the nAChRs in the ventral tegmental area (VTA) have been shown to be essential for the potentiating properties of the drug. While the nAChR study provides an understanding of the direct effects of nicotine, it does not provide a complete understanding of all the mechanisms involved in nicotine addiction. Therefore, there is an urgent need to expand our understanding of the effects of nicotine on the brain by evaluating endogenous proteins that may also mediate these processes. The recently discovered negative allosteric modulator of the endogenous nAChR, Lynx1, represents an interesting new target for modulating nicotine addiction because, unlike agonists and antagonists, allosteric modulators do not directly affect receptor function. In contrast, allosteric modulators mediate receptor activity in the presence of an agonist. Thus, Lynx1 is a key allosteric regulator to consider for nicotine potentiation as it has been shown to reduce nicotine-induced nAChR activity.

Methods:To determine whether constitutive knockout of Lynx1 alters the expression of related proteins, we first examined the baseline expression of the nAChR subunit using RT-qPCR. Lynx1 knockout mice and their wild-type littermates were then tested on self-feeding to determine if there were differences in their ability to press the reward-related lever. Following self-feeding, the mice were implanted with a jugular vein catheter and tested for low, moderate and high doses of intravenous nicotine self-administration. We next assessed Lynx1 expression in the VTA as it is a brain region necessary for nicotine potentiation. Using stereological quantification methods to examine cell activation patterns using the c-fos marker, we also found that male and female Lynx1 knockout mice had increased cell activation patterns in the VTA in response to a low dose of nicotine. Finally, we used viral vector microinjection to disrupt Lynx1 transcripts in the VTA to study the effect of Lynx1 ablation on nicotine self-administration. In all studies, male and female mice were used in an independent measurement design,N= 5-13. For food and nicotine self-administration studies, we used a between-subject design and analyzed the data using a Geisser-Greenhouse-corrected two-way repeated-measures analysis of variance (ANOVA). We used Sidak's multiple comparisons test for our multiple comparisons, with individual variances calculated for each comparison. For the c-fos and RT-qPCR studies, we used a between-subject design and analyzed the data using unpaired two-tailedT-oxen.

Results:Interestingly, we found that Lynx1 global knockout led to increased nicotine self-administration at low doses, with male mice showing this increase over a wider range of doses compared to female mice. Lynx1 knockout mice in the VTA exhibited similar nicotine intake behaviors, demonstrating that ablation of Lynx1 in the VTA is sufficient to increase nicotine self-administration at lower doses. Consistent with these findings, we also found that male and female Lynx1 knockout mice had increased cell activation patterns in the VTA when given a low dose of nicotine. By examining the expression of the basal subunit in the VTA, we found that male Lynx1 knockout mice, but not female mice, had increased expression of the α4 subunit of the nAChR, indicating a possible mechanism for sex-specific effects. The criterion of significance α < 0.05 was adopted.

Conclusions:Together, these findings indicate that Lynx1, both globally and specifically in the VTA, plays an important role in nicotine potentiation and cellular activation patterns. Although the net effect of Lynx1 on nicotine self-administration was similar in both sexes, we found gender-specific effects. Importantly, to our knowledge, we are the first to show that site-specific endogenous allosteric modulation can have a significant effect on nicotine-reinforcement behaviors.

Research supported by NIH NIDA R00 DA032543 (CF), TRDRP High Impact Pilot Award T30PI0931 (CF) and NIH NIDA F31DA050436(SIM).

Keywords:Nicotine addiction, negative allosteric regulator, drug self-administration, self-administration, nicotine

Revelation:Nothing to disclose.

Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, Florida, USA

Bottom:Repeated methamphetamine use induces long-term changes in gene expression in areas of the brain associated with reward processing and drug-seeking behaviors, and recent evidence suggests that methamphetamine-induced neuritis may also be involved in behavioral and molecular responses to the drug. Microglia, brain-resident immune cells, are the main drivers of neuroinflammatory responses, but the role of these cells in regulating methamphetamine potentiation, especially relapse, is poorly understood. Therefore, our aim was to test whether pharmacological ablation of microglia would have an effect on methamphetamine-induced reinstatement in a mouse model of intravenous methamphetamine self-administration (IVSA).

Methods:IVSA methamphetamine was first established by training male C57BL/6 mice to respond to food rewards using a reinforcement schedule where 5 presses of the active lever resulted in the delivery of the food reward and the presentation of a 20-second cue light. Light lever presses were recorded during the presentation of the clues, but they did not contribute to the award. Animals were trained in daily 1-hour sessions until meeting the criteria (>30 food rewards per session) for IVSA methamphetamine. The mice self-administered intravenous methamphetamine infusions for 5 consecutive days at a dose of 0.01 mg/kg/injection, then the dose was increased to 0.05 mg/kg/injection for a further 10 days. After completion of the IVSA, the mice were allowed to extinguish the methamphetamine search for 21 consecutive days, during which time lever presses did not result in drug delivery or presentation of the paired beacon light. After expiration, methamphetamine probe reinstatement was performed by presenting the cue of the paired drug and recording the active bar press during the 1-hour session. An increase in active bar pressing during a recall session relative to extinction response rates was interpreted as reinstatement in search of methamphetamine.

Pharmacological ablation of microglia was achieved by administering the CSF-1 inhibitor, PLX5622 (1200 ppm;N= 7) for food during extinction training, while control mice (N= 6) received standard food during this period. The effect of microglial ablation on the reinstatement of methamphetamine seeking was analyzed after completion of the methamphetamine reinstatement by comparing the percentage change in active rod pressure (extinction versus reinstatement) between PLX5622-treated and control mice.

Results:Pharmacological ablation of microglia in mice by PLX5622 (N= 7) attenuated methamphetamine recovery compared to control-fed mice (N= 6) (PI number= 0.0101; oddT-test). Interestingly, each mouse in the PLX5622-treated group (N= 7) showed a decrease in lever pressing during the recovery session compared to the baseline of animal extinction (PI number= 0.0055; oddT-test). We also did not observe a significant effect of PLX5622 on operational food training, nor on the number of awards won, nor on the rate of achievement in a separate cohort of animals (N= 8 per group).

Conclusions:The abolition of methamphetamine reuptake after microglial ablation suggests that microglia may play an important role in modulating the motivational aspects of methamphetamine seeking. Furthermore, although preliminary and limited to men, these results suggest that neuroinflammatory mechanisms may dictate vulnerability to methamphetamine relapse and thus provide new therapeutic avenues for the treatment of addictive disorders.

Keywords:Microglia, methamphetamine, intravenous drug self-administration, stimulus restoration, neuritis

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:Neurophysiological responses to drug stimuli remain poorly understood in heroin use disorder (HUD). The use of naturalistic dynamic stimuli (e.g., movies) has several advantages over traditional fMRI tasks, including improved ecological validity, participant engagement, and assessment of functional connectivity between brain regions. In addition, measures of inter-individual correlation (ISC) and functional connectivity (ISFC) of responses to naturalistic stimuli successfully predicted behavioral measures such as learning and memory. However, neural responses to heroin-related naturalistic stimuli have not been studied in HUD. Here, we use subject-to-subject correlation (ISC) and functional connectivity (ISFC) methods to characterize brain responses to HUDs in a video featuring naturalistic heroin use and HUD narrative.

Methods:We recorded BOLD activity in 30 treatment-seeking HUD participants (40.51 ± 9.06 years, 23 M) and 16 age, gender, and race-matched healthy controls (HC; 43.79 ± 10.33 years, 10 million) , while watching the first minute of Trainspotting, which includes scenes of overt heroin use, as well as scenes particularly important but not directly related to heroin use. Importantly, the overall narrative structure of the clip focuses on drug addiction, including complex HUD-related elements (e.g., the social, emotional, and financial challenges of addiction). For our analyses, the video clip was broken down into distinct scenes, which were then classified as drug or non-drug based on the presence of drugs or conversations about drugs. After watching the film, participants freely recalled the details of the film with their personal reflections and rated the scenes based on their emotional response and heroin craving. To assess neurophysiological responses, we calculated the ISC and ISFC of the whole-brain BOLD signal during filming and compared the HUD and HC groups for drug and non-drug scenes.

Results:Consistent with the response inhibition and performance specialization (iRISA) model of drug addiction in which over-motivation is attributed to the drug rather than non-drug cues/reinforcers, we hypothesize increased SSI during drug-related scenes in the HUD group in reward, meaning, habit networks, executive, self-directing and memory. We also expected SSI in rewards/recognitions, habits, and self-guided networks during drug-related scenes to be related to the desire for those scenes. Similarly, we expect ISCs resolved across scenes in salience and memory networks to correlate with scene recall. Since video content should preferentially elicit self-referential thinking in the HUD group, we expect subjects to show decreased SSI in core sensory areas but increased ISFC in the default mode network and hippocampus time-locked to drug-ambiguous scenes. This increased ISFC should predict the duration of the self-referential statement when subjects freely recall the movie. Consistent with previous studies using wrapped Hollywood films, the initial full-length film brain voxel analysis for the HUD group revealed significant ISCs in ~50% of the gray matter voxels, including the large occipital, temporal, and parietal lobes, as well as the medial and lateral frontal cortex, orbitofrontal cortex, hippocampus, amygdala, and striatum (FDR adjusted, q<0.001).

Conclusions:Insights from naturalistic exposure to drug cues may better characterize the neural mechanisms of reactivity to drug cues in heroin addiction.

Keywords:Heroin, addiction, cross-sectional correlation, naturalistic drug cues, fMRI

Revelation:Nothing to disclose.

University of Michigan, Ann Arbor, Michigan, United States

Bottom:The signal tracking/target tracking model provides a means to separate the prognostic and motivational associations created by Pavlovian cues. "Signal Tracker" (ST) rats reliably approach and interact with a reward cue (i.e. an upcoming food reward (i.e. a cup of food). For the ST, but not the GT, the offer becomes a reward in itself. As might be expected, ST shows greater susceptibility to reinstatement or "relapse" to drug abuse compared to GT, making it a valuable model for understanding predisposition to addiction-like behaviors. appears to be essential for the development of cue-following behavior and incentive efficiency.However, little is known about the sources of afferent glutamatergic neurons that modulate NAc activity and impart individual variation to associative learning.The NAc is densely innervated by the ventral hippocampus (vHPC), and earlier work in our lab has shown that vHPC changes impair the acquisition of tracking signals. However, it is unclear whether this effect is exerted by direct NAc modulation. We tested the hypothesis that vHPC-NAc projection has a functional role in determining the ST/GT phenotype.

Methods:We used an in vivo dual-vector approach with bilateral injection of Cre recombinase into the NAc and an inhibitory Cre-dependent designer drug-only activated Gi-linked receptor (DREADD) into the vHPC to selectively target the vHPC-NAc projection. These viral injections were performed by stereotactic surgery in male Sprague Dawley rats (7-8 weeks old). Five weeks after surgery, all rats underwent six daily sessions of the Pavlovian Conditioning Approach (PCA) procedure with clozapine N-oxide (CNO, 3 mg/kg, i.p.) or vehicle (6% DMSO, i.p.) on the board, followed by a treatment test session cross. Each session consisted of 25 presentations of a retractable lever (CS) extending into the chamber for 10 seconds and 25 deliveries of sucrose-free banana granules (US) into a food cup. Animals were classified based on whether their conditioned responses were directed preferentially to the index lever (ST) or to the food cup (GT).

Results:We found no significant differences in bar pressure and number, latency, or probability of entering a food cup between NOC (N= 24) and the vehicle (N= 22) of treated rats in all six training sessions, suggesting that vHPC-NAc projection does not affect the acquisition of cue-following and goal-following behaviors. Based on these six training sessions, all rats were classified as either ST or GT based on their average behavioral error during sessions 5–6. We found that after vHPC-NAc inhibition during a cross-treatment test session (vehicle administered instead of CNO), mice classified as ST (N= 4) showed a significant decrease in the number of barbell presses compared to the last training session (parT-test:T(3) = 14,72,PI number<0.001). On the other hand, rats are classified as GT (N= 7) showed no changes in lever or food cup oriented behaviors after vHPC-NAc inactivation. In addition, inhibition of vHPC-NAc projection appears to have an effect on signal monitoring and target expression when the behavior has been learned under normal conditions such as ST (N= 4) e GT (N= 7) who received NOC for the first time in a crossover treatment session showed no significant changes in lever and cup oriented behaviors.

Conclusions:Our data suggest that vHPC-NAc projection may not be necessary for the acquisition of cue-seeking and goal-seeking behaviors. However, it may have a more complex and selective role in the execution of signal-following behavior in ST, as chronic inhibition of vHPC-NAc affected expression/performance of signal-following. More experiments need to be conducted to further explore this role and shed light on the neural circuits responsible for influencing ST/GT behavior. Ultimately, this will lead to a better understanding of increased susceptibility to signal-induced psychopathologies such as addiction and PTSD.

Keywords:Pavlovian conditioning, ventral hippocampus, oblique nucleus, signal sensing, chemogenetics

Revelation:Nothing to disclose.

Virginia Commonwealth University, Richmond, Virginia, United States

Bottom:Psychedelic serotonergic agonists such as lysergic acid diethylamide (LSD) and psilocybin are increasingly being studied for their potential therapeutic effects in the treatment of various neuropsychiatric disorders, including substance use disorder (SUD). Opioid use disorder affects more than two million people in the United States, and more than 120,000 deaths worldwide are attributed to opioids. SUDs generally have a recurrence rate of 40-60%, with current drug therapies failing and producing their own set of undesirable side effects. Psychedelics alter perception and cognition by activating the serotonin 2A receptor (5-HT2AR), but have not been found to lead to addiction or fatal overdose and are considered non-reinforcing. Serotonin has a regulatory effect on the mesenchymal pathway that is implicated in the neurobiology of addiction. Clinical findings have demonstrated the ability of psilocybin to reduce alcohol consumption in heavy drinkers and increase smoking cessation in adults. Our previous findings suggested that psychedelics produce a long-lasting and potent effect on synaptic plasticity in the frontal cortex of mice. Aspects of SUD can be modeled in preclinical rodent models such as conditioned place preference (CPP). This pilot study was designed to evaluate the ability of the psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI) to reduce oxycodone-induced CPP in adult male mice.

Methods:The use of animals was approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6 adult male mice (N= 19–21/group) were tested in the six-day CPP paradigm. The first day included a familiarization session where basic behavior was recorded. Days 2-4 were conditioning sessions where mice received twice daily (AM/PM) (sc) injections of oxycodone (3 mg/kg) or 0.09% saline, followed by a pre-treatment period. - treatment for 15 minutes before being placed on the paired side of the drug chamber for 20 minutes. On day 5, mice were untreated and placed in the neutral chamber for 5 minutes, followed by 15 minutes of recorded exploration to assess the time spent on the drug-paired side (CPP oxycodone). After this session, mice received (i.p.) DOI (2 mg/kg) or saline (0.9%) and returned to their cages. On the last day, 24 hours after DOI injection, the mice were retested for 15 minutes to assess the CPP of oxycodone. The time spent on the drug-saline side of the test days compared to baseline scores was used to calculate the overall preference score.

Results:As expected, all oxycodone-treated mice showed a preference for the drug-paired side over the vehicle.T-test:PI number< 0.0001). It is important to note that when using a two-sided paired StudentT- a test to assess changes in individual behavior, our data showed that mice treated with DOI after oxycodone conditioning had a decrease in CPP (PI number= 0.038), while saline-treated mice did not (PI number= 0,208).

Conclusions:Our results support the notion that psychedelics can alter SUD-related behaviors in preclinical rodent models. To our knowledge, this study is the first to determine the extent to which a psychedelic can alter opioid-seeking behavior in preclinical models. The DOI's ability to reduce CPP 24 hours after administration suggests that psychedelics may have long-term therapeutic effects associated with addiction. These findings have translational significance as they suggest that psychedelics may be a potential treatment for LDS once abuse patterns have begun. Following this pilot study, future experiments will be conducted to determine whether this effect is observed in female mice with other psychedelics and whether this behavioral change is mediated by 5-HT2AR. These experiments will be critical to understanding how psychedelics modulate SUD-related behaviors, which in turn leads to better addiction treatment.

Keywords:Psychedelics, substance use disorders, opioid abuse, conditioned place preference, behavioral pharmacology

Revelation:Nothing to disclose.

South Carolina Medical University, Charleston, South Carolina, United States

Bottom:The current opioid crisis has created a need for further research to elucidate the maladaptive effects of neuroadaptation after long-term opioid use. Activation of the nucleus accumbens tetrameric synapse (NAcore), consisting of pre- and post-synapses, astrocytic processes, and the surrounding extracellular matrix (ECM), is associated with increased relapse susceptibility. This process is mediated by the downregulation of the glutamate transporter (GLT-1) after repeated exposure to opioids, which promotes the diffusion of glutamate into extrasynaptic compartments. Several studies focus on reversing glutamate diffusion after chronic opioid use, restoring GLT-1 function and/or expression with N-acetylcysteine ​​(NAC), thereby reducing heroin signal and primary restoration. In addition, the search for opioids is associated with rapid and transient plasticity in each compartment of the tetrameric synapse. In particular, matrix metalloproteinase (MMP) activity can be localized to specific D1 and D2 medium spiny neurons (MSNs), where D1 MSNs show transient and reversible MMP-9 activity after cue-induced heroin search, while D2 MSNs show increased constitutive MMP-2 gelatinolytic activity after heroin withdrawal, which is transiently reduced after 15 minutes of signaled recovery. While it is clear that opioid relapse induces MMP activity, it remains to be seen whether NAC is effective in preventing changes in MMP activity and whether this is cell type specific.

Methods:Male Sprague Dawley rats were trained to self-administer heroin, after which the animals were withdrawn during extinction training and then reintroduced with heroin-dependent cues. Brain slices of the nucleus accumbens were obtained after blanking training or 15 minutes after heroin exploration began and compared with saline-trained control rat slices. Following heroin self-administration, we measured MMP-2.9 gelatinolytic fluorescence after FITC gelatin microinjection under heroin-seeking conditions associated with vehicle-induced extinction or chronic high-dose systemic administration of NAC (5 consecutive days during extinction, 100 mg/kg, p.i.). To further evaluate the effect of NAC on MMP activity in heroin-naïve conditions, systemic administration of NAC was administered at various time points and under various pharmacological conditions to identify potential mechanisms in the ECM that contribute to the observed effects of NAC. The results were analyzed by one-way and two-way ANOVA followed by post hoc Bonferroni tests for multiple comparisons.

Results:Chronic high-dose NAC treatment (5 consecutive days, 100 mg/kg, i.p.) did not reduce heroin-seeking MMP-2/9 activity in the NA nucleus of reintroduced animals, despite significant suppression of active stress compared to NAC-naive animals. Interestingly, chronic NAC treatment increased gelatinase activity in heroin-naïve animals, which peaked 7 days after treatment but persisted until 10 days after treatment and returned to baseline after 15 days. These effects were not seen after acute NAC treatment. Interestingly, this sustained but reversible increase was induced by MMP-2, consistent with the constitutive functionality of MMP-2 activity. This increase was not mediated by a nitric oxide-dependent pathway, suggesting an alternative, indirect pathway that activates MMP-2 activity. Further studies are needed to determine whether these changes are specific to D1 or D2 MSNs in naïve animals and whether heroin exposure disrupts the homeostatic balance of MMP activity between these cell types.

Conclusions:These data reveal a potential therapeutic agent that can suppress heroin-seeking behavior through a unique mechanism involving ECM activation. While much remains to be done to fully characterize how the four synaptic compartments interact to regulate plasticity, it is clear that each of the four compartments is altered by opioid use, and therapeutic agents may play specific roles in each dwelling.

Keywords:Matrix Metalloproteinase-9 (MMP-9), Heroin, N-acetylcysteine, Extracellular Matrix

Revelation:Nothing to disclose.

Vanderbilt University, Nashville, Tennessee, Stany Zjednoczone

Bottom:Substance use disorder is a neuropsychiatric disorder that is typically characterized by persistent cycles of drug use, withdrawal, drug seeking, and relapse. The neural basis of these long-term drug-related behaviors has been linked to neural circuit function through changes in neurotransmission and receptor-based changes in brain reward circuitry. However, the underlying molecular mechanisms that contribute to the persistence of these drug-induced changes in neural function remain relatively poorly understood. Previous work from our lab and others identified cocaine-induced changes in transcriptomic and proteomic profiles in the nucleus accumbens (NAc). To understand how drugs such as cocaine cause permanent behavioral changes, it is necessary to correlate neuronal activity and changes in gene expression. One possible pathway is through epigenetic adaptations where DNA-protein interactions are modified to alter the availability and likelihood of targeted gene expression. For example, histone acetylation is induced after various cocaine treatments, particularly in a subclass of D1-expressing medium spiny neurons (MSNs) in the NAc. KAT2A is a histone acetyltransferase known to regulate activity-dependent transcription and memory in the hippocampus. Although several KAT2A histone targets are modulated by cocaine, the role of KAT2A in cocaine-related behavior remains unknown. We therefore hypothesize that cocaine-induced gene networks underlie the recruitment of various histone modifications, a subset of which is regulated by KAT2A.

Methods:Mice were trained to self-administer cocaine (1 mg/kg/injection) or saline according to reinforcement regimens VR3 and VR5. NAc tissue was collected after acute (1 day) or chronic (10 days) self-administration of cocaine. Using histone-specific DDA mass spectrometry, we identified post-translational histone modifications induced by: 1) acute cocaine, 2) repeated cocaine, or 3) persistent 24 h after self-administration. In addition, using targeted expression of loss-of-function KAT2A variants in NAc D1-MSNs, we determined whether KAT2A function is necessary and sufficient for cocaine-related behaviors and changes in D1-MSN activity during self-administration. Finally, using calcium-dependent fiber photometry, we recorded D1-MSN activity during the acquisition and maintenance of repeated cocaine self-administration.

Results:We identified specific temporal changes in histone H3 post-translational modifications, including KAT2A-regulated phosphoacetylation, after chronic self-administration of cocaine. Furthermore, we showed that loss of KAT2A function in D1-MSN alters cocaine sensitivity and motivation. Finally, we establish the D1-MSN cocaine self-administration activity profile, which is further modified by altered KAT2A function.

Conclusions:The NAc has been linked to drug-related rewards and behaviors. Several epigenetic changes in the NAc have also been associated with an altered NAc response to abused drugs. The results of these studies provide evidence for persistent cocaine-induced epigenetic adaptations and are the first step towards establishing a mechanistic link between epigenetic adaptations and changes in neuronal firing. In addition, we provide data linking these changes in epigenetic state to cocaine-seeking behavior. Future studies will identify the causal link between changes in epigenetic gene regulation and NAc circuit function during cocaine-seeking behavior.

Keywords:Epigenetics, cocaine self-administration, nucleus accumbens

Revelation:Nothing to disclose.

Virginia Commonwealth University, West Haven, Connecticut, Stany Zjednoczone

Bottom:Zonisamide is an anticonvulsant drug with a multi-pronged pharmacological mechanism that has shown potential efficacy in reducing alcohol consumption and treating alcohol use disorder (AUD). Zonisamide is a widely available generic drug approved by the FDA as an anticonvulsant. We evaluated the effectiveness of zonisamide in a sample of people with AUD who reported regular heavy drinking.

Methods:We conducted a 16-week, three-centre, double-blind, placebo-controlled, randomized clinical trial (two in Connecticut and one in Virginia) in which 159 patients with AUD were randomized 1:1 to receive either zonisamide or placebo. Zonisamide was flexibly titrated over 7 weeks to a daily dose of 500 mg. Medical management was used as a behavioral platform. Alcohol consumption was measured using the Timeline Follow-back method. The analysis was carried out using mixed models in SAS.

Results:There was a significant drug effect on the prespecified primary outcome (average number of drinks per day) in the sample in both the last 8 prespecified weeks of the study [eat(1,680) = 6,20,PI number= 0.013] and all 16 weeks of study participation [eat(3,1655) = 4,47,PI number= 0.035]. Patients on placebo reported an average of 0.72 [95% CI = (0.54-0.93)] more drinks per day than patients on zonisamide. Significant effects of gender and drug x gender interaction effects were also observed [eat(1,680) = 5,87,PI number= 0.016], so that men drank 43% fewer drinks per week when receiving zonisamide compared to placebo, while there was no difference between women. There was also a significant primary drug effect on binge drinking days per week [eat(1,822) = 4,62,PI number=> There was also a significant drug x time interaction on binge days per week [eat(3,1929) = 3,73,PI number= 0.011], with an effect slope greater in the zonisamide group than in the placebo group. Finally, there was a trend for a gendered interaction in binge drinking, with men experiencing a greater reduction in the risk of binge drinking when taking zonisamide compared to placebo than women (males: OR=0.26, CI 95%=0.07-0.94 women: OR=0.74, CI 95%=0.16-3.58).

Conclusions:Zonisamide is an effective treatment for AUD and appears to be more effective in men than women, although this and other potential predictors of response to treatment with zonisamide require further study.

Keywords:Alcohol use disorders - treatment, pharmacotherapy, clinical trials

Revelation:Nothing to disclose.

Massachusetts General Hospital, Boston, Massachusetts, Stany Zjednoczone

Bottom:Cannabis' main psychoactive cannabinoid, Δ9-tetrahydrocannabinol (THC), induces intoxication that impairs cognition mainly by affecting prefrontal cortex (PFC) function. There are currently no established methods for detecting cannabis intoxication or deficiency. We investigated whether functional near-infrared spectroscopy (fNIRS) could be used to detect changes in resting-state PCF connectivity (RSC) associated with damage from cannabis intoxication.

Methods:Study participants were healthy adults who reported using cannabis at least once a week. Participants completed two study visits where they received either a single dose of dronabinol, an FDA-approved synthetic THC ingredient in MARINOL® capsules, 5-80 mg, or an identical placebo in random order. THC was dosed as determined by the study physicians at the dose most likely to cause intoxication without side effects, based on the expected degree of tolerability, taking into account the participant's average dose, the frequency and type of cannabis use, and the level of intoxication and any adverse effects experienced with each use, gender, height, weight, BMI and baseline blood pressure. Before dosing and every 20-25 minutes after dosing, participants measured their heart rate and blood pressure and completed the Drug Effects Questionnaire (DEQ), a 100mm visual analog scale, to assess how well participants (1) experienced any THC effect(s). and (2) seemed high. Participants underwent two fNIRS sessions. one pre-dose and again approximately 100 minutes post-dose, representing the average peak of dronabinol pharmacokinetic effects. During each session, 6 minutes of resting state functional data were collected using a continuous wave NIRS device in which 8 sources and 7 detectors were placed on the front, resulting in 20 channels spanning the PFC regions. The fNIRS analysis was performed using the CONN toolkit (https://web.conn-toolbox.org). Impairment was defined by convergent classification with clinical consensus scores and an algorithm based on post-dose tachycardia and self-rated DEQ as "high" and comparison with the results of extensive field sobriety tests conducted by police drug recognition examiners. was certified approximately 120 minutes after the study drug.

Results:181 participants were randomized and 169 participants who had a post-dose fNIRS scan during the study visit where they received THC were included in this analysis. Of these participants, 164 completed the placebo visit. Participants who had a placebo visit but not a THC visit were excluded from this analysis. After the active study drug (THC), 80 participants had consistent clinical and algorithmic scores indicating impairment/intoxication from the study drug (mean THC dose: 35.6 mg ± 11.5). 57 of these participants had adequate scores without worsening (mean THC dose: 34.8 ± 16.1). Participants rated as impaired from THC showed greater subjective (DEQ) and physiological (heart rate) signs of impairment than those who were not impaired from THC. RSC was significantly reduced from pre-THC to post-THC in participants after THC damage (PI number<0.001; Fixed with FDR). This reduction was observed in multiple channels throughout the PFC. Those not injured by THC showed no significant changes in CSR. Those with reduced THC disorder had significantly lower PFC CSR after THC than after placebo (PI number<0.01; revised FDR). Those without THC disorders showed no significant difference in CSR between THC and placebo scans, even after receiving equivalent doses of THC.

Conclusions:These findings suggest that the PFC RSC response may indicate cannabis attenuation independent of THC dose. Future work is needed to determine the specificity of acute THC damage and determine whether this method can objectively detect impairment at the individual level.

Keywords:THC, cannabis, fNIRS, neuroimaging, resting state connectivity

Revelation:WO 2018/027151: Patent (own)

University of Minnesota, Minneapolis, Minnesota, United States

Bottom:Social changes and tensions caused by the COVID-19 pandemic have led to a deterioration in mental health and poorer health behaviors (e.g. poor sleep, unhealthy diet, restriction of movement, increased alcohol consumption). This has been well documented in several studies that used cross-sectional surveys of subjects. However, traditional scientific research has limitations in that it is not ecologically sound, and group-level analyzes can obscure individual-level relationships. These two problems can be addressed with intensive longitudinal data (ILD), which includes real-time information collection through active collections, such as surveys, and passive collections, such as wearable devices. Typical data collection takes place 3-4 times a day for a period of 30 days. While traditional ELD analyzes are able to identify correlations between different measures, they are unable to determine the underlying causal structure between measures. Identifying this causal structure at the individual level is critical to developing tailor-made interventions. Causal structure can now be revealed through causal discovery methods that analyze the statistical properties of observational data to construct a reliable causal structure. In this study, we apply causal discovery methods to ILDs collected in the COVID-19 study to investigate causal factors contributing to drinking behavior at an individual level.

Methods:We used data from a publicly available dataset identified by Boston College collected during the COVID-19 pandemic, collected from March 20 to June 23, 2020 (Cunnigham et al., SciData, 2021). Daily surveys were conducted using REDCap with a variety of questions about mood, activities such as exercise, social interaction, alcohol consumption, and sleep. We selected 9 variables of interest for this analysis: 1) total sleep time (TST), 2) time spent in bed (TIB), 3) sleep efficiency (SE), 4) depression (PHQ-9), 5) PANAS- PositiveAffect, 6) PANAS-NegativeAffect, 7) Anxiety, 8) Stress, 9) Alcohol use (alcohol_bev). Available data from 1,518 people was filtered for useful data and at least 27 daily entries. This left a total of 86 entities for analysis. For each subject, a series was created for each daily entry consisting of 9 variables for that day. Variables from the previous day (lagging variables) are also added to each row. The Greedy Fast Causal Inference (GFCI) algorithm from the py-causal python package was applied to each subject's data and the generated edges were examined. Individuals who had causal variables associated with alcohol use were selected and the causes investigated.

Results:Of the 86 subjects, 15 had one or more causal variables associated with alcohol use. Causes included anxiety (6), sleep (6), negative emotions (3) and depression (2).

Conclusions:Using causal discovery methods, we were able to identify variables with causal associations with drinking behavior in 15 individuals. Variables identified included anxiety, sleep, negative impact, and depression. These variables were consistent with those reported in the literature as contributing to drinking behavior. Because these variables have been identified as causal at the individual level, they provide intervention goals tailored to the individual. Future work will extend the analysis beyond the immediate boundaries to consider the overall chart structure for each individual.

Keywords:EMA, sleep inconsistency, alcohol consumption, stress management

Revelation:Nothing to disclose.

University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States

Bottom:Quality of life (QoL) is an important measure of how health conditions and treatments affect patients' lives and includes mental health, physical health, and social well-being. Cannabis is increasingly being used as a therapeutic agent for chronic and acute conditions where traditional treatments may not be effective or well tolerated. Previous studies evaluating the effects of medical marijuana on health-related quality of life have been inconclusive. Therefore, the purpose of this study was to understand the conditions under which patients are certified to use medical marijuana, the perceived effectiveness of marijuana in relieving symptoms, and the quality of life of those certified to use medical marijuana.

Methods:A total of 210 patients certified for Pennsylvania's state medical marijuana program were recruited to participate in the 12-month study. The surveys were completed by telephone at 4 time points (baseline, 30 days, 6 months, 12 months) and included demographic information, eligibility conditions, current medications, and treatment symptoms. The data presented here is from a baseline assessment collected between May and October 2020. For each symptom, participants were asked to rate the severity of "when not using medical marijuana" and "while using medical marijuana" on a scale of 1 (mild) to 3 (severe) . Quality of life was assessed using the FACIT-PAL questionnaire, which consists of the original FACT-G questionnaire and the palliative care subscale. The FACT-G contains 27 items divided into four domains: physical well-being (PWB), social/family well-being (SWB), emotional well-being (EWB) and functional well-being (FWB). The Palliative Care subscale contains 19 items for people with life-limiting conditions. The subscale scores were added to form the FACT-G Total and FACIT-PAL Total score (FACT-G Total plus the palliative care subscale). Higher scores indicated a better quality of life.

Results:Overall, the sample mostly self-identified as White (70%), Female (54.3%), and Non-Hispanic/Hispanic (85.7%), and most participants had used marijuana before completing this survey (78.1% ). The most common medical conditions confirmed for medical marijuana were pain (48.6%), anxiety (36.7%), and post-traumatic stress disorder (PTSD, 15.7%). About 23% of the sample (N= 48) reported using benzodiazepines, opioids and/or other anxiolytics. The most commonly reported symptoms were anxiety and pain, followed by sleep disorders and depression (65.2%, 56.7%, 38.6% and 31.4%, respectively). Unsurprisingly, anxiety was more common among those who attested to using medical marijuana to treat anxiety and post-traumatic stress disorder, but nearly 50% of those certified in pain also reported anxiety. Although pain was less common among those certified for anxiety and PTSD, 30% and 21.1% supported pain, respectively. Compared to normative data, the current sample reported lower FACT-G Total T-scores, EWB and SWB (i.e. T-Scores < 45). Compared to those with a pain certificate, those with an anxiety certificate reported lower scores on the SWB and Palliative Care subscales (ps 0.015 and 0.02, respectively). Similarly, those who reported using opioids/benzodiazepines reported lower quality of life on all subscales (except SWB and FWB) compared to those who did not (p < 0.01). There were also significant negative correlations between each FACIT-PAL subscale and the total number of symptoms and the total number of self-reported conditions (ps<0.01).

Conclusions:Current data provides much-needed information on the characteristics and symptom profiles of patients enrolled in the state's medical marijuana program. Pennsylvania is one of the few states where stress is a requirement for medical marijuana certification. Despite mixed evidence regarding the effectiveness of cannabis in treating anxiety, more than a third of the current sample self-identified as using medical cannabis to treat anxiety. Although patients reported a reduction in anxiety when using cannabis, those who certified anxiety also reported a lower quality of life compared to those who certified pain, particularly SWB. Finally, lower quality of life was associated with more self-reported comorbidities, higher total symptom scores, and opioid and/or benzodiazepine use, suggesting that there is a range of severity to your overall well-being in this medical marijuana-certified population. . Follow-up surveys will provide critical data on the trajectory of these symptoms related to cannabis use and quality of life.

Keywords:Cannabis, quality of life, anxiety, pain, medical marijuana

Revelation:NovoNordisk, Inc: Grant (car)

Arizona State University, Tempe, Arizona, United States

Bottom:Circular RNAs (circRNAs), a class of non-coding RNAs generated by reverse splicing of pre-mRNAs, play a key role in brain development and synapse formation. Although the function of most circRNAs is still unclear, these molecules are known to act as sponges for the sequestration of microRNAs (miRNAs) and RNA-binding proteins (RBPs). We previously found that circRNA levels derived from various synaptic proteins, such as circHomer1, were significantly downregulated in the nucleus accumbens (NAc) of cocaine-conditioned male mice. circHomer1 is found in synapses where it regulates HOMER1B mRNA levels, homeostatic plasticity, and learning and memory. This circRNA also binds to the neuronal HuD RNA-binding protein, a protein related to synaptic function and drug addiction.

Methods:To investigate the relationship between circHomer1 and cocaine motivation, men (N= 13) and solitary (N= 13) Sprague Dawley rats were trained to use cocaine lever (0.75 mg/kg/injection, IV) and light/tone cues in a 5-stage alternating schedule or received saline related to cocaine-primed rat responses. Then, after either 1 or 21 days of abstinence, the rats underwent a 1-hour stimulus reactivity test during which pressures produced stimulus presentations, but no cocaine was available. Mice were sacrificed immediately after the test and circHomer1 levels were measured in synaptosomal fractions of the NAc shell using RT-qPCR. As a follow-up to the study of competition between circRNAs and mRNAs for binding to miRNAs, we developed a new bioinformatics pipeline to predict endogenous mRNA-miRNA-circRNA (ceRNA) competing endogenous RNAs in the striatum of HuD knockout mice.

Results:Lever pressing during the stimulus reactivity test was increased from 1 to 21 days of abstinence to support the incubation effect. In contrast, preliminary data on circHomer1 expression revealed a strong downward trend from days 1 to 21 of abstinence in male cocaine-administered rats, while female rats showed low levels of circHomer1 after days 1 and 21 of abstinence, suggesting that the expression of this circRNA is inversely related to cocaine motivation in male rats only. HuD knocks out mice (N= 3) showed altered cerRNA networks regulating genes related to neuronal development and synaptic plasticity compared to wild-type mice (N= 3).

Conclusions:These findings suggest novel post-transcriptional mechanisms in the control of drug-seeking behavior.

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Keywords:Non-coding RNA, cocaine-seeking behavior, Homer1, circRNA

Revelation:Nothing to disclose.

Tufts University, Medford, Massachusetts, USA

Bottom:Evaluating the neural mechanisms of fight motivation remains a challenge for preclinical models, particularly in inbred mouse strains. It has been hypothesized that the influence of alcohol on the decision to engage in sociosexual aggressive behavior is based on medial prefrontal cortex-ventral tegmental area (PFC-VTA) connections that are different from those required to actually perform these tasks. - and anti-social behavior.

Methods:Adult male B6 mice showed sexual competence while bearing offspring. When confronted multiple times with an invading male in their cage, they exhibited chasing, threatening, and aggressive behavior in less than 5 minutes. The mice were then conditioned to respond to a lighted instrument in an experimental chamber attached to their home cage. the conclusion from the 10 responses (Constant Ratio 10, FR10) was reinforced by access to a female or male intruder presented in the resident's cage. Brains were focused at the moment of choosing between simultaneously available aggressive and prosocial options. C-fos immunostained in 10 brain regions of interest were analyzed by fluorescence microscopy. The effect of ethanol self-administration on aggressive behavior selection and c-fos expression in the mPFC-VTA pathway was investigated.

Results:Six out of 50 mice consistently selected the FR 10 response option reinforced by aggressive behavior towards the male intruder, while they did not respond to the concomitantly available option reinforced by sociosexual contact with the female. Self-administration of alcohol (1-1.5 g/kg) increased the aggressive choice response in most mice. By choosing the aggressive but not the sociosexual option, the prefrontal region of the PFC revealed greater c-fos activity.

Conclusions:The new methods and results guide future research into neural networks to decide between pro-social and anti-social behavior. Gene expression data show that neural clusters in the prefrontal cortex are associated with alcohol-sensitive aggressive choices.

Keywords:Alcohol, prefrontal cortex, aggression, immediate early gene, decision making

Revelation:Nothing to disclose.

University of Texas Medical Department, Galveston, Texas, United States

Bottom:The United States has tripled the annual number of cocaine overdose deaths in just the last five years. In the absence of an overdose death, continued cocaine abuse can develop into cocaine use disorder (CUD). This is perpetuated by the lack of FDA-approved drug treatment options for CUD. Those wishing to remain abstinent from cocaine are challenged by the environmental context and cues associated with prior drug use (e.g. paraphernalia). The medial prefrontal cortex (mPFC) is neuroanatomically positioned to provide top-down control of the striatal reward circuits and to modulate the incentive and incentive properties of cocaine-related cues. However, chronic cocaine exposure induces extensive neuroadaptations within the mPFC, resulting in executive function maladaptation and increased susceptibility to relapse. The present study was designed to identify transcriptional differences in mPFCs associated with increased cocaine-seeking behavior in order to identify novel gene targets for future CUD drug development.

Methods:Male Sprague-Dawley rats trained to consistently self-administer cocaine (0.75 mg/kg/injection) were evaluated for cocaine seeking within 10 days of abstinence. Rats were phenotyped as high-signal (HC) or low-signal (LC) responders based on the median split of bar presses to previously associated cocaine cues. After behavioral phenotyping, we isolated the mPFCs and processed the mRNA for transcriptional profiling (N= 5/phenotype). Interpretation of the transcriptome data was done using statistical gene pool enrichment methods (e.g., Cytoscape ClueGO), where differentially expressed genes are compared to gene pools associated with a specific function or biological pathway.

Results:Comparative analyzes revealed the expression of 309 transcripts in the mPFC of HC mice that were significantly higher or lower than in LC mice. Functional gene enrichment analyzes identified 15 behaviorally relevant biological processes that were overrepresented in mPFC HC vs. LC (e.g. kinetic exploration). Heatmap analysis of expression change values ​​visualized samples with similar transcript profiles that broadly grouped HC or LC mice. Finally, we used ingenuity pathway analysis to generate a pathway of putative regulatory interactions of selected genes.

Conclusions:The results of this study identify unique differences in the mPFC transcriptional landscape between high and low responders and provide priority candidates (e.g., HTR2C, PENK) for future pharmacotherapy aimed at maintaining cocaine abstinence and preventing CUD relapse.

Keywords:Cocaine, transcription, signaling reactivity

Revelation:Nothing to disclose.

University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States

Bottom:Self-administration of cocaine in rats leads to structural and functional deficits in astrocytes in the nucleus accumbens (NAc), a key region of brain reward circuitry. Specifically, cocaine impairs the ability of NAc astrocytes to maintain glutamate homeostasis, a balance between extrasynaptic and synaptic glutamate levels. This disorder is due in part to decreased expression of the astroglial glutamate transporter GLT-1, which is responsible for 90% of synaptic glutamate uptake. Glutamate uptake incurs a high energy cost, which is covered by the mitochondria. Mitochondria colocalize with GLT-1 in astrocytes, and inhibition of mitochondria in cultured astrocytes reduces glutamate uptake. In addition, the degradation of astrocytic processes has been attributed to decreased mitochondrial function in cultured astrocytes. Given the cocaine-induced impairment of glutamate homeostasis and altered astrocyte morphology, and the role that mitochondria may play in both processes, we hypothesize that astrocyte mitochondrial function may be impaired following self-administration of cocaine.

Methods:To test this hypothesis, confocal imaging of core NAc astrocytes was performed using two AAV5: the mitochondria-targeted enhanced green fluorescent protein as well as the membrane-targeted tomato fluorescent protein Td, both under the control of an astrocyte-specific promoter in male rats, 24 h after 12 days self-administration of short-access cocaine (2 hours per day). The morphological properties of the mitochondria were assessed using the MitochondriaAnalyzer ImageJ plug-in. Oxidative stress was assessed by immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG) co-located with astrocyte mitochondria.

Results:The results indicate that after self-administration of cocaine, the mitochondria in NAc astrocytes show significantly increased volume, surface area and diameter compared to those in saline-treated animals (PI number< 0.05 por Nested ANOVA;N= 10-12 rats per group. 2-6 cells per mouse). This probably indicates oxidative stress. However, further analysis is required. Interestingly, this effect was not observed in a separate cohort of rats trained for 12 days of self-administration and 16-17 days of extinction, suggesting that this effect is related to early abstinence as reported for dendritic mitochondria (Chandra et al..., Neuron 2017 ).

Conclusions:Structural analysis of mitochondria in astrocytes shows swelling at the onset of deprivation but not after the extinction training period. While there is some degree of transient swelling under normal circumstances, excessive swelling may reflect oxidative stress and impaired mitochondrial function. Experiments are underway to test the effect of gender on this observation, as well as the expression of mitochondrial stress-related genes and markers of oxidative stress in astrocytes.

Keywords:Astrocytes, mitochondria, cocaine, nucleus accumbens

Revelation:Nothing to disclose.

Yale University School of Medicine, New Haven, Connecticut, Stany Zjednoczone

Bottom:Neurobiological drugs for the treatment of alcohol use disorder (AUD) target AUD-associated phenotypes such as craving, anxiety, and withdrawal. Treatment of AUD by altering the pharmacokinetics (PK) of alcohol has been less studied. Genetic variation in the enzymes that control alcohol metabolism is associated with a reduced risk of AUD. Aldehyde dehydrogenase (ALDH) is the enzyme responsible for the conversion of acetaldehyde to acetate and has isozymes expressed mainly in the cytoplasm (ALDH1) and mitochondria (ALDH2). Disulfiram, the first drug approved for the treatment of AUD, non-selectively inhibits ALDH1 and ALDH2. This mechanism alters the pharmacokinetics of alcohol, but also causes side effects attributed to the peripheral accumulation of acetaldehyde due to ALDH1 inhibition. Motivated by preclinical studies showing that selective inhibition of ALDH2 can prevent signal-induced alcohol recall and reduce drug-induced dopamine stimulation, ANS-6637 was developed as an orally active selective and reversible ALDH2 inhibitor for study as a treatment for AUD and other substance use disorders. In a phase 1 study of the interaction of ethanol with ANS-6637 in healthy men, the most common adverse effects were hot flushes and increased heart rate, expected effects due to the accumulation of acetaldehyde. Participants who received ANS-6637 also reported reduced alcohol preference compared to those who received placebo. To further assess the potential value of ANS-6637 in the treatment of AUD, the current Phase 2 study included a human laboratory study of alcohol craving, integrated with a 5-week outpatient study that assessed safety and alcohol drinking behavior depending on the treatment I am looking for people with AUD. .

Methods:A 3-arm, double-blind, randomized, placebo-controlled proof-of-concept laboratory, included in a 5-week multicenter clinical trial, tested 2 doses of ANS-6637 against placebo. Patients with elevated liver function tests at screening were ineligible (AST/ALT > 2.5x ULN and/or bilirubin > 1.5x ULN). Participants were treatment-seeking men and women who were at least 21 years of age and met criteria for moderate to severe AUD. Eligible participants were randomized to receive placebo, ANS-6637 200 mg or 600 mg daily. After the first week of taking the study medication, participants completed a cue reactivity session where hunger was assessed in response to the sight and smell of participants' commonly consumed alcoholic beverage and under water control conditions. During the 5-week treatment period, weekly alcohol consumption, alcohol-related adverse effects, sleep, mood, and safety outcomes were obtained, and secondary efficacy endpoints were analyzed over the last 4 weeks of treatment. 81 participants are planned.

Results:The study was terminated shortly after enrollment of 43 participants (ns: placebo = 15; 200 mg = 15; 600 mg = 13) due to clinically significant elevations in liver enzymes in 3 women after at least 3 weeks of ANS-6637 treatment, which resolved upon drug discontinuation . 2 were randomized to 200 mg and 1 was randomized to 600 mg. Induced adverse events consistent with ALDH2 inhibition were dose related and included palpitations/palpitations (placebo 6.7%; 200 mg=67%; 600 mg=69%) and hot flushes (placebo 33.3%; 7% 0 mg; 7%= 00; = 92%). For the primary efficacy endpoint, strength of desire for exposure to the laboratory alcohol stimulus, differences between the groups were not statistically significant (LS mean + if: 7.27 + 1.25 for placebo, 3, 29 + 1.30 for 200 mg , 6.97 5 for the 200 mg/600 mg group). Secondary endpoints measured during the clinical trial were also not statistically significant. However, small to moderate effect sizes were observed for all drinking outcomes at the 600 mg dose compared to placebo (eg, effect size % days binge drinking 0.43; abstinence 0.54). Effect sizes on the PROMIS Alcohol Consequence Scale were medium (0.69) for the 200 mg group and large (1.05) for the 600 mg group. At the end of the 5-week study, the magnitude of the effect on craving was 0.42 for the 200 mg group and 0.58 for the 600 mg group compared to placebo. Sleep quality was higher in the placebo group compared to ANS-6637 treatment. In the mood state profile, the ANS-6637 groups had greater decreases in intensity and increases in vigor compared to placebo (effect sizes ranged from 0.28 to 0.71).

Conclusions:This study, which included a human laboratory study in a 5-week clinical trial, provided critical safety data related to unexpected liver toxicity that led to the early termination of the study. In addition, although the effect on alcohol craving as measured in response to the alcohol exposure paradigm after 1 week of dosing was small, data from participants treated for 5 weeks provide tentative evidence to suggest that selective inhibition of ALDH2 may reduce alcohol consumption, alcohol effects, and appetite. However, these results should be interpreted with caution as effect sizes may be unreliable due to the small sample size. In conclusion, although the risk of liver damage limits the value of ANS-6637 in the treatment of AUD, as a proof-of-concept study, the results suggest that other molecules targeting ALDH2 inhibition may require further study.

Keywords:ALDH-2, Alcohol use disorders - treatment, pharmacotherapy, craving

Disclosures:ASCP ACTIVE Workgroup patrocinado pela Alkermes, Amygdala Neurosciences, Arbor Pharmaceuticals, Dicerna, Ethypharm, Indivior, Lundbeck, Mitsubishi i Otsuka: Honoraria (Self)

Dicerna, Alkermes, Opiates: Advisory Board (Auto)

Novartis, Amygdala, AstraZeneca: other financial or in-kind support (own)

Emmes Corporation: Fees (alone)

University of Florida School of Medicine, Gainesville, Florida, United States

Bottom:Chronic cocaine users often make maladaptive decisions, overestimating the rewards and underestimating the potential negative consequences of their choices. Previous work has shown that chronic self-administration of cocaine to young adult male rats results in an increase in risk-taking that persists until abstinence, suggesting that cocaine alone can induce lasting changes in risk-based decision making. The current study sought to extend these previous findings in two directions. First, we determined whether the risks associated with cocaine risk-taking were due to the voluntary nature of self-administration compared to the pharmacological properties of the drug (i.e., whether passive administration of cocaine produces effects similar to self-administration). Second, we determined whether the effects of cocaine on risk behavior extend to female rats.

Methods:Four separate experiments assessed the risk preferences of operating theater rats in a risky decision task (RDT). In this task, the rats made discrete choices between two levers, one providing a small "safe" food reward and the other providing a large and "dangerous" reward accompanied by a mild foot shock, the probability of which increased from 0%. up to 100% in each test session. In Experiments 1 and 2, rats were tested on RDT, followed by 2 weeks of passive cocaine injections or extended access to self-administration, followed by 3 weeks of abstinence and retesting of RDT. In Experiments 3 and 4, rats underwent 2 weeks of extended-access cocaine self-administration or passive cocaine injections, followed by 3 weeks of abstinence and RDT testing. Data were analyzed by multivariate ANOVA, with drug use status and gender as between-participant variables and shock probability as between-participant variables.

Results:In Experiments 1 and 2, neither passive cocaine administration nor cocaine self-administration affected risk behavior in rats. These negative results were in stark contrast to previous findings. However, cocaine administration in the current studies was initiated at a much later age (15–25 weeks) than in previous experiments that used a similar experimental design but initiated cocaine self-administration at an earlier age (11 weeks). Experiments 3 and 4 were conducted to assess the possibility that rats are more sensitive to the effects of cocaine at a younger age, starting cocaine administration between 9 and 11 weeks of age. Under these conditions, risk-taking was increased by both passive cocaine injections (eat(4,80) = 7,01,PI number< 0.01) and cocaine self-administration (eat(4,76) = 8,04,PI number< 0.05) compared to control conditions, and the effect was similar in male and female rats. Importantly, these increases in risk-taking could not be attributed to cocaine-induced differences in shock reactivity or motivation to eat. Finally, acute amphetamine administration decreased the preference for a large and risky reward in both cocaine-exposed and control rats.

Conclusions:The results echo previous work showing that chronic self-administration of cocaine can result in a steady increase in risky behavior and extend previous results for both rats passively administered cocaine. In addition, the results suggest a limited period of time that cocaine can affect risk-taking, as starting cocaine exposure at or before 11 weeks of age resulted in a sustained increase in risk-taking, while starting cocaine exposure at 15 weeks of age or later had no effect. Finally, acute amphetamine administration reduced cocaine-induced increases in risk-taking, indicating a pathway by which maladaptive risk-taking may be reduced in substance use disorders.

Keywords:Cocaine, risky decisions, development, gender differences, punishment

Revelation:Nothing to disclose.

South Carolina Medical University, Charleston, South Carolina, United States

Bottom:Self-administration of methamphetamine (meth) reliably causes deficits in object recognition memory due to drug-induced plasticity in the paraolfactory cortex (Prh). Prh projections are numerous and include the prefrontal cortex (PFC) and nucleus accumbens (NA), two major nodes involved in the biology of relapse. In the first study, we assessed interconnection activity between Prh and PFC during an object recognition memory test. In a second study, we measured activation of Prh neurons during relapse induced by novel or methamphetamine-related stimuli. We have previously shown that animals exposed to methamphetamine (long-term access, LgA) continue to respond to methamphetamine-related cues without paying close attention to a new signal presented in the same environment, and the importance of the new signal is enhanced by stimulation (positive mGluR5 allosteric modulator, GqDREADDs ) Pr.

Methods:To assess the role of the Prh-PFC reverse circuit in object recognition memory, male and female mice were injected with a reverse adeno-associated virus encoding recombinant GFP-tagged Cre into Prh or PFC. Three weeks later, the mice were tested for OR memory. On the day of the test, one group studied both known and new objects. The second group studied only familiar objects. Brain tissue was processed for confocal microscopy to visualize GFP and c-fos expression in PFC or Prh to determine the extent to which cells in the pathway of interest were activated during the exploration of new and familiar objects. To test Prh activity in response to novel cues, mice were subjected to 1-hour (short access, ShA) or 6-hour (LgA) methamphetamine self-administration and abstinence. They were reintroduced into the chamber in the presence of a methamphetamine-related lever (methamphetamine reinstatement) or had an additional novelty lever in the chamber, as well as a new beacon (group of new clues). In this study, c-fos+ cell expansion in Prh was quantified and compared between groups.

Results:The mice spent more time discovering new objects than familiar ones. When exploring new objects, the cortical neurons that receive information from Prh were not activated outside of exploring familiar objects. However, basolateral amygdala, Prh, PVT, and ventral hypogastric tissue neurons that provide PFC inputs were activated during novel object exploration, indexed as an increase in GFP + c-fos + /GFP +  cells relative to object-exploring litters. In addition, the % of fos+ cells in Prh was positively correlated with recognition index (a measure of recognition memory). After self-administration of methamphetamine (ShA or LgA), rats were tested for methamphetamine seeking in response to a methamphetamine-related lever and/or novel lever. LgA rats responded preferentially to methamphetamine-related leverage. whereas ShA methamphetamine rats responded to both novel and methamphetamine-related leverage. Induction of c-fos in general Prh neurons (non-specific output) was greater in ShaA rats exposed to novel cues compared to methamphetamine cues and home cage controls.

Conclusions:In summary, we found that Prh neurons receiving cortical stimuli are more active during object recognition memory compared to the reverse circuit. These data suggest a more complex circuitry underlying recognition memory than previously indicated by anatomical studies or lesions. This information provides the basis for future work to understand the role of Prh and PFC and their connections in methamphetamine addiction. Established findings show that exposure to novelty increases c-fos activity in Prh, here we extend this finding to animals experiencing methamphetamine. Furthermore, we show that the relevance of the new clue is limited to the ShA of methamphetamine exposure and not to LgA metheexperiencers, who may exhibit a more normally "addicted" phenotype. This research is one of the first to characterize and identify the importance of Prh in memory recognition and methamphetamine addiction.

Keywords:Novelty response, novelty seeking, methamphetamine seeking, drug addiction, recognition memory

Revelation:Nothing to disclose.

University of California, Irvine, Irvine, California, United States

Bottom:Tobacco addiction remains one of the greatest preventable causes of disease and death worldwide. Unfortunately, currently available treatments are only moderately effective in helping individuals achieve long-term abstinence. Therefore, there is a critical need to identify new targets for therapeutic intervention. Recently, nicotinic acetylcholine receptors (nAChRs) and dopamine D3 receptors (D3Rs) have been shown to form heteromeric complexes in dopaminergic neurons, and a new compound, HyNDA-1, may enhance the interaction between the nAChR-D3R complex. Therefore, in these studies, we sought to investigate whether HyNDA-1 modulation of the nAChR-D3R complex could serve as a new target for therapeutic intervention to promote nicotine cessation.

Methods:In the first study, male mice (N= 12) were tested for the effect of HyNDA-1 on nicotine intake with an IV nicotine self-administration protocol. Subjects were tested with varying doses of HyNDA-1 (0-30 mg/kg) within the subject using the Latin square method. Based on these findings, we next investigated whether HyNDA-1 would alter the overall response of caregivers to a food reward in a separate cohort (N= 5). Finally, the third group of mice (N= 6) was tested in a conditioned place preference protocol (CPP) to determine whether HyNDA-1 induces rewarding or aversive properties at an effective dose for nicotine self-administration. Data on self-administration of nicotine and food were analyzed by mixed-effects model analysis with adjustment for multiple comparisons, followed by Tukey's post hoc test, respectively. The CPP data was analyzed by the pairT-test.

Results:We found that pre-administration of HyNDA-1 reduced nicotine self-administration in a dose-dependent manner. Specifically, HyNDA-1 at 30 mg/kg resulted in a statistically significant reduction in the number of nicotine injections obtained compared to vehicle (PI number< 0.01) and lower doses (PI number< 0.05). Interestingly, the effective dose of HyNDA-1 was ineffective in changing self-feeding (PI number> 0.05) and did not induce ventricular preference in the CPP test (PI number> 0,05).

Conclusions:These data demonstrate that modulation of the D3R-nAChR complex by HyNDA-1 reduces nicotine self-administration. Importantly, these effects were specific to nicotine, as HyNDA-1 treatment did not alter food self-administration. In addition, the HyNDA-1 compound by itself does not appear to infer rewarding or aversive properties as no differences were found with CPP. Taken together, these findings reveal that modulation of the D3R-nAChR complex has the potential to become an effective new smoking cessation target.

Supported by the National Institute on Drug Addiction (NIHDA039658for CDF) and the Related Diseases Research Program (TRDRP T30FT0967 for VL).

Keywords:Substance/nicotine use disorder, dopamine D3 receptors, nicotinic acetylcholine receptors, self-injecting drug use, residence preference

Revelation:Nothing to disclose.

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Bottom:Cocaine use disorders impose a significant economic and health burden on society. However, there are currently no effective treatments. Although preclinical models have identified numerous interventions that can disrupt the primary reinforcing effects of cocaine to reduce self-administration, these interventions have had very limited clinical success. Some hypothesize that the compulsive nature of Substance Use Disorder (SUD) leads individuals to continue using whether the drug is reinforcing or not. Thus, over-reliance on habit-based decision-making circuits can lead to drug use, whether or not the treatment reduces the value of the drug. Thus, classical preclinical models using fixed ratio (FR) reinforcement schedules may not be effective models of the compulsive nature of the disorder found in humans. A model with the potential for better predictive validity is the Intermittent Access (AI) model, which has been shown to produce greater motivation and drug seeking than punishment in rats, characteristics that may indicate that drug seeking has become a habit. However, this has not been directly tested or published comparisons in women. In addition, it is unclear whether AI self-administration changes the effectiveness of potential SUD treatments.

Methods:Male and female Sprague-Dawley rats were trained to self-administer cocaine on a FR or AI reinforcement schedule. We tested subsets of both groups for Progressive Relationship (PR) and penalized responses. Another group of rats was tested for habit-based decision making by injecting alpha-flupentixol, a dopamine receptor antagonist, into the dorsolateral striatum (DLS) prior to a cocaine-motivated search trial. We then tested the effectiveness of cue extinction learning by dividing the animals in each training session into groups exposed to either 0 or 120 cues. The stimulus-induced recall was tested the next day. Finally, we performed an exploratory analysis of dopamine release and neural activity in the dorsal striatum and DLS of rats during a training period that should include drug seeking as it transitions from goal orientation to habituation via photometry. Optical fiber with fluorescence index dLight and RCaMP.

Results:Rats trained in the AI ​​program showed increased intake, higher PR retention rates, and increased response to punishment compared to FR-trained rats. We found that the increases in PR cutoff points were from women, while the increases in penalized responses were from men. These results suggest that men are more prone to habitual drug seeking. Indeed, we also observed that drug seeking was affected by DLS DA receptor antagonism more strongly in males. Furthermore, while signal blanking was equally effective in FR and AI-trained females, signal blanking was ineffective in AI-trained males. Finally, fiber photometric data suggest that increased neural activity (Ca 2+ signal) is observed in response to the cocaine signal in both DMS and DLS at the beginning of training, but dopamine signal is only observed in DMS during targeted cocaine seeking. Detailed statistics: Data were analyzed by ANOVA. Self-administration: We matched all cohorts to group sizes: FR male=10, FR female=9, AI male=31, AI female=25. The groups were divided intoN= 4 or 5 for each gender and inclusion status or extinction group. Analysis of the action of the DA antagonist wasN= 8/group. Infusions obtained: day x time of interaction (eat(9,639) = 5,96,PI number< 0.001), a post hoc comparison showed more infusions in the AI ​​group on day 10 than on day 1 (PI number< 0.0001). PR: access model (eat(1,14) = 11,43,PI number= 0.005), dose (eat(3,42) = 29,77,PI number< 0.001), gender (eat(1,14) = 5,46,PI number= 0.03) and gender x dose interaction (eat(3,42) = 4,27,PI number= 0.01). Penalty Reaction: Dose Effects (eat(1,14) = 43,21,PI number< 0.001), access model (eat(1,14) = 6,93,PI number= 0.02) and the access x gender interaction model (eat(1,14) = 5,57,PI number= 0.033). A post hoc analysis showed a greater response in male AI rats (PI number= 0.01). Experiments with DA antagonists: interaction between treatment and training (eat(1,14) = 4,67,PI number= 0.049), a post-hoc analysis showed an antagonistic effect driven by male AI (PI number= 0.026). Extinction Proposal: Effects of Extinction (eat(1,29) = 11,61,PI number= 0.002) and the access model (eat(1,29) = 4,83,PI number= 0,036).

Conclusions:These studies reveal that within specific reinforcement schedules, motivated cocaine seeking may be moderated by goal-directed or habitual response strategies, and that AI schedules differently influence the propensity of men and women to exhibit habit-based drug seeking. Therefore, effective treatment may require different strategies tailored to the particular brain circuits and signaling systems that drive drug use.

Keywords:Intermittent self-administration, cocaine, punishment, progressive ratio test, gender differences

Revelation:Nothing to disclose.

University of Washington, Seattle, Washington, United States

Bottom:Alcohol withdrawal is a medical emergency and a behavioral barrier to reducing alcohol abuse. Unlike other drug withdrawal, there is ample evidence that alcohol withdrawal worsens after several cycles of drinking and withdrawal, increasing the risk of serious adverse complications including seizures, delirium, and death. Moreover, the negative reinforcement inherent in alcohol withdrawal and relapse cycles complicates any behavioral intervention aimed at reducing alcohol abuse.

We hypothesize that chronic and intermittent exposure to alcohol alters the way microglia, innate immune cells found in the brain, regulate neuroimmunity and neuronal synaptic signaling.

Methods:Using a model of chronic intermittent exposure to ethanol vapor, we collected ribosome-bound RNA of striatal microglia using a conditional transgenic RiboTag after five cycles of alcohol exposure and abstinence. We performed RNAseq on the microglia 'translationome' and analyzed the results with DeSeq2, GSEA and WCGNA.

Results:We found that alcohol deprivation induces oxidative stress in microglia and activates the "unfolded protein response" (UPR), a mechanism involved in cellular stress responses, leading to neuroinflammatory signaling and microglial dysfunction that can lead to cell death. In particular, Ddit3 (CHOP), a transcription factor that is a key UPR mediator, was induced in microglia after ethanol withdrawal. We confirmed these observations with combined fluorescent in situ hybridization and immunohistochemistry in a second cohort.

Conclusions:Activation of the UPR can induce a strong neuroinflammatory response and can change the homeostatic to apoptotic response to cell injury. Recognition of the potential role of UPR in microglial responses to ethanol tolerance and withdrawal may lead to new therapeutic targets for the treatment of alcoholism. Our current study aims to determine the effect of conditioned microglial CHOP knockout on ethanol withdrawal behavior and subsequent ethanol drinking propensity.

Keywords:Ethanol, RNA sequencing, RiboTag, alcohol withdrawal, microglia

Revelation:Nothing to disclose.

University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States

Bottom:Alcohol use causes neuroplastic changes in the central serotonin (5-hydroxytryptamine, 5-HT) systems that can potentially lead to compulsive drinking and contribute to alcohol use disorder. Previous work in mice has shown that chronic alcohol exposure alters the activity of the orbitofrontal cortex (OFC) and leads to loss of 5-HT function in this region. This is important because a decrease in 5-HT in the forebrain is causally linked to a loss of inhibitory control, which can lead to overeating. In the current experiments, we characterize binge-induced changes in OFC 5-HT signaling in mice and non-human primates and investigate their role in promoting compulsive and compulsive drinking.

Methods:Binge drinking was modeled in mice using the two-bottle selection in the dark (DID) paradigm. Briefly, mice were given three cycles of restricted access to 20% v/v. alcohol solution in addition to water. Each DID cycle consisted of a period of 2 hours of access to alcohol every Monday through Wednesday and 4 hours of access every Thursday. In rhesus monkeys (Macaca mulatta), reliable self-administration of alcohol (4% w/v) was established using shape-induced polydipsia. After induction, alcohol consumption took place in open access conditions for 12 months (22 h/day). In this process, non-human primates (NHPs) develop different typologies of drinking and self-select distinct and stable drinking categories (e.g., low drinking, LD, and heavy drinking, BD). Whole cell clamp electrophysiology was used to assess the plasticity of alcohol-induced 5-HT signaling in OFCs in mice and NHPs. Fluorescent in situ hybridization was used to quantify the expression levels of 5-HT1A and 5-HT2A mRNA in OFCs in mice. To conditionally delete 5-HT1A receptors in OFC pyramidal neurons (PNs), AAV5-CaMKIIa-GFP-Cre (or GFP control) was injected into the OFCs of male and female Htr1a-perfused mice. In addition to DID, mice were tested for compulsive or resistant alcohol aversions. Here the alcohol was adulterated with the bitter taste of quinine (100 µM) and the mice had access to this solution for 4 hours. To test the specificity of the 5-HT1A deletion during alcohol consumption, mice were challenged with a 4-hour sucrose preference test (3%).

Results:In PN OFCs, repeated DID cycles induced plasticity in 5-HT signaling at both acute (24h) and prolonged (7–10d) withdrawal time points, leading to a profound loss of 5-HT inhibitory control over OFCs. In control mice in water, the use of 5-HT baths of highly hyperpolarized OFC pyramidal neurons, but not in alcohol-exposed DID mice (5-HT induced total membrane potential (mV) in abstinent mice for 24 hours) and 7-10 H2O mouse days,PI number< 0.01). Similarly, 5-HT-induced hyperpolarization of PN OFCs was absent during binge eating compared to low-drinking rhesus macaques (PI number< 0.05 LD compared to BD). In male and female mice, we found that Htr1A mRNA expression was significantly reduced after DID, while 5-HT2A mRNA remained unchanged. These findings suggest that the loss of 5-HT-induced inhibitory signaling in OFCs is due to downregulation of 5-HT1A. We next sought to directly test the role of 5-HT1A in driving binge drinking by selectively and conditionally removing 5-HT1A in OFC PN. We found that 5-HT1A deletion in OFC PN increases binge drinking in men (alcohol consumption (g/kg/4h) and preference,PI number< 0.05 Cre vs GFP), but not female mice, and promotes aversion-resistant alcohol (quinine) intake in male and female mice (alcohol intake (g/kg/4h), Cre vs GFP,PI number< 0.05 for both sexes. alcohol preferences, Cre vs GFP:PI number< 0,05 M ePI number< 0,01eat), but did not affect sucrose intake.

Conclusions:Our cross-species physiology shows that there is conserved plasticity in frontal cortex 5-HT signaling induced by excessive alcohol intake. Specifically, we show that excessive alcohol consumption results in loss of 5-HT-mediated inhibitory control of OFCs in mice and NHPs. Downregulation of 5-HT1A receptors may serve as a possible mechanism for this phenomenon, as in mice excessive alcohol consumption strongly downregulated mRNA expression levels of this receptor. Furthermore, our work shows that loss of 5-HT1A in OFC PN promotes compulsive and binge drinking. Thus, it is possible that a reduction in 5-HT1A levels induced by excessive alcohol consumption may promote and sustain a person's alcohol abuse and thus increase the risk of developing an alcohol use disorder. Taken together, our work highlights the therapeutic potential of targeting OFC 5-HT1A signaling in the treatment of binge drinking and alcohol use disorders.

Keywords:Serotonin, serotonin 1A receptors, orofrontal cortex (OFC), rhesus monkeys, excessive alcohol consumption

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:There is growing evidence that a diverse gut microbiome is essential for good mental health. Maladaptive changes in the gut microbiome have been shown in a number of psychiatric conditions, including autism spectrum disorders, depression, and substance use disorders. Mechanistic studies in animal models have shown that changes in the microbiome result in altered behavioral and synaptic plasticity in several domains. Recently, animal studies have shown that reducing the microbiome with antibiotics can have an effect on reward and drug intake. In preclinical studies of opioid rewards, microbiome-depleted mice have impaired morphine locomotor sensitization and conditioned place preference. In addition to affecting opioid reward, disrupting the microbiome dramatically altered gene transcription in the nucleus accumbens (NAc). The current study assessed the impact of microbiome depletion on opioid uptake in a self-administration paradigm that is more translationally appropriate. In addition, we used a global NAc proteomic analysis to determine the influence of the microbiome on opioid-induced protein expression.

Methods:Adult male Sprague-Dawley rats (weight -250 g) were housed in pairs upon arrival. Cages were randomly assigned to drink either plain water (H2O) or antibiotics (Abx, containing vancomycin, neomycin, bacitracin and pimaricin) to disrupt the microbiome. Rats received the appropriate oral solutions 2 weeks prior to the onset of the behavior and remained on these oral solutions throughout the study. Rats were run in three independent cohorts with different experimental objectives for each. In all experiments, rats were implanted with a jugular catheter one week prior to the start of the experiment and trained to self-administer fentanyl or saline according to the FR1 schedule for 10 days before continuing with the procedures described below. Experiment 1 measured total intake during self-administration of a high dose (5 mcg/kg) of fentanyl (H2ON= 6, AbxN= 6). For Experiment 2, rats were trained on a lower dose of fentanyl (2.5 mcg/kg) before undergoing a 6-day FR-enhancement study (2 days in FR2, 2 days in FR3, and 2 days in FR5) and completing a two-day progression factor (H2ON= 6, AbxN= 7). For Experiment 3, prior to evaluating the dose-response curve, rats were trained at a dose of 2.5 mcg/kg. The rats were given each of the following doses for two consecutive days: saline, 0.025 μg/kg, 0.25 μg/kg, 0.79 μg/kg, 7.9 μg/kg or 25 μg/kg. The doses were given in random order (H2ON= 7, AxN= 7). Finally, to gain insight into the underlying neurobiological differences in motivation between H2O and Abx mice, the NAc from Experiment 2 mice was excised and snap frozen. Independent, objective data collection by mass spectrometry was used to determine protein expression differences in the NAc of intact and depleted microbiome mice. All animal procedures were approved by IACUC Mount Sinai and in accordance with the "Guide for the Care and Use of Laboratory Animals".

Results:For Experiment 1, microbiome Abx depletion reduced high-dose fentanyl intake during intake (linear mixed effects analysis, significant session effects (PI number< 0.001) and beverage type (PI number= 0.0053)). However, in Experiment 2, there was no difference in the acquisition or administration of fentanyl at a dose of 2.5 mcg/kg (linear mixed effects analysis, main effect significant only for sessions (PI number< 0.0001)). In contrast, Abx rats worked harder to obtain fentanyl in RR gain and progressive factor tasks (FR gain: linear mixed effects, main session effectsPI number= 0.0006), type of beverage (PI number= 0.0104) and a significant interaction (PI number< 0.0001); progressive proportion: independent samplesT-test,PI number= 0.012). Since Abx rats were administered less fentanyl than H2O rats at the high dose and equal amounts of fentanyl at the lower dose, Experiment 3 evaluated the effect of Abx on fentanyl dose response in rats. The knockdown of the microbiome induced a shift to the left in the dose-response curve (linear mixed-effects analysis, significant dose effect (PI number< 0.001) and significant interaction (PI number= 0.0109)). Finally, NAc proteomic analysis showed that pathways related to G protein signaling, cAMP signaling, opioid receptor signaling and nitric oxide production were upregulated only in Abx mice, and synaptogenesis pathways were upregulated only in H2O mice.

Conclusions:The current study showed that Abx oral microbiome alterations affect fentanyl self-administration, causing a leftward shift in the dose-response curve and resulting in an increase in motivation for moderate fentanyl administration. Proteomic analysis of the NAc shows that microbiome depletion affects multiple pathways related to drug-taking behavior. Future studies will explore the relationship between altered protein expression in these pathways and altered opioid self-administration induced by microbiome depletion.

Keywords:Fentanyl, the gut microbiome, self-administration

Revelation:Nothing to disclose.

National Institute on Drug Abuse, Baltimore, Maryland, USA

Bottom:Peripheral metabolic biomarkers play a role in energy homeostasis and eating behavior. It has also been suggested that they may modulate central reward pathways important to alcohol and drug seeking behaviors. Metabolic dysregulation can lead to changes in reward pathways and alcohol-related behaviors. An increase in metabolic markers such as triglycerides has been linked to changes in reward-related neural regions, a decrease in dopamine turnover in the reward nervous system, and a decrease in reward-related behavior. Our recent findings revealed that triglyceride and HDL cholesterol levels significantly influenced neural activity in the anterior insula during anticipation of potential monetary loss, and that this effect was not mediated by alcohol consumption.

Methods:The initial analysis consisted of sixteen male and female participants from a previously collected dataset. Participants were treatment seekers who had been detoxified with AUD (N= 7), heavy drinkers not responding to treatment (N= 3) and controls without AUD (N= 6), with clinically abnormal levels of at least 3 of the following parameters: blood pressure, triglycerides, glucose, HDL and BMI Participants completed the neuroimaging version of the Monetary Motivation Delay (MID) task. Mediation models used metabolic biomarkers to predict neuronal activation in the ROI during the prediction phase (e.g., high gain, high loss, low gain, low loss, outcome: gain, gain loss, loss, loss avoidance), and AUDIT was introduced as a potential mediator in this relationship.

Results:Abnormal metabolic status had a significant effect on ACC activation in anticipation of winning a high value prize (b=-0.945, 95%, CI=-1.96-(-0.14)PI number= 0.02), high loss (b = -2.656, 95%, CI = -3.44 - (-1.87)PI number= 0.01) without the effect of alcohol consumption. Alcohol consumption evoked a low value expectation of reward loss (b = -85.48, 95%, CI = -178.230 - (-7.90)PI number= 0.03). Abnormal metabolic status also significantly affected activation of the right anterior insula (AI) in anticipation of high-value reward loss (b=-2.158, 95%, CI=-2.73 - (-1.58)PI number=>PI number= ,02).

Conclusions:We found that abnormal metabolism had a significant effect on reward pathways, however, alcohol consumption mediated this relationship only in anticipation of low-value reward loss. We previously reported that normal triglyceride and HDL levels had a significant direct effect on neuronal activity in the left AI in response to anticipation of potential losses, however, this had no effect on the severity of alcohol consumption. These preliminary findings highlight the potential role of normal and abnormal metabolism in reward and incentive pathways in the drinking population and suggest that the interplay of alcohol use, peripheral metabolism, and reward processing may be more pronounced in individuals with clinically impaired metabolic function. normal. Additional analyzes are being conducted to extend the sample.

Keywords:Alcohol use disorders, metabolism, reward processing

Revelation:Nothing to disclose.

Boston University School of Medicine, Boston, Massachusetts, Stany Zjednoczone

Bottom:Projections from the prefrontal cortex to the striatum are proposed to play a key role in the pathological binge drinking that characterizes alcohol use disorder, with these inputs to the striatum becoming stronger during alcohol exposure. Here, we investigated the role of pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor, PAC1R, in binge drinking in male and female mice in the circuit from the prefrontal cortex (PrL) to the nucleus accumbens (NAcc). We hypothesize that over-activation of this circuit in this circuit causes binge drinking after exposure to alcohol.

Methods:We first characterized the PrLCAPAP population and its screening targets using in situ hybridization (N= 2) and virus control (N= 3), respectively. After 8 weeks of exposure to alcohol or water using the Intermittent Access to Two-Bottle Choice (IA2BC) method, we quantified the amount of PACAP and PAC1R and the activity of cells expressing these markers in PrL and NAcc as measured by co-expression of FosB (N= 4-6/group). We then used a cre-dependent caspase-mediated viral approach to dislodge PrLCAPAP neurons and measure the effect on alcohol consumption in male and female mice (N= 11/group, 5-6/gender). We extended these results by sharply inhibiting these PrLCAPAP neurons with inhibitory DREADDs, after 8 weeks of IA2BC exposure (N= 14.7/gender). We next investigated the role of NAccPAC1R by measuring ethanol intake after administration of PA-8, a small molecule PAC1R antagonist (N= 18.9/sex) followed by more chronic manipulation, with knockdown of the short hairpin RNA (shRNA) of the receptor in the NAcc (N= 9-11/group). Finally, we used chodopsin channel-related circuit mapping (CRCM), a combination of optogenetic stimulation of input neurons PrLCAPAP and whole-cell rectification recordings of NAccD1R +/- neurons to confirm functional connectivity between the two populations (N= 4).

Results:Using fluorescent in situ hybridization, we identified the PrLCAPAP population as long-range glutamatergic cells projecting to layer 2/3. We characterized the projections from PrLCAPAP neurons and observed visible fibers in the NAcc and other downstream regions associated with alcohol consumption. We then used the Intermittent Access to Two-Bottle Choice (IA2BC) method to model chronic alcohol consumption. In mice exposed to alcohol for eight weeks, we found an increased collocation of FosB and PACAP in PrL (T(7) = 4,159,PI number≤ 0.01). There was also an overall increase in PACAP (T(7) = 3,118,PI number≤ 0.05) e PAC1R (T(4) = 3,664,PI number≤ 0.05) in PrL and NAcc, respectively, suggesting activation of this neuropeptidergic system after alcohol exposure (8 weeks IA2BC). When we used cre-dependent caspase virus to selectively ablate PrLCAPAP neurons prior to alcohol exposure, we observed a reduction in chronic alcohol use that was specific to male mice (Virus X Time X Gender:eat(13, 221) = 2,83,PI number≤ 0.001; Men - Viruses:eat(1,8) = 5,65,PI number≤ 0.05, virus X time:eat(13,104) = 1,96,PI number≤ 0.05, Women - Virus:eat(1.9)=0.24, ns, Virus X Time:eat(13117) = 1.74, n.s.). We then used a designer-receptor approach to study the effects of inhibition of PrLCAPAP neurons on alcohol consumption in male and female mice after 8 weeks of IA2BC alcohol exposure. We found that inhibition of PrLCAPAP neurons reduced alcohol intake, again only in male mice (dose X sex:eat(2,32) = 45,9,PI number<0.001; Men - Dose:eat(2,16) = 6,505,PI number≤ 0.01, dose X Time:eat(4,32) = 2656,PI number≤ 0.05. Women - Dose:eat(2.16)=0.16, n.s., Dose X Rate:eat(4.32)=0.27, n.s.). Research is underway using a mouse model of PACAP to determine whether PACAP in PrL neurons is specifically responsible for increased alcohol consumption. In addition, we are currently using fiber optic photometry to investigate whether alcohol exposure modulates the endogenous activity of PrLCAPAP neurons. Pharmacological antagonism of PAC1R, through the drug PA-8 administered systemically to alcohol-exposed animals, also reduced alcohol consumption, again only in male mice (dose X sex:eat(2,36) = 2,15, n.s. [PI number= 0.13], Men - Dose:eat(2,22) = 27,88,PI number≤ 0.001, Dose X Time:eat(4.44)=0.57, n.s., Women - Dose:eat(2.16)=0.25, n.s., Dose X Rate:eat(4.32)=0.76, n.s.). We followed up this finding by assessing whether PAC1R knockdown would prevent binge drinking by injecting a short hairpin RNA targeting the PAC1R receptor via a viral vector into the NAcc of male mice prior to alcohol exposure. We found that PAC1R knockdown reduced ethanol consumption compared to control virus (virus:eat(1,18) = 11,34,PI number≤ 0.01, virus X time:eat(6108) = 1.45, n.s.). Hodopsin channel assisted circuit mapping (CRCM) experiments demonstrated functional connectivity at PrLCAPAP synapses with NAccD1R+/-. Electrophysiological experiments are underway to determine the effect of ethanol exposure on this junction, as well as the possible modification of glutamatergic transmission at these synapses through the use of PACAP and PAC1R antagonism.

Conclusions:Taken together, these data describe the projection of PACAP-expressing cells onto PrL in the NAcc nucleus and show that these neurons play a key role in abnormal alcohol consumption in a mouse model of chronic alcohol exposure that appears to be specific to male mice.

Keywords:PACAP, alcohol use disorder, nucleus accumbens, prelimbic cortex

Revelation:Nothing to disclose.

Wake Forest School of Medicine, Winston-Salem, North Carolina, United States

Bottom:Recent evidence suggests that heroin produces rewarding and addictive effects, at least in part, through activation of the mesolimbic dopamine system. In particular, it has been shown that heroin craving can be alleviated by systemic administration of downstream regulator antagonists of the dopaminergic system, such as kappa opioid receptors (KORs) or presynaptic D3 receptors (D3Rs), thereby increasing dopamine levels. Therefore, it is possible that withdrawal from chronic heroin exposure leads to a hypodopaminergic state in the nucleus accumbens (NAc), as previously observed after withdrawal from chronic stimulant and alcohol use. Therefore, this study aimed to (1) investigate changes in terminal dopamine function after heroin self-administration in male and female rats and (2) identify the mechanism of hypodopaminergy after chronic heroin self-administration.

Methods:Adult male and female Long Evans rats were trained to self-administer heroin (0.05 mg/kg/inf) and then placed in long-term access (LgA, FR1, 6-hour session, unlimited infusions, 0.05 mg/kg/inf) inf). heroin itself - an example of administration to induce increased heroin consumption. After LgA, rats were used for neurochemical analysis - in vivo microdialysis and ex vivo fast cyclic scanning voltammetry (FSCV) in the NAc.

Results:The results support previous literature in which, after LgA, male and female rats had reduced baseline extracellular dopamine levels as well as reduced dopaminergic response to heroin challenge (0.1 mg/kg/inf, IV) in the NAc during heroin withdrawal. -management. FSCV results revealed that heroin-exposed rats had reduced dopamine release during single-pulse stimulation, but increased phasic dopamine release during stimulation trains (5-pulse, 5–100 Hz) compared to the corresponding heroin-free rats. Furthermore, we found that presynaptic D3R and KOR activity in the NAc increased after LgA in both male and female mice.

Conclusions:These results reveal a marked reduction in dopaminergic system function after heroin exposure and identify a potential mechanism in the NAc that may lead to the hypodopaminergic state seen during heroin withdrawal.

Keywords:Heroin self-administration, dopaminergic system, rapid scan cyclic voltammetry, microdialysis, opioid abuse

Revelation:Nothing to disclose.

Johns Hopkins University School of Medicine, Baltimore, Maryland, Stany Zjednoczone

Bottom:Vaping of cannabis and hemp extracts containing D-9-tetrahydrocannabinol (THC, the main psychoactive component of cannabis) is on the rise. The development of animal models using vapor exposure is important for further understanding the rewarding and aversive effects of vaporized cannabinoids. There is currently limited data on the conditioned reward effects of THC vaporization in rats, and there are no studies to date that have assessed gender differences.

Methods:Male and female Sprague-Dawley rats (N= 96; 12 by gender/drug) were conditioned after exposure to THC vapor (3 exposure conditions) or vehicle (propylene glycol, PG) for two series of 8 daily sessions (total 16 days). THC vapor conditioned rats received vehicle vapor (PG) and one of 3 alternating days of exposure to THC vapor (Conditions: 5 puffs of 100 mg/ml THC, 5 puffs of 200 mg/ml THC, or 10 puffs of 200 mg/ml ml THC). Vehicle-conditioned rats were exposed to PG vapors daily. Rats received passively administered vapor for 30 minutes immediately prior to daily 30-minute conditioning sessions. Place preference tests were performed after the 8th and 16th conditioning sessions and continued daily until extinction occurred. After extinction, the rats underwent a drug recall session.

Results:Male and female rats showed an exposure-dependent preference for the THC vapor chamber. The lowest THC vapor exposure conditions tested (5 puffs of 100mg/ml THC) did not result in conditioned place preference. The highest exposure conditions tested for THC vapor (10 puffs of 200mg/ml) gave priority to both men and women (PI numbers < 0.05). Gender differences were observed, with men preferring the THC vapor chamber when exposed to fewer puffs (5 puffs of 200mg/ml THC). This exposure condition was not sufficient to produce conditioned place preference in females (Sex × THC interaction.PI number< 0.05). The preference for the vaporized THC chamber (10 puffs of 200mg/ml THC) disappeared faster in men than in women. Re-exposure to THC vapor (i.e. main drug) did not result in recovery in either sex.

Conclusions:This is the first study to examine the conditioned reward effect of THC vapor in male and female rats, and the first to examine the extinction and reinstatement of THC CPP vapors. No conditioned place aversion was observed in any of the THC vapor conditions tested. The use of vaporized THC in preclinical models is important for translational value and for better understanding the beneficial effects of cannabis components and the consequences of vaping exposure.

Keywords:THC, couple, conditioned place preference, reward and aversion, gender differences

Revelation:Nothing to disclose.

University of Minnesota, Minneapolis, Minnesota, United States

Bottom:Non-invasive brain stimulation methods to modulate brain activity using transcranial technologies are increasingly popular in studying the relationship between modulated brain areas and stimulation outputs. As one of the most widely used technologies, transcranial direct current stimulation (tDCS) has shown promising results in modulating brain activity/connectivity. However, inter-individual variability for tDCS made it difficult to detect the effects of the intervention at the group level. Animal and human studies have shown significant variability in the electrical dose received by different regions of the brain at an individual level during a fixed stimulation protocol. The collection of multimodal MRI data (i.e. structural and functional MRI) helps investigate how dose-response modulates brain function in response to tDCS. In this study, we present pre-recorded data from our study (NCT03382379) on the correlation between electric fields (assessed by subject-specific computational head models) and brain response (changes in BOLD signals) in a group of participants with methamphetamine use disorder (MUD). We suggest here that the dose-response relationship can be studied at least at 4 different levels.

Methods:We collected data in a randomized, triple-blind, sham-controlled study with two parallel arms. Sixty participants with MUD were randomly assigned to sham or active tDCS (N= 30 per group, 2 mA, 20 min, increase/decrease by F4/Fp1). Structural and functional magnetic resonance imaging (including high-resolution T1 and T2 MRI, resting-state fMRI, and met-cued vs. neutral meth reactivity tasks) were collected immediately before and after tDCS. T1 and T2 weighted MRI data were used to create head models for each patient to simulate the electric fields. Correlations between electric fields (dose) and changes in brain function (response) were studied at four different levels: (1) voxel level; For each of the voxels, a general linear model was used to model brain activation in response to a post-neutral local contrast level (2) (atlas-based graph). The Brainnetome atlas has been used to subdivide cortical head models and task-based functional maps, (3) cluster level (active clusters as opposed to interests); Significant clustering masks obtained over time by neutral contrast group interaction analysis were used to determine the correlation (the functional activity after and before LME stimulation (3dLME, AFNI) was compared. A family error (FWE) was found in the Monte Carlo simulation - based on correction multiple comparison (3dClustSim, AFNI) with alpha < 0.1.PI number< 0.005 and cluster size >40) and (4) network level (task networks and networks in sleep mode). Independent component analysis was used to extract masks from large-scale resting-state networks, and regional homogeneity (ReHo) was calculated for each subject. The normalized average ReHo and average electric fields were extracted from the network. Task frontal connectivity (generalized psychophysiological interaction) in the executive control network was also calculated. A sample size of 30 participants per arm provided 80% power to detect an effect size (δ Cohen) of 0.74. Pearson's correlation coefficient with the FDR correction was used to examine the associations.

Results:Our results showed that (1) at the whole-brain voxel level, block design analyzes showed no significant relationship between electric fields and BOLD signal change — post minus prestim (PI numbercorrected >0.05; 9.74% of small effect size voxels (0.1<|R| <0.3), 1.36% with a mean effect size (0.3 <|R| <0.5) and only 0.09% with a large effect size (0.5 <|R| <1), the total number of voxels was 8,530,021, and the Pearson correlation coefficient (|R| ) was taken as a measure of effect size). (2) at the regional level of the whole brain, no significant correlation survived FDR correction (24.29% of small effect size regions and 3.33% of medium effect size regions, total number of regions was 210 cortical regions). (3) at the cluster level, our results showed no significant correlation between changes in functional activity and electric fields in clusters (PI numberFDR>0.05 corrected? 40% of clusters with low effect size and 40% with medium effect size. the total number of groups was 5). (4) at the network level, a significant negative correlation was found between the electric field and ReHo in the network in default mode (R= -0.46 (average effect size),PI numbercorrected = 0.018). For network-level task-based fMRI data analysis, frontal connectivity showed a significant positive correlation with the electric field at the site of frontal stimulation (R= 0.41 (average effect size),PI numbercorrected=0.03).

Conclusions:The proposed pipeline provides a methodological framework to analyze the effects of tDCS in terms of dose-response relationships at four different levels to directly relate the variability of the electric field (dose) to the variability of the neural response to tDCS. The results suggest that network-based analysis may be a better approach to provide new information on the dependence of the neuromodulatory effects of tDCS on the local brain current dose in each individual. Dose-response integration may be informative for dose optimization/adjustment or deriving predictive biomarkers/treatment response in future brain stimulation studies.

Keywords:Transcranial DC stimulation, computer modeling, functional magnetic resonance imaging (fMRI), substance abuse disorders, methamphetamine

Revelation:Nothing to disclose.

Harvard Medical School/McLean Hospital, Belmont, Massachusetts, Estados Unidos

Bottom:Marijuana is one of the most commonly used drugs in the world, especially among teenagers and young adults. Repeated and long-term use of marijuana can lead to marijuana use disorder or CUD, cognitive impairment, anxiety, and psychosis in susceptible individuals. The magnitude of the neuropsychological consequences is related to the dose, frequency, duration and age of onset of use, with adolescent onset of use being associated with increased risk. Recent trends in youth marijuana use, consumption of higher potency marijuana strains or marijuana-based concentrates, and increased daily use increase these risks. The negative effects of marijuana are attributed to THC, the most important phytocannabinoid in the marijuana plant. Although both THC and cannabidiol (CBD) are present in marijuana plant varieties (e.g. anxiety, cognitive deficits). When combined with THC, CBD is said to attenuate, soothe, attenuate, or have no effect on THC-induced memory impairment, anxiety, psychosis, or neuroadaptation. Since the full range of behavioral and molecular consequences for adolescents exposed to high levels of THC on a daily basis remains unknown, we previously studied the effects of THC administered alone or in combination with CBD in adolescent primates using behavioral and pharmacokinetic parameters. The THC phenotype has been nominally attenuated by CBD in non-human primates and is likely related to the disruption of THC metabolism or entry into the brain by CBD. The current study focused on the molecular changes in the brain caused by daily THC or THC+CBD given over four months to adolescent primates. We initially focused on profiling the amygdala transcriptome, an area of ​​interest related to reported anatomical, functional and behavioral changes in people using marijuana.

Methods:Twelve adolescent male squirrel monkeys (aged 2.3–2.5 years) were divided into three groups: vehicle control, THC, or THC+CBD (N= 4/group). They were first treated with low doses of THC or THC+CBD weekly for four weeks. Then, at the beginning of the test regime, animals received either THC (1mg/kg) or THC+CBD (1mg/kg + 3mg/kg) daily for 4 months. mRNA levels in selected brain regions were assessed by bulk RNAseq and candidate markers were confirmed by RT-qPCR. Gene ontology analysis using the Ingenuity Pathway Analysis software investigated whether genes regulated by THC or THC+CBD compared to controls are involved in pathways related to specific diseases or brain functions. Candidate markers were confirmed by qPCR, and statistical differences between the treatment groups were calculated by two-way ANOVA.

Results:RNA sequencing revealed that over 1,000 genes were differentially expressed in each of the groups tested (control vs. THC, control vs. THC+CBD, THC vs. THC+CBD). Of these, 81, 92 and 32 genes were modified >1log2-fold. Downregulated genes have been linked to pathways related to GPCR, CDK5, synaptogenesis, dopamine, opioids, endocannabinoids, netrin, ephrin and other signaling pathways. THC downregulated genes are implicated in schizophrenia, suicidal tendencies, bipolar disorder, vomiting, with CBD attenuating some of the upregulated genes. Of interest for adolescent development, dopamine D2 receptor downregulation was confirmed by qPCR (PI number= 0.05). Gene expression patterns differed in the amygdala and hippocampus, with CBD more potently suppressing THC-regulated changes in gene expression in the hippocampus.

Conclusions:Adolescent primates exposed to a high daily dose of THC revealed altered mRNA expression of genes involved in key neuronal pathways and mental disorders. Our findings provide clues for investigating the importance of adaptive changes to the functional and pathological consequences of frequent heavy marijuana use by adolescents. Ongoing research is applying this methodology to other brain areas involved in adolescent brain development, such as the prefrontal cortex.

Keywords:Marijuana, tonsils, ripening

Revelation:Nothing to disclose.

Johns Hopkins University School of Medicine, Baltimore, Maryland, Stany Zjednoczone

Bottom:Interest in classic psychedelics has increased in recent years, in part because of their potential therapeutic use. Some of this work has shown how the nature of the subjective drug experience is related to short-term and long-term health outcomes. These studies have largely shown that greater mystical experiences (i.e., a construct reflecting oneness, sacredness and intelligence, positive mood, transcendence of time and space, and ineffability) are associated with greater positive clinical benefit. Previous studies have relied on standardized self-report scales to assess subjective experience, and the extent to which similar relationships may exist using alternative means of assessment is unclear. The purpose of this secondary analysis was to use linguistic analysis to compare narrative drug experiences completed after double-blind administration of psilocybin (a classic psychedelic) and the active comparator drug, methylphenidate. A secondary objective was to assess the relationship between language features and traditional subjective affect scales.

Methods:Healthy participants (N= 36) completed a double-blind crossover study in which oral psilocybin (30 mg/70 kg) or optically compatible oral methylphenidate (40 mg/70 kg) were administered in counterweight order. Standardized questionnaires of subjective drug effects, including mystical experiences and provocative effects, were completed. Participants were also asked to complete an open-ended narrative describing the session. Computational text analysis was performed using Linguistic Inquiry Word Count (LIWC) 2015 software, and the results covered linguistic processes (total words, words/sentences) and word categories, including psychological processes (emotional language) and higher-order language domains (analytic thinking) , impact, authenticity and emotional tone). Early analyzes compared psilocybin and methylphenidate sessions with inpatient useT-tests with sum effect sizes as Cohen's dz. Text features identified as significantly different between the groups were then compared using the sampleTtests on samples of normative expressive writing data (6,179 files of 2,526,709 words) collected in experimental (non-psychedelic) studies where participants were asked to write about deeply emotional topics (e.g., personal depression). Finally, significant text features were assessed for their relationship to the mystical and suggestive experience questionnaires using the Spearman correlation.

Results:The narratives were longer for the psilocybin sessions (average word count = 1,784 words) than for the methylphenidate session (average word count = 1,149 words) (PI number= 0.004, div = 0.52). Comparisons by condition showed that the psilocybin narratives contained lower scores in the construct of higher-order analytical thinking (PI number= 0.002, div = −0.57). The psilocybin narratives also had a higher occurrence of impersonal pronouns (e.g., é, é;PI number< 0.001, dz = 0.82), auxiliary verbs (e.g. will, have;PI number= 0.003, dz = 0.53) and certainty language (e.g. always, never?PI number< 0.001, dz = 0.78) and lower occurrence of kinship language (e.g.PI number= 0.003, dz = -0.53), work language (e.g. work?PI number= 0.009, dz = -0.46) and entertainment language (e.g. movie.PI number<0.001, div=-0.70). Comparisons of psilocybin values ​​with normalized samples of "expressive writing" showed that the former had higher analytical writing scores (PI number< 0,001,Hello= 1.02) and fewer auxiliary verbs (PI number= 0,002,Hello= -0.55), the language of relativity (PI number= 0,003,Hello= -0.53) and work language (PI number< 0,001,Hello= -3.51). Higher experience scores in challenges were associated with lower analytical thinking (R= -0,46,PI number= .01), a less relational language (R= -0,37,PI number= .04) and more impersonal pronouns (R= ,40,PI number= 0.03). The results of mystical experiences were not significantly correlated with language features (absoluteRvalues ​​= 0.05 to 0.33).

Conclusions:The qualitative narratives of psilocybin experiences contained linguistic features drawn from both the comparator drug (methylphenidate) and a large sample of normative data. In particular, these language features showed only slight overlap with standardized quantitative scales, suggesting that they capture unique aspects of the subjective drug experience. Future analyzes may use this narrative analysis framework as a tool to understand aspects of the psychedelic experience in order to evaluate the potential mechanisms underlying the positive psychological effects of psychedelics towards the broader goal of optimizing treatment scenarios and designing new drugs.

Keywords:Psychedelics, psilocybin, methylphenidate, language

Revelation:Nothing to disclose.

Vanderbilt University School of Medicine, Nashville, Tennessee, Estados Unidos

Bottom:Value-based decision-making underlies almost all motivated behavior and requires the ability to link results to specific actions and make adaptive decisions about future behavior. Underlying value- and reinforcement-based decision-making is the nucleus accumbens (NAc), which is integrally involved in learning, selecting, and performing goal-directed behaviors. The NAc is a heterogeneous population composed mainly of D1 and D2 spiny projection neurons (MSNs) believed to have opposing roles in behavior, with D1 MSNs promoting reward and D2 MSNs promoting aversion. However, this framework is largely based on ex vivo recordings showing cell type-specific plasticity after reward/drug exposure. Here, we focus on defining the temporal dynamics of MSNs D1 and D2 in response to various stimuli in contexts to determine how information is processed in these populations.

Methods:We tested the role of D1 and D2 MSNs in behavioral paradigms that require processing of stimulus valence, salience, prediction and timing using optogenetics, patch electrophysiology, fiber photometry and cytosolic calcium imaging. We first tested whether activation of MSNs D1 and D2 is enhanced by an optogenetic intracranial self-stimulation task. We then recorded cellular activity at the population and single neuron level during operant and Pavlovian learning tasks with rewarding and aversive effects. In addition, we investigate how inhibiting these two populations at specific time points (clue vs. output presentation) affects learning. Finally, we tested whether aversive learning induces plasticity in D1 and D2 MSNs using patch-clamp electrophysiology.

Results:First, we found that visual stimulation of MSN D1 and D2 populations supported intracranial self-stimulation. Subsequently, using patch-clamp electrophysiology, we found that D1 and D2 MSNs underwent plasticity after aversive learning, demonstrating that plasticity in these populations was not determined by the valency of the experience. To define the information encoded in these populations, we record their in vivo activity during reinforcement schedules and Pavlovian learning paradigms that separate stimulus value, outcome, cue learning, and action. We showed that D1 MSNs responded to the presence and intensity of unconditioned stimuli - regardless of valency. On the other hand, D2 MSNs responded to the presentation of prognostic cues regardless of whether those cues signaled positive or negative outcomes. We also found that learning was disrupted when D1 MSNs were inhibited at the time of the outcome and D2 MSNs were inhibited during cue presentation, indicating a causal role for these signals in learning.

Conclusions:Taken together, these results provide key evidence for the distinct information encoded in MSN D1 and D2 populations in the NAc. The information encoded in these populations goes beyond simple valence coding and shows that these populations do not have opposing actions. These results will greatly enhance our understanding of the involvement of MSN subpopulations in the NAc in both basic learning and memory, and how these neurons contribute to the development and maintenance of substance use disorders.

Keywords:Median spinal neuron, nucleus accumbens, Valence, associative learning

Revelation:Nothing to disclose.

Ponce University of Health Sciences, Ponce Research Institute, Ponce, Puerto Rico

Bottom:The role of endogenous estrogens in the male brain in relation to drug addiction is unknown. Women are more likely to abuse cocaine, resulting in higher rates of cocaine use and an earlier onset of drug use in adolescence than men. In females, estrogen facilitates extinction of cocaine-seeking behavior, suggesting that estrogen may play a key role in mediating extinction learning. Men also use estrogen in the brain by converting aromatase to circulating androgens. However, it is unclear whether estrogen mediates extinction in men as it does in women. We hypothesize that blocking estrogen synthesis in the male brain should result in impaired extinction learning, leading to increased cocaine-seeking behavior.

Methods:We use two different paradigms for cocaine addiction, cocaine-dependent place preference (CPP) and self-administration followed by extinction and reintroduction. After preparing the CPP (12 days) for 20 min. daily (6 cocaine, 6 saline) male rats were injected with the strong aromatase inhibitor fadrozole (FAD, 0.5, 1 or 2.5 mg/kg) or vehicle prior to each day of extinction. CPP (30 minutes a day). We also examined the effect of FAD on the extinction and reintroduction of SA cocaine. During 10 days of short (2 h) sessions of self-administration of cocaine with two levers, one active and one inactive, the rats learned that the active lever was linked to cues such as light and tone, and to cocaine injection. After the last self-administration session, the rats were divided into three groups and the male rats received an injection of FAD (1 or 2.5 mg/kg) or vehicle for 30 minutes. before each disappearance. In the extinction phase, rats were exposed to the same operant chamber as before, but without any stimulus or cocaine infusion, for two hours a day for 15 days. Drug-seeking memory was then recalled using cocaine-primed recall and cues.

Results:Our data show a dose-dependent effect of FAD on CPP cocaine blanking as 1.0 mg/kg facilitated CPP cocaine blanking and 2.5 mg/kg decreased it compared to the vehicle group. On the other hand, preliminary data show no effect of FAD on cocaine extinction from SA. FAD also exhibits a dose-dependent effect on stimulus reinstatement. 1.0 mg/kg reduced active lever presses, while 2.5 mg/kg produced a similar number of presses compared to the vehicle group. Interestingly, both doses of FAD reduced operant conditioning after cocaine-induced reinstatement. In addition, no effect on food restoration was observed at a dose of 1.0 mg/kg, but a dose of 2.5 mg/kg attenuated it.

Conclusions:Our findings indicate that estrogen modulates cocaine-CPP extinction in a dose-dependent manner. Preliminary data also show that a lower dose of FAD during SA extinction attenuates cue- and cocaine-induced reinstatement, suggesting that aromatase inhibition improves extinction memory and thus reduces cocaine-seeking behavior. These findings suggest that estrogen plays a role in regulating extinction learning in male rats. Another experiment is underway to add more mice to all groups to ensure statistical validity.

Keywords:Cocaine, aromatase, self-administration, air-conditioned room preferred

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:The revival of drug-seeking behavior is often caused by persistent recall of conditioned responses previously associated with drug environments, and this maladjustment becomes evident when individuals fail in extinction-based treatment. Thus, the inability to transfer and apply extinction learning when subjects revisit the original drug context is associated with changes in brain reward circuitry, including the nucleus accumbens (NAc). Unfortunately, extinction and its accompanying drugs have shown limited success in the clinic, perhaps due to a lack of understanding of the neurobehavioral mechanisms underlying the transfer of extinction memory to drug-related contexts.

Methods:Here we use a rat contextual model of drug self-administration (SA; conditioning) (AAA or ABA) where rats acquire cocaine or saline (controls) SA in context A and then extinguish in the same context (A) or another panel (B) , followed by a re-exposure test in panel A (renewal). The percentage change from conditioning to extinction/refresh rolls is used as an indicator of extinction learning. We combine this behavioral paradigm with the use of novel transgenic mouse strains expressing Cre recombinase in medium spiny neurons expressing dopamine 1 or 2 receptors, genome-wide chemogenetic and transcriptional profiling of NAc subregions to understand the effects of extinction/renewal on behavior, cellular and transcription levels.

Results:As expected, we observed for the first time that 100% of rats exposed to the AAA paradigm showed extinction learning, however, a heterogeneous distribution was observed in the ABA group (>50% extinction and >40% recovery). Along with this, the group of rats that did not receive extinction learning (withdrawal from the cage) resulted in >70% recovery. Ongoing transcription experiments on these resulting phenotypic clusters will reveal so-called core genes encoding specific extinction- or turnover-related phenotypes in NAc (core vs. cortex) subregions. Complementing this data set, we used RNAscope, slice electrophysiology, pharmacology, and behavior in D1- and D2-Cre mice. Preliminary results confirm the cell type-specific expression and recombination of Cre in both mouse strains. In addition, we found that chemogenetic inhibition of D1 cells in the NAc reduced renewal-induced relapse, suggesting a critical role for D1 cells in the recovery and viability of these mice as a tool for cell-specific characterization (i.e., circuitry and transcription). .

Conclusions:Together, these approaches will provide behavioral and molecular clues as to how extinction, withdrawal, or renewal transcriptionally reprograms the NAc to develop new molecular sites to facilitate extinction transfer and prevent recovery/relapse.

Keywords:Drug relapse, cocaine addiction, extinction, transcription

Revelation:Nothing to disclose.

Vanderbilt University, Nashville, Tennessee, Stany Zjednoczone

Bottom:The mesolimbic dopaminergic system is involved in the expression of gender-specific behavior and is a critical mediator of many mental illnesses, including schizophrenia, anxiety, depression, and substance use disorder (SUD). Particularly in the area of ​​LDS research, work has focused on gender differences in the anatomy of dopaminergic neurons and relative levels of dopamine between men and women. Interestingly, an important feature of dopamine release from axon terminals in the nucleus accumbens (NAc) is that it is rapidly regulated by local regulatory mechanisms independent of physical activity. One of the strongest modulators of the final dopamine function are nicotinic acetylcholine receptors (nAChR). In the NAc, dopamine is released in a tonic (slow and steady) and phasic (short, bursts/spikes) manner, which are highly modulated by cholinergic interneurons (ChATs), which signal through nicotinic receptors containing α4β2* located directly at its dopamine terminals. However, our understanding of the interaction between ChAT and dopaminergic systems is almost complete in men, and our data show that this interaction is fundamentally different in women.

Methods:Using ex vivo fast scanning cyclic voltammetry in conjunction with pharmacological antagonism of α4β2*-nAChR with Dhβe in male NAc sections (N= 7 - 10 slices), natural circle (intact,N= 7 - 10) and oophorectomized women (N= 7 - 10), we measured dopamine release in fractions of a second after a series of tonic (1 pulse and 5 pulses at 5 Hz) and phasic (5 pulses at 10, 20 and 100 Hz) stimuli. Furthermore, we assessed the interactions between the effect of 17β-estradiol (E2) and the effect of Dhβe on the α4β2*-nAChR by measuring the potentiating effect of E2 on dopamine release mechanisms with and without Dhβe in NAc slices. Finally, we determined the sex-specific effect of ChAT interneuron activity on reward-seeking behavior with Gq-DREADD injected into the male NAc (N= 13) and solitary (N= 15) ChAT-cre +/− mice for selective activation of ChAT interneurons during sucrose positive reinforcement conditioning.

Results:We found that the regulation of dopamine release by the α4β2*-nAChR is absent in female mice under most conditions. The estrous cycle had no effect on nAChR regulation deficits of dopamine release in intact females. However, they were saved by oophorectomy, which indicates that ovarian hormones play an important role in this process. Critically, we found that E2 strongly increases dopamine release, an effect that is blocked by antagonizing the α4β2*-nAChR. Finally, behavioral studies of Gq-DREADD revealed that male mice learned faster than intact females when ChAT interneurons were activated, indicating that the regulation of ChAT dopamine release and reward learning is enhanced in males with little or no effect in females.

Conclusions:In conclusion, we show that circulating ovarian hormones alter the ability of α4β2*-nAChRs at dopamine terminals to regulate dopamine release from the NAc, suggesting that sex differences in ChAT regulation of dopamine neurotransmission underlie sex-specific variation in reward learning. Going forward, it will be crucial to directly link these gender differences to reward processing and reinforcement learning in order to develop gender-specific pharmacotherapies for the treatment of SUD as well as various psychiatric conditions.

Keywords:dopamine, nicotinic acetylcholine receptors, sex differences, cyclic fast scan voltammetry

Revelation:Nothing to disclose.

Rutgers University, Piscataway, New Jersey, United States

Bottom:The orexin system is a promising target for the treatment of substance abuse disorders. An important component of these disorders is relapse, which can result from reduced executive control coming from areas of the brain such as the prefrontal cortex. Orexin neurons project into the medial prefrontal cortex (mPFC), but little is known about orexin signaling in this region during substance addiction. Previous studies have shown that the orexin 1 mPFC receptor regulates alcohol intake, as microinjection of SB334867 into this region reduces binge drinking and has also shown reactivation of ethanol seeking in mice. However, it is not known whether orexin signaling by the mPFC promotes cocaine self-administration, increased consumption, or motivation for drug use.

Methods:Male rats underwent jugular vein catheterization followed by training in cocaine self-administration procedures. Briefly, we trained rats to self-administer cocaine according to the FR-1 schedule, followed by basic economic demand curve behavioral assessments, followed by short-access intermittent self-administration training. Finally, we re-evaluate rats in a behavioral economics paradigm to characterize changes in demand elasticity, an inverse measure of drug motivation. After behavioral evaluation, brain tissue was harvested and processed for immunohistochemical analysis and mPFC in situ hybridization.

Results:In line with previous research, preliminary data show that intermittent self-administration of cocaine significantly reduces demand elasticity (t6=3.5,PI number< 0.05), which indicates an increased motivation to respond to the drug. Tissue analyzes show that intermittent access training increases the expression of orexin-a fibers in the mPFC compared to naive rats (t4=2.23,PI number< 0.05). Our ongoing research is also investigating whether there are changes in orexin receptor expression in the mPFC or the number of orexin neurons projecting into this region after self-administration of cocaine.

Conclusions:These results indicate that cocaine addiction increases orexin signaling in the prefrontal cortex, which may promote relapse. We investigate how chronic cocaine self-administration alters orexin signaling in this brain region and whether there is a causal role for cortical orexin innervation plasticity in addictive endophenotypes. This research will provide new insights into the role of orexin signaling in the brain during substance addiction.

Keywords:Median prefrontal cortex, orexin, cocaine addiction, orexin receptor, intermittent access, self-administration

Revelation:Nothing to disclose.

Yale University School of Medicine, West Haven, Connecticut, Stany Zjednoczone

Bottom:Converging evidence has shown that rapid delivery to the brain increases drug abuse potential. The rate of nicotine release is a key design feature of e-cigarettes – which can be used recreationally for their stimulating and pleasurable effects, or therapeutically to ease the urge to smoke. However, the trade-off between abuse potential and the therapeutic effects of nicotine as a function of administration rate remains poorly understood. We developed a new laboratory paradigm for humans involving intravenous administration of nicotine at discrete rates to determine dose-response curves for potential abuse and suppression of smoking urges. We hypothesized that the likelihood of nicotine abuse would increase gradually with faster rates of delivery, but the effect of delivery rate on craving suppression would be minimal.

Methods:This human laboratory study included twenty-six nocturnal smokers who did not seek treatment (50% female, age 28 ± 3.7 years) who smoked ≥ 5 cigarettes per day in the past year. Participants completed five testing sessions in which they were randomly assigned to receive a saline infusion or a nicotine dose of 1 mg per 70 kg of body weight - administered intravenously over 1, 2.5, 5, or 10 minutes, at a rate of 1, 0.4, respectively. .2 or 0.1 mg/min. Biomarkers of nicotine use, including serum cotinine and nicotine levels, were measured at baseline to confirm smoking and overnight abstinence, respectively. The primary outcomes were the likelihood of nicotine abuse, as measured by the Drug Effects Questionnaire (DEQ) subscales of excitatory and pleasurable effects. and willingness to smoke as measured by Factor 1 (willingness to smoke) and Factor 2 (relief from abstinence) of the Shortened Willingness to Smoke Questionnaire (QSU-B). Each measure was analyzed using a mixed-effects model, with individual factors of delivery rate, time, and the interaction between delivery rate and time.

Results:At the start of the study, the average serum cotinine level of participants was 203 ± 137.2 ng/mL, which corresponds to daily smoking, and the mean pre-session nicotine level was 2.9 ± 3.7 ng/mL, confirming overnight abstinence from smoking . Preliminary results indicate a significant effect of nicotine release rate on the DEQ stimulant (eat(4,67,1) = 8,46,PI number< 0.0001) and pleasant effects (eat(4,89,9) = 5,35,PI number= 0.0007), so that a faster rate of delivery produced a stronger stimulus (Hello= 0.6) and pleasant effects (Hello′ =0.5), compared to a lower delivery rate – indicating a higher likelihood of fraud. For BQSU Factor 2, there was no significant main effect of delivery rate. However, the desire to smoke is greater in the placebo group, with no significant differences between nicotine conditions (eat(4,77,3) = 3,00,PI number< 0,05) (Hello′ =0.5) – indicating that the rate of nicotine delivery did not affect its ability to suppress the urge to smoke.

Conclusions:We plot a delivery rate response curve for a dose of nicotine, which is approximately the amount of nicotine delivered when smoking a standard tobacco cigarette. Our findings reinforce the importance of the delivery index in assessing the trade-off between abuse potential and the therapeutic effects of nicotine. Future research should explore whether e-cigarette delivery rates (e.g., puff duration and puff strength) can be optimized to reduce their addictive potential while maintaining their ability to suppress the urge to smoke. This strategy promises to favor smoking cessation over the reinforcing effects of nicotine, thereby reducing the morbidity and mortality associated with nicotine addiction.

Keywords:Nicotine addiction, Electronic cigarette (e-cigarette), Abuse liability, Harm reduction, Nicotine abstinence

Revelation:Nothing to disclose.

University of Maryland School of Medicine, Baltimore, Maryland, Stany Zjednoczone

Bottom:Social reinforcement therapies are effective for many, but not all, addicts. We recently developed a functional mouse model that mimics the characteristics of such a treatment, a community-enhancement approach. In this model, rats uniformly choose social interaction over methamphetamine or heroin. Social preference-induced abstinence protects against the incubation of drug seeking that would occur during forced abstinence. Here, we determine whether these findings generalize to cocaine and whether delaying or increasing effort for social interaction may reveal potential individual differences in responding to people.

Methods:We trained male and female rats for social self-administration (6 days) and then for cocaine self-administration, initially for 2 hours per day for 4 days, then for 12 hours per day continuously or intermittently for 8 days. We assessed relapse to cocaine seeking at 1 and 15 days. Between trials, the rats underwent forced abstinence or social choice-induced abstinence. Once a stable social preference is established, we manipulate latency for both rewards or just the social reward or response (effort) requirements for the social reward.

Results:Regardless of cocaine access conditions and gender, functional social interaction inhibited cocaine self-administration and prevented cocaine seeking incubation. Social access preference was reduced by delaying both the reward and the social reward itself, or by increasing the response requirements of the social reward, with marked individual variation.

Conclusions:This selection process may identify mechanisms of individual differences in an animal model of cocaine use and thus aid in drug screening for individuals who are relatively insensitive to reward-based treatment for social interaction.

Keywords:Animal models, Social behavior, Drug relapse

Revelation:Nothing to disclose.

Augusta University of Georgia College of Medicine, Augusta, Georgia, United States

Bottom:Social behavior is crucial to maintaining a healthy pet lifestyle. Deficiencies or dysfunctional social behaviors are associated with several mental disorders, including addictions. The dorsal striatum (DS) has been identified as a key area of ​​the brain that controls context-dependent decision-making, including social behavior. In DS, the striatal and striatal circuits coordinate the go-and-go path and produce biased behaviors in exploration tasks according to contexts such as the presence of approach and avoidance conflicts. Interestingly, two distinct regions in SD, the dorsal and dorsolateral striatum (DMS and DLS) have been distinguished due to their different roles in context-dependent reward-seeking behaviors. However, although recent studies indicate that DS exhibits a variety of task-related temporal cellular signals, and that alcohol withdrawal induces abnormal DS activities leading to impaired social behavior, it is still unclear how coordinated activities in DMS and DLS encode social activities. behaviors and whether repeated exposure to alcohol affects this coordination.

Methods:Using multi-regional photometric imaging of calcium fibers, we simultaneously study spatio-temporal neurons and astrocytes in DMS and DLS that represent context-sensitive signatures in environments with different social conditions and their contribution to alcohol-induced damage. We selectively expressed GCaMP6, a genetically encoded calcium-dependent fluorescent marker, in median striatal (dMSN) spiny neurons of the intermediate pathway, middle striatal (iMSN) spiny neurons, and astrocytes from male transgenic mice that express GCaMP6 in a dependent manner. D1R-Cre, A2AR-Cre or ALDH1L1-Cre. For mice expressing enhanced green fluorescent protein (eGFP) in iMSN, we confirm that fluorescence changes in GCaMP6 mice are unlikely to reflect movement artifacts.

Brain calcium imaging acquired and combined with gait speed during voluntary movements in three-chamber social approach tasks. Mice were exposed to the chamber under three different social conditions: without a social agent, with a foreign mouse, or with a foreign mouse and a familiar mouse. "Social preference" was measured as a comparison of the time spent by the subject with a foreign mouse and a new object (sociability) or a familiar mouse (social recognition).

We also compared whether cellular profiles could be affected by exposure in the chronic intermittent ethanol (CIE) paradigm. Briefly, mice were exposed to air or vaporized ethanol in a vapor inhalation chamber for five weeks. Each day cycle consisted of 16 hours of ethanol vaping followed by 8 hours of abstinence in their home cage. This was repeated daily for 4 consecutive days followed by 3 days of abstinence.

Results:In freely moving mice expressing GCaMP6 in the DS dMSN, we typically see persistent, movement-associated increases in Ca2+ signaling in the DMS, independent of the presence of a foreign mouse. Interestingly, during locomotion activity with the foreign mouse, a decrease in Ca2+ signaling in the DLS was mainly induced, and changes in opposing cellular Ca influx in DMS and DLS during exploration with the foreign mouse were significantly reduced when the mouse was exposed to a choice condition with the foreign and familiar mouse together . In addition, machine learning analysis using a support vector machine (SVM) showed that the dMSN coordinated temporal dynamics in the DMS and DLS predicted which social environments the rats explored. Changes in Ca 2+ influx in iMSNs and astrocytes were also observed during voluntary locomotion, but were not fully dependent on social environmental factors.

We also observed that after 3 days of abstinence from repeated exposure to alcohol, dMSN activity was significantly reduced in DMS but not in DLS. In addition, abstinence from repeated alcohol exposure interfered with speed predictions based on the DMS dMSN temporal dynamics. When we inhibited the dMSN activity in the DMS of alcohol-naïve mice using chemogenetic approaches without any change in locomotion, the effect of alcohol deprivation was summarized by reduced social preference.

Conclusions:Our results show that the coordinated actions of striatal neurons in DMS and DLS are major distinct signatures of social context-dependent locomotion, and alcohol withdrawal disrupts these coordinated neuronal actions. These findings provide new insights into how the actions of striatal cells time-coordinate social context-dependent behavior and could be a computational model for predicting types of social behavior.

Keywords:Social behavior, dorsal striatum, neural coordination, alcohol abstinence

Revelation:Nothing to disclose.

Emory University School of Medicine, Atlanta, Georgia, United States

Bottom:Goal-oriented decision-making (i.e., taking action based on expected outcomes) is a fundamental adaptation to navigating everyday life, the failure of which is a central feature of many neuropsychiatric disorders, including addiction. Adaptive action selection requires coordinated integration of various cognitive processes across multiple timescales, including flexible coding, stable representation, and retrieval of learned action-outcome associations (potentially) to guide future choices. The orbitofrontal cortex (OFC) supports performance-based decision making across a wide range of behaviors. However, the coordinated neural circuits and cellular mechanisms by which OFC networks enable causal learning remain unclear.

Methods:We sought to identify OFC circuits that mediate causal learning and memory by examining the ability of mice to select actions based on their consequences using a food-reinforced instrumental choice task. We used virus-mediated selective chemogenetic manipulations to investigate the role of OFC circuits during the encoding of new contingency information and the subsequent retrieval of this learning during selected epochs (N= 108). We then use activity-dependent neural manipulations to determine whether a memory trace for learned decision variables is held by specific neural assemblies in the OFC (N= 43). In addition, we developed a transsynaptic feedback mapping method to locate OFC neurons based on their specific dissynaptic network connectivity properties and used it to quantify circuit-specific dendritic spine dynamics related to emergency learning (N= 24). Finally, we used a molecular-functional dissociation strategy to assess the role of neurotrophin tone in OFCs in coordinating neural circuitry function during the eras of encoding and memory retrieval.N= 63). Both male and female mice were used for this study. Biological sex was included as a covariate that revealed no statistically significant gender effects.

Results:We first show that chemogenetic inhibition of afferent projections from the basolateral amygdala (BLA) -> OFC stops the encoding, but not the retrieval, of new emergency memories. Furthermore, we show that chemogenetic stimulation of BLA->OFC projections is sufficient to enhance conditional learning, thus revealing that these projections bi-directionally control emergency memory encoding. We next show that chemogenetic inhibition of efferent projections from OFC->>dorsal striatum (DMS), but not OFC->BLA, interferes with emergency memory retrieval. We therefore hypothesized that OFC neural assemblies consistently represent contingency information learned over time and facilitate the retrieval of this learning during periods of choice. Here we show that reactivation of OFC neurons responding to new but unknown contingency information is necessary to retrieve new options to guide voting behavior. Finally, we reveal that emergency learning activates dendritic spine plasticity specifically in the BLA->OFC->DMS network and that neurotrophin tone in BLA->OFC and OFC->  circuits is required for emergency new learning.

Conclusions:We describe the directional transmission of learned causal information in a time-tuned amygdala-frontal-striatal network in which contingency memories are encoded by BLA->OFC inputs, represented in OFC complexes, and retrieved by OFC outputs. > DMS on future selection. We also reveal that the integration of these separate learning and memory processes requires circuit-specific neurotrophin tone and neuronal structural plasticity in the OFC. We thus identified the OFC as a critical site in the distributed amygdala-frontal-striatal network, providing a temporal link between encoding and memory retrieval, thus linking the initial learning of new information to its future application.

Keywords:Orbital frontal cortex (OFC), instrumental learning, encoding and memory retrieval, dendritic rays

Revelation:Nothing to disclose.

University of Pennsylvania, Philadelphia, Pennsylvania, United States

Bottom:We recently showed that alcohol metabolites contribute to brain histone acetylation by direct deposition of alcohol-derived acetate on histones. This reaction is catalyzed by the metabolic enzyme acetyl-CoA synthase 2 (ACSS2), which is nuclear and associated with chromatin in neurons. In ongoing research, we are now investigating the role of this pathway during voluntary alcohol intake as well as during prenatal alcohol exposure.

Methods:We used in vivo permanent isotopic labeling in mice to determine the involvement of alcohol metabolites in histone acetylation in adult and fetal brains. We also use a series of molecular (RNAseq, ChIPseq) and behavioral experiments to show that this deposition has important functional and behavioral consequences.

Results:We show that alcohol exposure leads to long-term impairments in histone acetylation, which may underlie alcohol use disorders and related comorbidities, including cognitive decline and neurodegeneration. In addition, we have shown that in pregnant rats, exposure to alcohol results in the incorporation of alcohol-derived acetate into the brains of pregnant fetuses. Deposition of this metabolite is dose-dependent and restricted to developmental time points where ACSS2 is expressed and is associated with chromatin. This novel mechanism thus defines a unique window of epigenetic sensitivity to alcohol metabolites in the pregnancy brain, which may underlie fetal alcohol spectrum disorders.

Conclusions:We recently discovered a new aspect of alcohol's effect on the brain, driven by the direct incorporation of alcohol metabolites into histone acetylation. We now show that this pathway plays an important role in fetal alcohol spectrum disorders, alcohol use disorders and related comorbidities such as neurodegeneration and cognitive decline. Targeting this pathway may therefore be a promising new therapeutic avenue.

Keywords:Alcohol, drug metabolism, epigenetics, spectrum of fetal alcohol disorders, cognitive decline

Revelation:Nothing to disclose.

University of California, Irvine School of Medicine, Irvine, California, United States

Bottom:Adversity in early life (ALS) is associated with susceptibility to reward-related problems, such as addiction to prohedonic opioid drugs. Females may be particularly sensitive, suggesting a sex-specific maturation disorder of the ALS reward circuit. However, the mechanisms by which this happens are poorly understood. To test the hypothesis that ALS interferes with the normal maturation and function of endogenous opioid systems, we used a naturalistic model of ALS in which litter and nest materials are restricted during the first week of life, and examined the effects of ALS on the expression and function of opioid ligands and receptors and search behaviors. opioid drugs in female rats.

Methods:Adult female ALS rats were tested for behaviors similar to opioid addiction, including binge eating and economic demand elasticity, to measure the motivation to take opioid drugs.N= 12/group). To obtain information on ALS-induced molecular changes in reward-associated regions, we used RT-qPCR for a set of molecular candidates (N= 8-9/group). Following our intriguing molecular findings, we pharmacologically manipulated endogenous opioid signaling during opioid self-administration to test the mechanisms of ALS-enhanced opioid seeking (N= 12/group).

Results:ALS resulted in greater motivation to take opioids in female rats, consistent with our previous findings (T(22) = 3,620,PI number= 0.0015). RT-qPCR revealed a selective reduction in delta opioid receptor expression after ALS in the basolateral amygdala (T(17) = 3,197,PI number= 0.0053) and no changes in the μ or κ receptor or endogenous ligands. Preliminary results from the pharmacological manipulation of the δ-opioid receptors in BLA during opioid self-administration suggest a potential mechanism by which ALS may confer addiction vulnerability.

Conclusions:ALS induces long-lasting changes in d-opioid receptor expression in the amygdala that may underlie the pro-addiction phenotype in female rats. Unlike μ and κ, which are expressed at the adult level in neonatal mice, expression of d matures later, potentially making the receptor vulnerable to adverse experiences in early life. Understanding the mechanisms by which ALS promotes vulnerability to opioid use disorder is critical to identifying those at high risk of addiction and developing effective interventions to prevent opioid-related morbidity and mortality.

Keywords:Early life adversity, opioid addiction, delta opioid receptor, basolateral amygdala, nucleus accumbens

Revelation:Nothing to disclose.

Penn State University, University Park, Pennsylvania, United States

Bottom:Somatostatin (SST) neurons have been implicated in various neuropsychiatric disorders such as depression and anxiety, but their role in substance use disorders, including alcohol use disorder (AUD), has not been fully characterized. In our previously published work (Dao et al., 2021 Neuropsychopharmacology), we found that repeated cycles of binge drinking in a drinking in the dark (DID) model led to hypofunctioning of SST neurons in the prefrontal cortex (PL) by reducing the firing capacity of the action potential and excitatory/inhibitory transmission dynamics. We found that by chemogenetically manipulating these neurons, we could reduce binge drinking in both male and female mice. Ongoing work in our laboratory aims to determine the role that SST peptide signaling itself plays in this circuit-dependent change in alcohol consumption.

Methods:All experiments were performed with permission from the Penn State Institutional Animal Care and Use Committee. Using a combination of behavioral pharmacology, we worked to determine the role of SST peptide signaling in the PL cortex. Virgin male and female C57BL/6J mice were used for electrophysiology. Mice were sacrificed under isoflurane anesthesia and crown sections of PL cortex were prepared. SST (1 μM) was added to the bath after determination of the constant resting membrane potential for each cell. For the cannula experiments, male and female C57BL/6J mice were anesthetized under isoflurane anesthesia and unilateral PL cortex-targeting cannulas (Plastics One, provided by Dr. Patrick Drew, Penn State) were implanted. Ongoing experiments use bilateral cannulas. Mice were allowed to rest for a week before assessing stress-like behavior. Statistical analyzes were performed using GraphPad Prism (T-tests and ANOVA as described below. mouse numbers/cell numbers given below for individual experiments).

Results:We first examined the effects of the SST peptide on pyramidal neurons in the PL cortex. Pyramidal neurons (identified by membrane properties and general morphology) were fixed in male and female mice, and after stabilizing the properties of the current clamp, 1 μM SST were bathed for 10 min (N= 15 cells for females, 19 cells for males). in pairsT-tests were used to account for bath use before and after SST application for RMP and rheobase, and ANOVA was used for voltage vs. current plots. SST significantly hyperpolarized the resting membrane potential in both sexes (RMP, females:T14 = 2,544,PI number= 0.0234; men:T18 = 3,171,PI number= 0.0053), as well as properties such as rheobase (pA, femaleT14 = 5,059,PI number= 0.0002; menT18 = 5,259,PI number<0.001). SST also reduced the number of action potentials evoked at various ongoing stages in both sexes (female ANOVA, SST effecteat1,18 = 10,71,PI number= 0.0042; current interaction x SSTeat20,276 = 9,51,PI number<0.0001; ANOVA SST male effecteat1,18 = 9660,PI number= 0.61; current interaction x SSTeat20,360 = 7,772,PI number<0.001). These results were also consistent when maintaining a common membrane potential of -70 mV. In a separate group of mice, retrograde markers were injected into downstream regions (the nucleus of the bed of the striatum terminal and nucleus accumbens, respectively) and SST modulation of these output pathways was confirmed. Ongoing segmental experiments include assessing whether these changes are reversible (using the SST antagonist, cyclosomatostatin).

To understand the functional significance of SST peptide signaling in this region, mice were implanted with unilateral cannulas (Plastics One) aimed at the PL cortex. In the initial mixed-sex cohort, mice received aCSF vehicle control (N= 4 animals) or Octreotide, an SST analogue (0.1 µg administered in sterile aCSF,N= 5 animals) 15 min before behavior. Administration of octreotide resulted in an increase in the distance traveled in the open-field test as well as an increase in the rate of open-arm entry in the elevated plus maze (T6 = 2,578,PI number= 0.0419). Ongoing experiments replicate this work in cohorts with bilateral cannulas. Specifically, we are investigating the role of SST signaling/octreotide administration in anxiety-like behavior in naïve mice as well as its effects on alcohol consumption.

Our work builds on our previously published work to define the role of SST peptide signaling in the PL cortex. We show that bath SST application reduces several measures of excitability of pyramidal neurons, including hyperpolarization of the membrane potential and decreased action potential firing. We translated this result into in vivo behavior by administering an analog of this peptide directly into the PL cortex. We found that unilateral administration had an anxiolytic effect in mice that showed no behavioral response (importantly, they were not anxious). Based on our previously published work showing a reduction in SST neuron function after excessive alcohol consumption, we expect these effects to be even more pronounced after exposure to alcohol or processes such as chronic stress.

Conclusions:Somatostatin neurons and the SST peptide itself serve as a promising therapeutic target for various neuropsychiatric disorders. Here, we seek to elucidate the mechanism by which SST alters neuronal signaling in the PL cortex (by reducing the overall excitability of pyramidal neurons, a key baseline population known to play a role in substance use disorders). In addition, we present tentative evidence for the use of an SST-like compound (octreotide) for anxiety-like behaviors and potential substance use.

Keywords:Somatostatin, precerebral cortex, alcohol

Revelation:Nothing to disclose.

National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland, USA

Bottom:Mitragyna speciosa ("kratom" in the US) is a plant containing over 40 bioactive alkaloids, some of which act on μ-opioid receptors as a seemingly "biased" partial agonist with dose-dependent stimulant and analgesic effects. Kratom alkaloids also have non-opioid mechanisms of action. Surveys of current users indicate that kratom is primarily used to self-medicate symptoms of pain, depression and fatigue, and to relieve/prevent opioid withdrawal. However, current user surveys may over-represent people with positive experiences (or people who cannot quit smoking). We are looking for former and current users. One of the many goals of the study was to collect formative data for a point-in-time study that requires knowledge of the temporal dynamics of use (e.g., whether each dose of kratom has acute effects or is used more as a maintenance drug).

Methods:The Amazon Mechanical Turk was used for crowd participation between September 2020 and March 2021. Of the 2,615 respondents who use the substance, 289 have ever used kratom. All were contacted again between April and May 2021 for this kratom survey, including updated data from previous surveys and pilot questions.

Results:Of the 289 lifetime kratom respondents, 134 (48%) completed our kratom survey. They were women aged 34.8 ± 8.4, female (52%), Caucasian (69%), with secondary or higher education (100%), employed (63%) and reported first use of kratom at age 29 .9 ± 8.8 years old. The majority (60%) used kratom >100 times and reported using kratom >4 times a week for an average of 61.9 ± 104.3 weeks (81%). Using the DSM-5 checklist, 23% qualified for kratom use disorder and 30% for kratom use disorder in the past year. The most frequently approved DSM criteria reflect tolerance or application to avoid withdrawal, compared to less frequently approved disruptions of functions or duties. In addition, 33% of respondents reported withdrawal symptoms due to discontinuation of the drug for >1 day. 18% for missing 1 regular dose.

Majority (N= 103.80%) had acute subjective effects with each dose of kratom. Of these, 54% said the results helped them in their daily responsibilities. 29% said the effects were compatible with (not necessarily useful for) everyday tasks. Less than 4% said the effects interfered with their daily activities. 40-50% reported using kratom to improve daily quality of life, increase energy, or treat anxiety, depression, fatigue, pain. 24.5% used it as an opioid substitute.

Less than half (42%) considered themselves "regular" kratom users. Respondent reported normal doses (in units preferred by respondents) were 5.4 ± 4.8 capsules, 4.6 ± 3.6 grams, 2.5 ± 2.7 tablespoons, 2.1 ± 1.0 cups of tea 6.1 tablespoons or 1.0 tablespoons. On days of use, respondents took an average of 2.6 ± 2.4 times, with this routine consistent for 65.0 ± 112.9 weeks. A significant minority (41%) reported consuming more kratom in the first hour after waking than at other times of the day. 54% said they preferred the first daily dose in the morning to other doses. Changes in dosing schedules during periods of use occurred "occasionally" (33%) or "rarely" (29%). From the first use of kratom, participants reported that their doses increased (26%), stayed the same (23%), decreased (19%), or stopped (21%).

None of the participants reported feeling the effects of kratom within seconds, and 83% reported feeling the effects within minutes. 12% in hours. The majority (92%) said they usually stopped feeling the effects of kratom within a few hours. Only 2 participants reported feeling the effects wear off within minutes. 7% were unsure because they had consumed more kratom before the effects wore off.

The average “very low” (no effect) dose was 3.96 ± 4.95 capsules (N= 50), 2,64 ± 2,44 grama (N= 45), 1.37 ± 0.96 tbsp (N= 19), 2.2 ± 2.17 tbsp (N= 5) or 1.57 ± 0.98 cups of tea (N= 7). The lower limit of the mean effective dose was 4.13 ± 3.31 capsules (N= 45), 3,19 ± 2,25 grama (N= 43), 2.33 ± 2.20 tbsp (N= 24), 2.00 ± 0.89 tbsp (N= 6) or 1.3 ± 0.67 cups of tea (N= 10). The mean upper dose limit (no side effects) was 5.88 ± 4.02 capsules (N= 43), 6,85 ± 4,58 grama (N= 40), 2.87 ± 1.58 tbsp (N= 23), 2.5 ± 1.58 tablespoons (N= 10) or 2.25 ± 1.16 cups of tea (N= 8). The average “very high” (“slightly high”) dose was 7.25 ± 4.24 capsules (N= 40), 8,68 ± 4,38 grama (N= 37), 3.39 ± 1.66 tbsp (N= 27), 3.57 ± 1.72 tbsp (N= 7) or 3.44 ± 2.01 cups of tea (N= 9).

The mean perceived efficacy of kratom across all reported indications (VAS, 0–100) was 72.8 ± 16.7. For discontinuation adverse events, the mean score was 53.0 (±24.1). After standardizing dose sizes with intraunit z-scores, we found that greater severity of smoking cessation effects was predicted by greater weekly use (β=5.24, 95% CI=-0.52, 11.0;PI number= 0.07), plus weeks of regular use (β = 6.74, 95% CI = 0.88, 12.60;PI number= 0.02) and at reduced doses (β = 20.88, 95% CI = 5.75, 35.99;PI number<0.001). Withdrawal from kratom was the strongest predictor of lower outcome ratings (β=−16.45, 95% CI=−25.9, −6.90;PI number<0.001). The amount consumed per week was positively associated with the assessment of favorable outcomes, but not significantly (β = 2.19, 95% CI = -1.52, 5.89;PI number= 0.25); similar for weeks of use (β=0.90, 95% CI=-2.98, 4.78;PI number= 0,65).

Conclusions:Kratom was usually associated with acute rather than chronic effects (unlike most psychiatric or maintenance medications), but the acute effects were generally compatible with or helpful in daily tasks (unlike many illicit drugs).

Keywords:Kratom, Partial Agonist, Mu Opioid Receptor Agonist, Opioid Side Effects

Revelation:Nothing to disclose.

Yale University School of Medicine, New Haven, Connecticut, Stany Zjednoczone

Bottom:In animal studies, nicotine, the main addictive component of cigarette smoking, binds to and activates the b2 subunit containing nicotinic acetylcholine receptors (b2*-nAChRs) in ventral tegmental neurons, which strongly facilitates ventral tegmental dopamine release. area. striatum and increases nicotine utilization. Chronic exposure to nicotine, however, decreases sensitivity and increases the number of b2*-nAChRs and reduces the dopaminergic response. Increases in b2*-nAChR levels and lower dopamine D2/3 receptor availability in the striatum have been reported in smokers and 7-day abstinent smokers, respectively, compared to non-smokers. Little is known about the role of striatal dopamine in smoking initiation or cessation. In addition, cognitive deficits and mood swings are hallmarks of nicotine withdrawal associated with the cholinergic and dopaminergic systems. Difficulties in quitting smoking can be attributed to cholinergic and dopaminergic dysfunction and the resulting changes in cognition and mood. The purpose of this positron emission tomography (PET) imaging study was to investigate the availability of b2*-nAChR and striatal dopamine function in recently quit smokers and non-smokers, and to investigate the cholinergic and dopaminergic correlates of cognition and mood.

Methods:The study involved 27 smokers (9F, age 38 ± 10 years) and 28 non-smokers (11F, age 30 ± 11 years). The smokers were moderately addicted to nicotine (Fagerström nicotine addiction test: 5.9 ± 2.1) and smoked 16 ± 7 cigarettes per day for 18 ± 10 years. Smokers received smoking cessation counseling and emergency management for approximately 2 weeks. [18F]Flubatin PET scans, which measure the availability of b2*-nAChR, were obtained 90–120 min after a bolus infusion of 254.9 ± 44.9 MBq [18F]Flubatin (Kbol 360 min) over 14 days of abstinence, and smokers 19 non-smokers smokers. Dopamine function was assessed in 19 smokers after 11 ± 9 days of abstinence and in 18 non-smokers using two PET scans after bolus injections of the dopamine D2/3 receptor agonist radioligand [11C]PHNO (scan 1, 443.1 sc 4.6 ± 1 ± 168.4 MBq): "baseline" scan before and 3 hours after amphetamine scan (0.5 mg/kg, PO). Subjects completed the 1-back working memory (CogState) task and completed the Center for Epidemiology Research Depression Scale (CES-D) on the [18F]Flubatin and [11C]PHNO scan days, respectively. PET measurements included volumes of distribution corrected for free fraction (VT/fp) [18F]Flubatin in frontal regions and striatum assessed by equilibrium analyses, unchanged [11C]PHNO (BPND) (dopamine D2/3 receptor accessibility) in striatum calculated using a simplified reference tissue model (cerebellum as reference) and amphetamine-induced percentage change in [11C]PHNO BPND (stimulating dopamine release) in the ventral striatum. Differences between groups based on PET scores were assessed using two-sample t-tests. Correlation coefficients (R) were calculated to examine the correlation of PET estimates with measures of cognition and mood. the meaning wasPI number< 0,05.

Results:In this preliminary sample, there were no significant differences between groups in the availability of b2*-nAChRs in the frontal cortex, caudate nucleus, or macula (ps>0.05). Greater availability of b2*-nAChRs in the frontal cortex was significantly associated with a longer average response time to correct responses in a single-backed task (i.e., poorer working memory performance) in smokers who were recently abstinent (N= 13,R= 0,68,PI number= 0.01), but not in non-smokers. Recently quit smokers showed a lower percent amphetamine-induced change in ventral striatal BPND than non-smokers (19 NS: 26.5 ± 6.5%, 18 AS: 19.6 ± 10.0%).PI number= 0.02). Higher CES-D scores (worse mood) were associated with less amphetamine-induced dopamine release in the ventral striatum in smokers who had recently abstinenced (N= 15,R= -0,63,PI number= 0.03) and not in non-smokers. Analyzes are underway to determine inter-individual relationships between b2*-nAChR availability and dopamine function.

Conclusions:There were no group differences in b2*-nAChR availability in this preliminary data set. However, greater frontal cortex b2*-nAChR availability was associated with poorer working memory in recent abstinent smokers, suggesting a potential neural correlate of cognitive impairment during early abstinence. Abstaining smokers had less amphetamine-induced dopamine release in the ventral striatum than non-smokers, the degree of which was associated with poorer mood. This is consistent with evidence of "dulled" dopamine release in people with other substance use disorders, and suggests that withdrawal anhedonia is related to dopamine dysfunction. Efforts are underway to fully elucidate the cholinergic and dopaminergic interactions during smoking cessation attempts.

Keywords:Smoking cessation, PET imaging, a4b2 nicotinic acetylcholine receptors, dopamine function, mood and cognition

Revelation:Nothing to disclose.

Yale University School of Medicine, Hartford, Connecticut, Stany Zjednoczone

Bottom:Disregulated circuitry in addiction engages several cognitive functions related to automatic and controlled processes. Unraveling these processes could benefit the development of targeted interventions related to these processes and the underlying dysregulated circuits found in substance use disorders (SUD). Both processes are activated after a power failure during a Go/NoGo task, resulting in potential-evoked components (ERP) called error-related negativity (ERN) and error-positivity (Pe). The ERN first indexes the initial (automated) error processing (e.g., recognition of a significant error event) and then the Pe, which indexes the post-error processing (e.g., top-down checking to increase accuracy in subsequent tests). Theta and delta frequency powers contribute to this ERP component as measured by the front-mid electrodes. Theta power indexes the initial automatic process, and its origin is in the anterior cingulate cortex (ACC). The post-fault secondary voltage process induces Pe, which is broad and diffuse compared to ERN and is measured by central parietal electrodes and largely explained by delta power. Frequency analyzes often reveal ERP components by identifying specific theta and delta contributions that help explain the underlying sequence of cognitive functions recorded with ERP. With the dysregulated circuitry associated with these automatic and controlled processes, we hypothesize that the SUD sample exhibits lower ERN theta power (a measure of automatic processing) and Pe delta power (a measure of controlled processing) relative to HC, suggesting a significant impairment in behavioral error recognition and regulation.

Methods:People from (N= 71) i bez (N= 82) LDS performed a Go/NoGo task during ERP recording. We used the 64-channel BioSemi ActiveTwo system for EEG data collection and Matlab, EEGLab, Psychophysiology Toolbox andRto analyze this data. The average amplitudes were calculated in the ERN window, from -30 to 150 ms, and in the Pe window, from 125 to 450 ms, relative to the power supply error. The time-frequency surfaces for the filtered band (3–9 Hz) and delta (below 3 Hz) were calculated, and the principal components (PC) were calculated. Nine electrodes were isolated and ERN and theta power (F1, Fz, F2, FC1, FCz, FC2, C1, Cz, C2) and Pe and delta power (FC1, FCz, FC2, C1, Cz, C2, CP1, CPz, CP2). These means were compared between groups withT-test. To better characterize the relationship between ERN amplitude and theta and delta power, correlation and regression analyzes were computed to elucidate the common and unique contributions of ERN and Pe amplitude from theta and delta computers, respectively.

Results:Compared to the HC group, the SUD group showed a lower amplitude in the Pe windowT(1,151) = 4,17,PI number< 0.01 but no difference in the ERN windowT(1,151) = -0,75,PI number>0.45. Supporting our hypothesis, we identified the SUD group, compared with the HC group, to have less theta power measured during the ERN,T(1,151) = 4,37,PI number< 0.01, and the measured delta power during Pe,T(1,151) =5,28,PI number< 0.01. Theta power was negatively correlated with ERN amplitude for both HCs,R(80) = -0,28,PI number= .01 and LDS groups,R(69) = -0,33,PI number< 0.01 and was positively correlated with the amplitude Pe, [HC]R(80) = ,34,PI number< 0,01, [SUD]R(69) = ,32,PI number<0.01; Delta power was negatively correlated with ERN amplitude, in the SUD group,R(69) = -0,40,PI number<0.01 but not HC group,PI numbers>0.24; two delta PC were correlated with Pe amplitude in both groups, [HC]R(80) = ,78,PI number< 0,01,R(80) = ,71,PI number< ,01; [SUD]R(69) = ,55,PI number< 0,01,R(69) = ,46,PI number< 0.01. Interestingly, significant regressions test the unique theta and delta power contributions explaining the ERN amplitude in HC, R2=0.16,eat(5, 74) = 2,47,PI number= .04, only one PC Theta identified,PI number< 0.03 and in the SUD R2 group = 0.21,eat(5, 65) = 3,52,PI number< 0.01, only one delta computer identified,PI number< 0,02.

Conclusions:Neural ERP correlates of automatic and controlled processes activated during the Go/NoGo task were identified as dysregulated in the SUD group relative to the HC group. Supporting our hypothesis, we measured fewer theta and delta ERNs in the SUD group compared to the HC group. In addition, unique theta and delta contributions to different ERN amplitude groups. The HC group showed the traditional theta power relationship and the SUD group showed a unique delta contribution to the ERN amplitude. Of course, the groups differed in theta power and its relationship to the ERN amplitude, which probably influenced the subsequent controlled process with the Pe index. This means that the dysregulation identified in the Pe window for the SUD group (i.e. lower delta power) starts with a decrease in theta power associated with the ERN, which reflects the dysregulation of the primary automatic process, which is transferred to the dysregulation of the secondary controlled process. These findings suggest that enhancing automated processing, and thus increasing theta power, through targeted interventions has potential for future SUD treatments. Using a circuit-based interventional approach, theta-generating ACC can be targeted by transcranial cortical magnetic stimulation to modulate theta power and potentially dysregulated automation in the SUD. Studies of specific neuromodulation mechanisms targeting this circuit are needed to evaluate it as a potential intervention.

Keywords:Substance use disorder, event probabilities, frequency over time, treatment goals

Revelation:Nothing to disclose.

National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland, USA

Bottom:While nearly 70% of smokers express a desire to quit, less than 10% succeed (1). This poor quitting performance has been linked to nicotine withdrawal syndrome (NWS). Many brain circuits and cognitive constructs are involved in NWS, including cognitive, emotional and reward processing (2-4). Although behavioral evidence suggests that NWS symptoms improve in the first week of abstinence and then subside, a mechanistic understanding of prolonged nicotine withdrawal is lacking.

Methods:As part of a longitudinal study to examine changes in cognitive control and emotional processing as a function of abstinence duration, we hypothesized that cognitive control processing would decrease and emotional processing would increase as a function of abstinence. We also hypothesized that NWS symptoms worsen during acute withdrawal and improve during prolonged withdrawal. Our analysis plan was pre-registered at the Open Science Forum (https://osf.io/xy26j/).

The study protocol included a total of 101 smokers. Functional MRI data were obtained during the parametric flanker task (PFT) and the emotional face task (AMY) at three time points: ad lib smoking (T1), acute abstinence (∼48 h, T2), and prolonged abstinence (~30 days, T4). T4 was only collected from participants enrolled in the quit smoking arm and receiving weekly CBT-based counseling. NWS symptoms were measured using the Wisconsin Smoking Cessation Scale (WSWS) at each time point, along with other psychometric measures including the Positive and Negative Affect Scale (PANAS).

Results:First, we perform an exploratory analysis on a subset (N= 68) dropouts and not seeking treatment who did not remain in the study after T2. Linear mixed-effects modeling analysis for voxels showed no major effect of smoking on AMY or PFT-induced activity. We then extracted 6 task-based ROIs from AMY (visual areas, bilateral fusiform gyrus, right inferior frontal gyrus, bilateral amygdala) and 5 from PFT (bilateral superior and inferior parietal cortex (SPC, IPC), right medial cortex (MCC)) and calculated the effect size of the contrasts of interest in each ROI to determine the limits of the smallest effect of interest (5).

In an unmasked sample of all subjects rated at T4 (N= 19), we tested the presence and absence of a recall effect on task activation in these previously obtained ROIs using a unilateralT-test and double-sidedT- test procedures to check the statistical difference and equivalence between T2 and T1 and T4 and T1, respectively (9). Non-parametric Friedman and Wilcoxon tests were used to assess abstinence-induced behavioral changes in PFT scores and WSWS scores.

PFT-induced brain activity in left SPC and IPC and MCC was significantly reduced in acute withdrawal (PI number’s=0.039, 0.0003, 0.009) and was statistically equivalent to the saturation conditions in the two right parietal ROIs. After long-term abstinence, PFT activation in four of the five ROIs was statistically equivalent to ad lib smoking and was not significantly different or equivalent in the right CPI. The accuracy of the corresponding low- and medium-demand PFT tests was significantly reduced in acute cases (PI numbers = 0.012, 0.05, 0.04), but not in prolonged abstinence, while no differences were found in high-demand studies at any time point. In addition, the error rate of omissions in cognitive demand levels increased significantly after acute withdrawal (PI number= 0.0045) and tended to increase further at T4 (PI number= 0,07).

Statistical equivalence was demonstrated in activation of the AMY task between T2 and T1 and T4 and T1, in 4 out of 6 ROIs. Although no significant difference or equivalence was observed in the bilateral amygdala at any time point. However, positive affect, as measured by the PANAS, decreased at T2 (PI number= 0.004), with a noticeable downward trend in T4 (PI number= 0,06).

Thirst did not differ between T1 and T2, although there was a significant decrease in T4 (PI number= 0.0005). Other symptoms of NWS increased as expected at T2: anger (PI number= 0.041), difficulty concentrating (PI number= 0.008), sadness (PI number= 0.047) and sleep disorders (PI number= 0.014); none differed between T1 and T4.

Conclusions:These preliminary results suggest that withdrawal-induced acute changes in cognitive processing tend to recover from long-term abstinence, also accompanied by a reduction in symptoms of NWS and drug craving. Further analyzes will assess the impact of prolonged nicotine abstinence on emotional processing.

This research was supported by the Intramural Research Program of the National Institute on Drug Abuse (USA) and the Department of Neuroscience, Imaging, Clinical Sciences of the G. d'Annunzio University (Italy).

Bibliographic references:

1. Centers for Disease Control and Prevention, 2020. Health effects of smoking

2.Ashare RL et al., 2014. Cognition during nicotine withdrawal: implications for the treatment of nicotine addiction. Neuropharmacology 76:581-91

3. Fedota JR et al., 2021. Time-variable functional connectivity decreases with acute nicotine withdrawal. Biol Psychiatry Cogn Neurosci Neuroimag 6: 459-469

4. Koob GF (2009) The dynamics of neural circuitry in addiction: reward, anti-reward, and emotional memory. Pharmacopsychiatry 42 Suppl 1: S32-S41

5.Lakens D et al., 2018. Equivalence Tests in Psychological Research: A Tutorial. Advances in Methods and Practices in the Psychological Sciences 1: 259-269

Keywords:Nicotine addiction, cognitive and affective neuroscience, flanker task, emotional facial processing, smoking cessation

Revelation:Nothing to disclose.

University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Bottom:The concomitant use of cocaine and alcohol is the leading cause of emergency hospitalization, highlighting the vital need to understand the mechanistic basis of this highly addictive and dangerous drug combination. Both cocaine (a dopamine, norepinephrine, and serotonin transporter inhibitor, DAT, NET, and SERT, respectively) and ethanol (EtOH) increase extracellular dopamine (DA), norepinephrine, and serotonin. However, we and others found no evidence of EtOH interacting with DAT, NET or SERT, suggesting that EtOH may be acting elsewhere to inhibit the uptake of these monoamines. Organic cation transporter 3 (OCT3) is emerging as an important player in the regulation of monoamine signaling. However, it remains unclear whether OCT3 contributes to the drug's effects, which work mainly by increasing extracellular levels of monoamines.

Methods:To prove the validity of this concept, we conducted studies to determine the ability of EtOH to inhibit the uptake of the [3H]1-methyl-4-phenylpyridinium protocation ([3H]MPP + ) in cultures of HEK293 cells stably overexpressing human OCT3 and found an IC50 of 4 mM, confirming the role of OCT3-dependent effects of EtOH and showing that concentrations of EtOH reaching the brain after behaviorally relevant doses may be sufficient to engage OCT3. We have previously found that cocaine has no effect on OCT3 (Mayer et al., 2018, PMID: 29773909), raising the possibility that EtOH may interact with OCT3 to inhibit monoamine uptake, possibly enhancing its addictive properties. these drugs. To this end, we used rapid chronoamperometry to study the effect of topical application of EtOH, cocaine and their combination on the clearance of exogenously applied DA from the extracellular fluid in the striatum of male and female OCT3+/+ and OCT3-/- mice in Vivo. In addition, we conducted behavioral experiments to assess conditioned place preference in response to administration of EtOH, cocaine, or both, to determine whether co-administration of EtOH and cocaine increases place preference compared to administration of each drug alone. All experimental protocols in animal studies have been approved by the Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals

Results:We found that in both men (N= 11-12/dose) and women (N(N= 8-10M and 8-10F/dose) mice. Behavioral data in men (N= 16/dose) of OCT3+/+ mice revealed that the combination of EtOH (1000 mg/kg) and cocaine (3.2 mg/kg), doses that do not induce conditioned place preference (CPP) by themselves, produce strong CPP. In OCT3+/+/+ mice, EtOH did not produce CPP, while cocaine did. In addition, cocaine CPP in women was enhanced by co-administration of EtOH. CPP studies in OCT3-/- mice are ongoing (N= 7M and 7F/dose), but the development of CPP has not yet been demonstrated, regardless of treatment.

Conclusions:Taken together, the results suggest that the enhancement of cocaine's neurochemical and behavioral effects with EtOH is dependent on OCT3. OCT3 may be a putative target for therapeutic intervention in the treatment of EtOH and cocaine abuse.

Keywords:Alcohol and substance use disorders, cocaine use disorders, organic cation transporters

Revelation:Nothing to disclose.

Salk Institute for Biological Studies, La Jolla, California, United States

Bottom:An individual's position in the social hierarchy is inversely related to alcohol consumption in rodents and primates, and to problem drinking in humans, highlighting the conservative influence of subordination on alcohol motivation. Social status also influences how individuals respond to challenges such as social isolation, a particularly profound stressor of increasing importance to humans. However, the neural circuitry mechanisms by which experiences of social stress trigger inappropriate alcohol consumption remain largely unknown.

Methods:In this study, we combined behavior, ex vivo whole cell electrophysiology, in vivo calcium imaging with cell analysis, and machine learning to identify circuits underlying social sorting and isolation effects in alcohol consumption.

Results:We found that the social position of adult male mice correlated with alcohol consumption (r2=0.25 by Pearson correlation; **PI number= 0,01;N= 26), and social isolation increases alcohol consumption (***PI number<0.001 per pairT-test;N= 14). Specifically, we identified a previously unknown association between prior social status and social isolation-induced alcohol consumption, with subordinates showing greater increases in alcohol consumption compared to dominant individuals (**PI number< 0.01 per uncoupledT-test;N= 3/group). These data suggest that behavioral factors resulting from dominance hierarchies may predict vulnerability to drinking due to social isolation. Using whole-cell ex vivo patch-clamp electrophysiology, we found that social isolation increased basolateral amygdala (BLA) excitability (*PI number< 0.05 by two-way ANOVA.N=   13–14 cells), highlighting the susceptibility of BLA to social isolation. Unilateral photoactivation of ChR2-expressing BLA terminals in mPFC increased alcohol (YFP: 138.5 licks and ChR2: 392.5 licks, ***PI number< 0.001 by two-way post hoc ANOVA.N= 6/group), but not sucrose or water, drinking, suggesting that social isolation may increase alcohol consumption via BLA projections to the mPFC. In fact, using single-cell calcium imaging, we found that BLA encodes alcohol (N= 180–109 cells), and the percentage of alcohol-coding neurons increases after social isolation (from 9.5% to 18.3%). Using the SVM classifier, we found that population-level BLA activity prior to binge drinking accurately decoded (>70%) whether alcohol or water was consumed, further supporting the role of BLA in alcohol coding.

Conclusions:Together, these findings suggest that low social status may be a strong risk factor for increased alcohol consumption after the challenge of social isolation, which may be mediated by corticobody circuits.

Keywords:Alcohol, social isolation, circuits

Revelation:Nothing to disclose.

University of Colorado, Anschutz Medical Campus, Aurora, Colorado, United States

Bottom:Opioid use disorder remains a major global health challenge requiring the development of more effective treatment strategies and a better understanding of the neural circuits that drive opioid use and relapse. Neuronal activity in the ventral pallidum (VP) is critical for opioid reward and opioid-seeking relapse induction, but the exact neural mechanisms mediating these behavioral states are not fully understood. The large population of VP neurons associated with drug relapse are characterized by the expression of the calcium-binding protein parvalbumin (PV). Throughout the brain, photovoltaic neurons are covered by specialized extracellular structures known as perineural networks (PNNs). However, despite the dense expression of PNNs in the PV, the role of these structures in the physiology of PV neurons and the search for opioids remains unknown.

Methods:To investigate the role of PNN in VP in opioid relapse, male and female mice (N= 17) were surgically implanted using indwelling jugular catheters and chronic cannulas over the PV. Mice were trained to pick up heroin in their nose (and a heroin alert light was delivered simultaneously) according to a fixed reinforcement schedule (FR1, 8d) and then progressed to variable rate schedules (VR3 and VR6, 3 days each), to reliably assess the acquisition of heroin reception. Mice then underwent extinction training, followed 24 hours later by microinjection of PNN ABC chondroitinase-depleting enzyme (ChABC) or vehicle (0.1% BSA) into the PV. The following day, relapse to drug seeking was assessed by a signal-triggered recall test, after which mice were injected to deplete VP PNN. Experimental procedures followed the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the University of Colorado, Anschutz Medical Campus Institutional Animal Care and Use Committee (IACUC).

Results:In the VP, most PNNs are located around PV neurons, but a subset of PNNs are associated with distinct, yet uncharacterized neurons. ChABC microinjection into the VP completely abolished the PNN in the area of ​​the microinjection needle. Compared to vehicle, microinjection of ChABC into the PV reduced cue-induced heroin reinstatement by selectively reducing active burn-in for heroin cues (two-way ANOVA, main effects of reinstatement:eat(1,15) = 20,33,PI number= 4.16 × 10^-4, treatment:eat(1,15) = 5833,PI number= 0.029 and re-interact x healing:eat(1,15) = 5837,PI number= 0.029) but did not cause any change to the inactive strings. Ongoing experiments characterize the role of PNNs in the physiology of VP neuronal subtypes, including the activation of PV neurons during relapse with or without PNN exhaustion.

Conclusions:These results confirm that PV is a critical regulator of opioid relapse and extend our understanding of the opioid addiction pathway by showing that heroin seeking requires intact PNNs in the PV. Given the importance of PNN in maintaining memory and behavioral plasticity, therapies aimed at modulating the structure of PNN may represent a useful new avenue for the treatment of opioid dependence.

Keywords:Heroin, ventricular pallor, perineural networks, parvalbumin neurons, relapse

Revelation:Kaleidescapes, LLC: founder (husband)

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:Irritability, a low threshold for anger and frustration in response to stress, is a common but understudied feature of many mental disorders, including cocaine use disorder (CUD). Recent reports have linked irritability to social stress and poorer psychosocial functioning, lower quality of life, and persistently increased suicidal ideation in adults with mood and/or substance use disorders. However, little is known about the impact of childhood trauma on irritability symptoms among people with CUD (iCUD). Furthermore, childhood trauma and CUD are associated with heightened immune states involving neuroimmune interactions with long-term effects on psychopathology, but how this dysregulation relates to irritability symptoms remains poorly understood. Our goal was to characterize irritability in CUD and elucidate the relationship between irritability, childhood trauma, and immune dysregulation in this population.

Methods:Clinical data were compared between 31 iCUD and 32 healthy controls (HC), who did not differ in age, sex, race and IQ, along with blood serum for analysis of inflammatory cytokines, chemokines and growth factors. A new composite measure of irritability, including the irritability dimension as both a trait and a state, was created using irritability-related items from the Emotion Regulation Difficulty Scale, the Cocaine Selective Severity Rating and the Perceived Stress Scale. The psychometric properties of this agent were tested using exploratory factor analysis and convergent validity through correlations between state and trait measures of anger, anxiety, and depression. In univariate analyses, we assessed the association between this new measure of irritability and measures of childhood trauma, life experiences, and levels of immune markers.

Results:In exploratory factor analysis, five irritability-related items were loaded into one factor (first eigenvalue = 2.44, others <0.16). The composite measure of irritability was strongly correlated with measures of anxiety (rs = 0.61,PI number< 0.0001) and trait anger (rs = 0.56,PI number< 0.0001). Irritability levels were significantly higher in the iCUD group compared to the HC group (CohenHello= 0,59;PI number= 0.02). Early life adversity of childhood emotional abuse, sexual abuse, and physical neglect were significantly associated with irritability in both groups (rs=0.34–0.41, allPI number< 0.01). Among HCs, there was a varied relationship between irritability and total negative life experiences (rs = 0.40,PI number= 0.028), but not in iCUD (rs = -0.01,PI number= 0.98). Among tests of immunological factors, macrophage-derived chemokine (MDC/CCL22) was associated with higher levels of irritability (rs=0.28,PI number= 0.026) and sexual abuse (rs = 0.244,PI number= 0.058, trend) between groups.

Conclusions:Using a composite measure of irritability, we found that childhood trauma and CUD were significantly associated with irritability. However, a differential relationship between irritability and overall negative life experiences has only been observed in CH, and the mechanisms behind this phenomenon will be explored in future analyses. People with CUD had a state of increased immune activation of MDC/CCL22, a pro-inflammatory cytokine involved in post-traumatic conditions, which is associated with irritability. These findings indicate a potential link between immunological, social, and psychopathological factors in cocaine addiction, providing a basis for future causal testing. Delineating the regional substrates of stress-related inflammatory signatures in relation to psychosocial conditions and psychiatric symptomatology in CUD may accelerate the development of new treatments to enhance efficacy and recovery.

Keywords:Irritability, cocaine use disorder, immune biomarkers, childhood abuse exposure, neuroimmune interactions

Revelation:Nothing to disclose.

Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil

Bottom:Early life stress (ELS) is an important environmental risk factor for cocaine use disorder (CUD). Due to the high plasticity of the brain during early development, exposure to ELS can lead to long-term changes in brain structure and increase susceptibility to CUD later in life. They seem to be more prominent in areas of the brain related to motivation, impulsivity and inhibitory control, such as the cingulate areas. We therefore studied the effect of CUD on the thickness of 4 sky regions in both hemispheres and checked whether ELS history could influence the effects of CUD.

Methods:Participants were 78 patients with CUD and 53 healthy participants (HC) without CUD or neuropsychiatric diagnosis. Magnetic resonance imaging (MRI) of annular thickness (anterior caudal, anterior caudal, posterior, and isthmus) was performed 2 weeks after admission to the rehabilitation department using a 3T Signa GE scanner. The Childhood Trauma Questionnaire (CTQ) was used to assess ELS and the Addiction Severity Index (ASI) was used to assess drug use patterns (alcohol, tobacco, marijuana and cocaine). Tooth regions with significant group differences and significant associations with CTQ score were entered into a mediation model where lifetime cocaine use, CTQ score, and tooth thickness were the independent variable, mediator variable, and outcome, respectively. All statistical analyzes were controlled for age and gender.

Results:All tooth areas in both hemispheres were significantly less thick in the CUD group compared to the HC group.PI number-values < 0.05). Moreover, the CUD group had a higher CTQ score than the HC group. Significant negative correlations were found between the CTQ score and the cylindrical isthmus (right hemisphere) and the anterior part of the spine (left hemisphere). In the mediation analysis, we found a significant negative direct effect of cocaine use on the cylindrical isthmus (b = -0.005, 95CI [-0.008, −0.002]) and an indirect negative effect of cocaine use on the cylindrical isthmus mediated by the CTQ score (b = -0.001, 95CI [ -0.002, -0.0001]), indicating partial mediation. Specifically, 17.5% of the model's total effect was explained by ELS-mediated indirect effects. Additionally, a marginal effect of sexual interaction was observed (PI number= 0.055). For the anterior vertebral crown, ELS had no significant indirect effect on thickness (b=-0.0004, 95IC [-0.001, 0.0004]).

Conclusions:We show that ELS is associated with CUD and together mediates the effect of CUD on reducing the thickness of the cylindrical isthmus in the right hemisphere. This supports the role of ELS as a key environmental stressor influencing brain developmental pathways related to drug addiction.

Keywords:Cocaine use disorders, brain structure, stress in early life

Revelation:Nothing to disclose.

University of Colorado, Anschutz Medical Campus, Aurora, Colorado, United States

Bottom:The paraventricular thalamus (PVT) has recently been identified as a key component of drug addiction neural circuits in humans and rodents. The PVT receives input from the cortical areas of the brain and has extensive connections to the subcortical areas. In particular, PVT is a major source of glutamatergic inputs to the nucleus accumbens (NAc), a region known to play a key role in drug-seeking relapses. One of the most intriguing roles of the PVT →NAc pathway is its ability to drive aversive bodily states experienced during opioid withdrawal, which can lead to relapse. We recently found that the PVT → NAc pathway is required for heroin relapse after withdrawal, but not after extinction training. We hypothesize that this pathway may be a substrate by which cognitive behavioral therapy (e.g., extinction training) may attenuate opioid withdrawal and thus reduce heroin seeking. We first tested the hypothesis that acute withdrawal from self-administered heroin induces mechanical hyperalgesia. We next investigated whether hyperalgesia is ameliorated by extinction training. We next tested whether chemogenetic inhibition of the PVT → NAc pathway could mimic extinction-induced analgesia during heroin withdrawal. We show that extinction, but not withdrawal, from heroin self-administration causes a loss of synaptic plasticity in NAc D1 but not D2 expressing medium spiny neurons (MSNs) that receive input from the PVT. In addition, chemogenetic inhibition of the PVT → NAc pathway reduced heroin relapse after withdrawal, but not after extinction training. We therefore implemented an in vivo long-term depression (LTD) protocol utilizing optogenetic stimulation of PVT terminals in the NAc in an attempt to recapitulate extinction-induced loss of synaptic plasticity in this pathway and thus reduce heroin relapse in abstinent rats. Fiber photometry was used to validate the in vivo LTD protocol by simultaneously stimulating PVT tips and recording NAc calcium activity from the same fiber in the same animal.

Methods:Our experimental procedures followed the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the University of Colorado, Anschutz Medical Campus Institutional Animal Care and Use Committee (IACUC). For the NAc chemogenetic experiments of male and female mice (N= 23), AAVrg-Cre was bilaterally injected and PVT was injected with AAV2-hSyn-DIO-hM4D(Gi)-mCherry. Other behavioral data from this cohort are published in Cell Reports (Giannotti et al., 2021, in press). Another group of male and female rats (N= 5) was used for in vivo LTD experiments. NAc was bilaterally injected with AAV9-hSyn-GCaMP6f and PVT was injected with AAV9-hSyn-ChrimsonR-tdT. Fiber optic cannulas were implanted bilaterally into the NAc. Rats were self-administered heroin for 12 days, and hyperalgesia was assessed after 14 days of abstinence (7 days of extinction or 14 days of home cage abstinence) using an electronic Von Frey apparatus, 15 minutes after J60 (0.1 mg/kg bw) or pre-treatment vehicle treatment (H2O). Another group of mice (N= 5) received bilateral stimulation in vivo according to the LTD optogenetic protocol (638 nm, 10 mW, 1 Hz - 15 min) or no stimulation, after 14 days of home cage abstinence, 45 min prior to relapse testing. Optogenetically induced calcium events (638 nm, 20 Hz - 1 s) were recorded every 5 minutes before (baseline pre-LTD; 20 min) and after (post-LTD; 40 min) the in vivo LTD optogenetic protocol. Chemogenetic experiments were analyzed using a two-way RM ANOVA followed by Fisher's LSD post hoc test. Fiber photometric data was analyzed using a duplex pairT-test.

Results:Withdrawal (14d) from self-administered heroin causes mechanical hyperalgesia (2-way RM ANOVA, treatment:eat(2,42) = 59,79,PI number<0.001; treatment x method of withdrawal:eat(2,42) = 3,437,PI number= 0.0415) in both the extinction and withdrawal groups compared to pre-heroin baseline (PI numbers < 0.001). However, extinction training significantly reduced heroin withdrawal-induced hyperalgesia compared with the withdrawal group (withdrawal vs. extinction:PI number= 0.0447). Finally, chemogenetic inhibition of the PVT➝NAc pathway after abstinence reduces hyperalgesia to levels similar to those seen after extinction training (withdrawal vs extinction:PI number= 0.8311), suggesting that extinction may alleviate opioid withdrawal by reducing activity in this pathway. In addition, our fiber photometry data indicate that the in vivo LTD protocol reduces the peak amplitude (PI number= 0.0055; in pairsT-test) and area under the curve (0-2 sec.PI number= 0.0093; in pairsT-test) of optogenously induced calcium responses in NAc MSNs. It should be emphasized that due to the size of the sample, these are preliminary data (N= 5) and pending histology. Therefore, we cannot conclude whether this in vivo LTD protocol is able to reduce relapse rates in non-heroin deprived rats.

Conclusions:Here we show that the PVT → NAc pathway promotes heroin withdrawal-induced hyperalgesia after withdrawal but not after extinction training. In addition, we successfully used fiber photometry to validate the in vivo LTD optogenetic protocol in the PVT→NAc pathway, as several parameters of calcium transitions indicate reduced synaptic activity in this pathway. Future studies will increase the sample size of this initial experiment and investigate whether the same in vivo LTD protocol can alleviate heroin withdrawal-induced hyperalgesia and subsequent relapse.

Keywords:Heroin self-administration, opioid withdrawal, hyperalgesia, chemogenetics, fiber photometry

Revelation:Nothing to disclose.

UC San Diego, La Jolla, California, United States

Bottom:A key question for cocaine addiction research remains the question of why occasional drug use turns into problem use associated with high motivation and compulsiveness, which characterizes substance use disorders in some individuals and not in others. Better characterization of individual differences in propensity to develop addiction-like behaviors could help identify new pharmacological targets, biomarkers, genetic variants and facilitate drug development. However, most studies used small sample sizes (N<8–20), examples of self-administration with limited access, or animal models with limited individual differences. Such limitations reduce the translational relevance of the results and limit the reproducibility and reproducibility of these studies. To address this issue, we characterized addiction-like behaviors with an advanced model of extended-access cocaine self-administration in large cohorts of heterogeneous rats (HS), a unique strain of rats with large individual differences.

Methods:HS mice (N= 567 with 275 females and 292 males) were allowed self-administration of cocaine under the expanded access model (2 weeks short duration 2 hours per day, 3 weeks long duration 6 hours per day, and 1 week additional trials). Animals were also tested for compulsive cocaine use using response and progression rate (PR) despite aversive consequences (potential shocks) and withdrawal-induced irritability-like behaviors (bottle brush test). Large cohorts (N= 46–60) was used to minimize group-specific effects, and response rates were normalized across cohorts.TTests and ANOVAs were conducted to determine significant effects. With so many animals, small effect sizes (Cohen'sHello= 0.2) can be detected with high power (1-B = 0.95, a = 0.05).

Results:Behavioral characterization showed large inter-individual differences in addiction-like behaviors, with little intra-individual variation. Although large gender differences were observed with women having a higher response rate (FR) (scaling of consumption,PI number< 0.01) and responsiveness despite foot shock (force,PI number< 0.001) than men, differences between individuals were significantly greater than between the sexes alone. Final cocaine intake showed a bimodal distribution, with 80% of the susceptible rats increasing their cocaine intake in the expanded access protocol and 20% of the resistant rats maintaining low consumption. In addition to scaling (PI number< 0.001), these groups also showed significant differences in FP response (motivation,PI number< 0.001) and coercion (PI number< 0.001), but not in deprivation-induced irritability. Principal component analysis of intake, motivation, compulsion, and abstinence showed that climbing, motivation, and compulsion clustered in the first major component (PC1), which explained 48% of the variance (eigenvalue > 1,R= 0.52 to 0.60). Irritability-like behavior was orthogonal to PC1 (R= 0.008). The addiction index was calculated by averaging the Z scores of the three dependent variables as the PC1 proxy for a comprehensive assessment of compulsive cocaine use. The addiction index was then used to classify vulnerable animals as resistant, mild, moderate, or severe addictive behaviors, with significant differences in scale.PI number< 0.001), motivation (PI number< 0.001) and coercion (PI number<0.001). The dependency rate cannot be predicted from behavioral measurements taken during short access sessions (FR, PR, latency time, loading phase intake (first 15 min), titration phase intake (last 60 min) and interval between injections, allR< | 0,12 | ,PI number= ns). Behavioral measures during the expanded access session progressively predicted an addiction-like phenotype starting on day 5 of drug intake (R= 0,18,PI number< 10-5) do 14 (R= 0,47,PI number<10-6). RP answers (R= 0.62), charging phase (R= 0.49), titration phase (R= 0.57), intervals between injections (R= −0.25) and the number of responses during the shock session (R= 0.22) also predicted the dependence factor (allPI number< 10-6).

Conclusions:To our knowledge, this is the largest study of intravenous cocaine self-administration in rats. We identified individuals with behaviors similar to resistant, mild, moderate and severe cocaine addiction in over 500 heterogeneous rats. Behavioral responses using the limited access model did not predict the development of addiction-like behaviors, highlighting the need for a chronic extended access model to better understand and predict addiction-like behaviors. Along the way, a wide variety of biological samples were collected and made available to the community through the Cocaine Biobank to facilitate the identification of biomarkers of addiction-like behavior and to facilitate repeatability and reproducibility.

Keywords:Psychostimulants, relatives of rats, substance use disorders

Revelation:Nothing to disclose.

National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, USA

Bottom:The diverse and far-reaching impact of the COVID-19 pandemic continues to unfold globally across individuals, communities, healthcare systems and the economy. Stress, social isolation, financial loss and uncertainty can significantly affect mental health and alcohol consumption. Therefore, it is of utmost importance to prospectively assess the impact of the pandemic on alcohol consumption and related behaviors and outcomes. To this end, we identified a group of participants who had previously participated in NIAAA clinical trials and invited them to participate in a longitudinal study to assess the impact of the pandemic on alcohol consumption and consequences. Specifically, we aimed to explore pandemic-related changes in alcohol consumption and underlying determinants such as alcohol craving, negative life events, and risk for alcohol use disorders.

Methods:Participants in previous NIAAA clinical trials covering the entire spectrum of alcohol use were contacted by telephone to participate in the study. After providing consent, participants completed a baseline survey to obtain pre-pandemic baseline and impact data. Participants then complete online or telephone surveys at weekly to semi-annual intervals for 2 years. Assessments include the Alcohol Use Disorder Identification Test (AUDIT) and questionnaires assessing alcohol craving (Penn Craving Scale), anxiety (Perceived Stress Scale), and negative life events (Life Events Questionnaire). Participants also complete the COVID-19 Scale, based on CDC and WHO questionnaires, which assesses the impact of the pandemic on medical and mental symptoms and behaviors, and stressors (work, family, finances, etc.). Responses from the COVID-19 scale were used to derive the COVID-19 Stress Score. Due to ongoing pre-reporting, impact surveys have been collected during different "waves" of the pandemic: Wave 1: March-July 2020, Wave 2: August-November 2020, Wave 3: December 2020 and beyond.

Results:In the period from June 2020 to March 2021, we registered 386 participants (180eat/206 M), in 3 groups: People treated with AUD (N= 116), AUD without seeking treatment (N= 54) and controls without AUD (N= 216). Main effect data analysis showed wide variation in pandemic-related changes in alcohol consumption, with 31% of participants reporting an increase, 32% reporting a decrease, and 37% reporting no change in AUDIT-Consumption scores. The magnitude of the change in AUDIT scores was inversely related to the pre-pandemic AUDIT score. COVID-19 stress scores showed significant racial differences, with the Black/African American group reporting greater financial stress related to COVID-19. Overall, greater COVID stress was associated with higher AUDIT scores. Additional analysis focused on group differences defined by pandemic-related changes in AUDIT scores. The group that showed increases in AUDIT had significantly higher measures of perceived pre-pandemic stress, negative life events and hunger compared to the group that showed decreases in AUDIT and no change in AUDIT scores (allPI numbervalues ​​ < 0.05). Predictors of increases and decreases in AUDIT scores were assessed using separate general linear models and included changes in negative life events, perceived stress and hunger, pre-pandemic AUDIT score covariance, AUD diagnosis, race, gender, and pandemic wave. The results showed that increases in negative life events and hunger were significantly associated with increases in AUDIT scores, while decreases in negative life events were significantly associated with decreases in AUDIT scores.

Conclusions:The results of this study indicate high heterogeneity and bidirectional changes in alcohol consumption associated with the pandemic. Changes in AUDIT scores were due to pre-pandemic alcohol consumption and were associated with changes in negative life events and famine. Further longitudinal data collection and analysis in this sample of individuals will extend these early changes in AUD consumption patterns, hunger and risk, and the role of COVID-related stress in these changes. The results will help identify potential targets for interventions to help people who are vulnerable after the pandemic and other major public health emergencies.

Keywords:Alcohol use disorder, COVID-19 pandemic, perceived stress, hunger

Revelation:Nothing to disclose.

National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, Stany Zjednoczone

Bottom:Blink rate has been proposed as a biomarker of the dopamine system that may be valuable in detecting and monitoring related neuropsychiatric disorders (e.g., SUD, Parkinson's disease) 1,2. Findings regarding the relationship between blink rate and dopamine receptors (DRD1, DRD2) are inconsistent3. Direct D1 and D2 receptor agonists may independently increase blink rate in primates4,5, but indirect agonists (e.g. cocaine) may decrease blink rate6. Simultaneous stimulation of D1 and D2/3 receptors by indirect agonists (e.g. cocaine or methylphenidate) may lead to variable effects on blink rate. Here, we investigate the dopaminergic basis of eye blink rate using a combination of resting eye tracking as well as PET measurements of DRD1 and DRD2 availability (following methylphenidate (MP) challenge).

Methods:PET scans of [11C]raclopride and [11C]NNC112 were performed on 32 healthy subjects (20-50 years old) on one of two scanners: High Resolution Investigation Tomography (HRRT) (N= 16; 7 women; Siemens AG; Germany) or CV PET/CT scanner (N= 16; 5 women; Siemens AG; Germany). Patients were scanned once with [11C]NNC112 (15 mCI, 90 min dynamic imaging) for DRD1 assessment at baseline (i.e. without drug challenge) and twice with [11C]raclopride on separate days: once 1 hour after dosing oral placebo (PL) to assess baseline D2/3 dopamine receptor availability and once 1 hour after 60 mg oral methylphenidate (MP) to assess changes in DA (single-blind series of counterbalanced sessions, 10 mCi, 60 min dynamic imaging) . The [11C]NNC112 scan was performed (10am) before the [11C]raclopride scan (13:00) on the same day or on different days. The binding potentials of DRD1 and DRD2 (BPnd) were calculated using a simplified reference tissue model using Magic Pipeline 7. Eye Tracking: Resting blinks were counted (approximately 2 h after PET and 3 h after PL or MP. We used a long-term ASL series of motion tracking cameras LRO eyeballs and ET7 software system (sampling rate = 120 Hz). For blink detection, the blink duration was set to a minimum of 0.1 s and a maximum of 0.4 s. Interfering eye-tracking sessions were eliminated, resulting in 25 tracking sessions (15 male, 10 women) from MP days and 17 eye-tracking sessions (10 men, 7 women) from PL days; 16 people had access to both MP and PL eye-tracking MRI: structural scans were collected before fMRI to isolate individual parts freesurfer brain, which were used to designate the putamen, caudate nucleus, and ventral striatum as ROIs.

Results:Subjects blinked less frequently and kept their eyes open longer during MP than PL (% of resting eyes closed: PL=%21.2 (15.1), MP=%4.06 (5.3), pairs -T(15) = 5,1,PI number<0.001; Blink rate (blinks/s): PL=0.56 (0.23), MP=0.42 (0.29), paired-T(15) = 2,48,PI number< 0.05). DRD2 BPnd in putamen (not caudate or SV) was positively correlated with blink rate in the MP session but not in the PL session (N= 25,R= 0,69,PI number<0.001). The baseline DRD1 BPnd in the caudal tip was positively correlated with the blink rate in the MP session, but not with the PL (N= 25,R= 0,53,PI number< 0.01) and negatively correlated with MP-induced changes in blink rate (PL-MP) (N= 16,R= -0,69,PI number< 0.01), which were also negatively correlated with DRD1 in the putamen (R= -0,56,PI number< 0.05) ROI. D2R availability in the striatum was significantly reduced by MP (reflecting the increase in DA) for the whole group (N= 32) (1 tailT-tests, tailedT= 3,31,PI number< 0.01 is badT= 7,27,PI number< 0.01), MP-induced "dopamine surges" were not associated with MP-induced changes in eye blink rate.

Conclusions:Our findings are consistent with a regulatory role for DA in firing rate, but point to distinct roles for DRD1 than DRD2 in the striatal regions. In particular, we showed that the effect of MP on eye blink rate depends on the baseline DRD1 but not on the baseline DRD2/3. Participants with low DRD1 in the putamen and caudate nucleus had a greater reduction in blinking from MP. Our findings also indicate that the associations between dopamine receptors and eye blink rate likely arose when D1R and D2/3R were simultaneously stimulated.

Keywords:Dopamina, DRD1, DRD2, flash, metylofenidat

Revelation:Nothing to disclose.

National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, USA

Bottom:Alcohol use disorder (AUD) is the most common substance use disorder, with 3.3 million deaths worldwide each year. Taste and smell are reportedly impaired in people with AUD, and some studies have shown that people with severe AUD suffer from an inability to distinguish between taste and smell. The purpose of this analysis was to investigate reports of possible chemosensory dysfunction in AUD in an ongoing COVID-19 impact longitudinal study. This was important because the COVID-19 pandemic had a significant impact on many factors related to AUD, including access to alcohol and changes in social and environmental variables (e.g., social isolation and access to health care).

Methods:Analyzes were performed on taste and smell measurements from an ongoing COVID-19 longitudinal impact study that began in June 2020. As there were only nine participants who tested positive for COVID-19, we chose to exclude data from these participants to focus on the impact taste and smell of alcohol consumption in the context of the COVID-19 pandemic. Of the 307 participants with available diagnoses, there were 140 AUD (89 men/51 women, 46.4 ± 13.5 years) and 167 controls without AUD (75 men/92 women, 43.4 ± 14.5 years). All participants were asked to complete a series of online surveys over 24 months. Taste and smell data were recorded using an online VAS (0-100: higher score, better sense of taste/smell). Specifically, in this analysis, we examine three waves of the pandemic. This allowed us to look at the different points of onset of the pandemic and the evolution of symptoms over time. The three COVID waves were defined as (i) Early Pandemic (March 11 to July 31, 2020), (ii) Mid Pandemic (August 1 to November 22, 2020), and (iii) Late Pandemic (August 23, 2020) .) ). from November 2020). We hypothesized that there would be a decline in taste and odor scores related to drinking behavior during different waves of COVID. Clinical Tracks#NCT04391816

Results:We analyzed mean differences in olfactory and taste scores in the AUD and non-AUD groups during the three waves of COVID. Contrary to our hypothesis, AUD participants showed an increase in olfactory scores at week 4 from baseline (PI number= 0.05) at the beginning of the pandemic. Whereas in non-AUD participants, we observed a decrease in the average odor rating in the midst of the pandemic at week 8 from baseline. Additionally, at baseline and week 4, we observed that participants with AUD had a significantly higher mean olfactory score compared to those without AUD (PI number< 0.05). However, this difference was only observed at the beginning of the wave of the COVID pandemic. This result indicates an increase in olfactory perception in people with higher alcohol consumption. A significant decrease in the average taste score of people with AUD was observed at different time points from the onset during different waves of COVID: (a) mid-pandemic data revealed a decrease in the average taste score at weeks 4, 8 and 12 (PI number< 0.01), (b) while late pandemic data showed a decrease in mean taste rating at week 12 compared to baseline (PI number< 0,01).

Conclusions:Some of the previous literature on changes in olfactory perception in heavy drinkers has reported conflicting results. While some have reported impairment of olfactory perception with increasing consumption, others have reported preserved olfactory abilities. Preliminary results provide evidence of increased complications associated with COVID-19 signs and symptoms, including changes in taste perception in those with higher alcohol consumption. Interestingly, we observed significantly higher mean odor and taste scores after six months in the AUD group compared to the non-AUD group. This was only observed during the late COVID wave. Since approximately 80% of AUD patients sought treatment, the higher olfactory and taste scores at the end of the study period may reflect the effect of the treatment these participants were receiving. Limitations of the study included self-report bias, small sample size due to attrition, and lack of psychophysical measures to provide an objective assessment of taste and smell. As we demonstrated in our meta-analysis (Hannum et al. 2020), objective measurements are a more sensitive method of detecting loss of smell and taste due to SARS-CoV-2 infection. As part of our future direction, we plan to add objective chemosensory measurements to this ongoing study when participants can return to NIH. To better understand the observed changes in olfactory scores, we planned to analyze how the smoking status of subjects in both the AUD and non-AUD groups affected gustatory and olfactory scores. Additionally, we recognize that there were unique social and environmental variables among the different waves of COVID, such as individuals with limited access to alcohol and at risk of alcohol withdrawal. Therefore, we also plan to build on our findings and explore other COVID-related factors, including alcohol consumption patterns among people with AUD.

Keywords:AUD, taste, smell, COVID-19

Revelation:Nothing to disclose.

Binghamton University, Binghamton, New York, USA

Bottom:Alcohol use disorder (AUD) is one of the leading preventable causes of death worldwide. The central amygdala (CeA) acts as a unifying center for negative emotional responses related to anxiety, stress, and AUD. The norepinephrine (NA) system mediates stress responses, and its dysfunction is associated with craving and relapse risk in abstinent alcoholics.

Methods:Here, we investigated whether alcohol dependence (ethanol) and withdrawal (abstinence) alter amygdala noradrenergic regulation in rodents. Male rats were housed under control conditions, chronically intermittently exposed to ethanol vapor (CIE) to induce addiction, or withdrawn from CIE for two weeks, and ex vivo electrophysiology, in situ hybridization, biochemistry (Quantification of catecholamines by HPLC system). and behavioral, site-specific brain pharmacology studies were performed.

Results:We found that NA increased basal GABA CeA release to 212.6 ± 24.0% of baseline in 9 out of 15 cells [T(8) = 4,69,PI number< 0.01 per sampleT-test] by α1-adrenergic receptors in control rats, and this relationship recruits β-adrenergic receptors to inhibit NA release. Despite functional recovery from the effects of NA on GABA signaling after 2 weeks of withdrawal, CeA cells expressing α1 and β1 receptor RNA are still impaired. In addition, the withdrawal significantly reduced the concentration of NA in CeA. We then used CeA microinjections of prazosin or propranolol to determine the role of these receptors in alcohol consumption in independent (moderate alcohol consumption) and dependent (high alcohol consumption) rats. We found that α1 receptors mediate low to moderate levels of alcohol consumption associated with independent rats.PI number< 0.05 vs. vehicle according to Sidak's post hoc test after two-way ANOVA with independent vs. Department:eat(1,26) = 4,98,PI number< 0.05; Prazosyna:eat(3,78) = 5,79,PI number<0.01; rat x dose interaction:eat(3,78) = 2,46,PI number> 0.05], while β-receptor activity leads to excessive alcohol consumption depending on [PI number<0.001 vs. vehicle with Sidak's post hoc test after two-way ANOVA without depth vs. Department:eat(1,26) = 12,80,PI number< 0,01; propranolol:eat(3,78) = 6,86,PI number<0.001; rat x dose interaction:eat(3,78) = 2,65,PI number> 0,05] .

Conclusions:Thus, CeA α1 and β adrenergic receptors are the major neural substrates of AUD. The identification of these novel and complex mechanisms as important factors influencing alcohol consumption, especially in the state of alcohol dependence, could lead to promising ongoing drug development.

Keywords:Alcohol addiction, noradrenergic system, GABA, central amygdala

Revelation:Nothing to disclose.

UCLA, Los Angeles, California, United States

Bottom:The self-medication hypothesis suggests that individuals use psychoactive drugs to treat psychiatric symptoms, including negative emotional states. However, it is not known whether people prone to such negative affective states experience the subjective effects of drugs differently, nor is it known to what extent this relationship may vary between classes of drugs. Here we investigate the relationship between the affectivity of negative traits and the subjective effects of five different psychoactive drugs, including psychedelics (LSD), amphetamines (d-amphetamine and MDMA), cannabinoids (THC), and opioids (buprenorphine).

Methods:Healthy adult volunteers completed the Multidimensional Personality Questionnaire (MPQ) and then completed at least two drug testing sessions during which they received a placebo or one of the following: LSD (13 μg), MDMA (1.5 mg/kg), d-amphetamine ( 20 mg), THC (15 mg) or buprenorphine (0.2 mg). At regular intervals during the sessions, participants rated how much they felt the drug was working and whether they liked the results. These ratings were tested against the Negative Emotionality (NE) scale of the MPQ.

Results:People with a high NE trait gave higher ratings of "feeling good" under the influence of LSD (N= 41;R= 0,37,PI number< 0.05), buprenorphine (N= 58;R= 0,35,PI number< 0.05) and d-amphetamine (N= 379;R= 0,11,PI number<0.05), but not THC. However, during these sessions, they showed no higher "drug-like" ratings, and for d-amphetamine, those with a higher NE trait gave significantly lower "drug-like" ratings (R= -0,14,PI number< 0,01).

Conclusions:Higher NE was associated with greater sensitivity to experiencing the interoceptive or subjective effects of drugs. This increased sensitivity did not appear to be associated with higher drug taste ratings. It will be interesting to investigate to what extent drug vulnerability is related to the risk of drug use or abuse.

Keywords:MDMA, LSD, buprenorphine, THC, personality

Revelation:Nothing to disclose.

University of North Carolina at Chapel Hill/School of Medicine, Chapel Hill, North Carolina, United States

Bottom:Alcohol use disorder (AUD) is a chronic, relapsing condition that affects approximately 14.1 million Americans. Despite the prevalence of this disorder, we lack a full understanding of the underlying neurobiological mechanisms. One of the major mechanisms contributing to the development of AUD is believed to be dysregulation of central stress systems. The hypothalamic-pituitary-adrenal (HPA) hormonal axis is one of the body's major stress response systems and is strongly involved in the risk and development of AUD. The HPA axis is activated by the release of corticotropin-releasing hormone (CRH) from CRH neurons in the paraventricular nucleus of the hypothalamus (PVNCRH). Dynamic changes in the HPA axis are associated with alcohol abuse, but changes in PVNCRH neurons that excite the HPA axis have not been thoroughly studied after alcohol consumption. PVNCRH neurons are of particular interest because, in addition to initiating the HPA axis, they have been shown to mediate rapid stress-related behavioral responses, independent of CRH release. Therefore, we tested the hypothesis that chronic alcohol consumption alters coping behaviors and the function of PVNCRH neurons.

Methods:Male C57BL/6J mice were subjected to long-term voluntary drinking of water or alcohol (20% w/v) consisting of three 24-hour sessions per week for a total of six weeks. In acute 24-hour abstinence, mice with access to water and alcohol were divided into a non-stressed or stressed state (2-hour restriction) to determine their response to alcohol.N= 13-14/group). Immediately after the stress, spontaneous behavior in the cage was recorded for 15 minutes. The mice were then placed in an open field test for 5 minutes. To investigate the physiological function of PVNCRH after chronic alcohol consumption, a second experiment was conducted in male and female CRF reporter mice subjected to the same paradigm of long-term voluntary alcohol consumption. In acute withdrawal, ex vivo electrophysiology experiments were performed to measure synaptic transmission and excitability. To determine whether spontaneous homecage behaviors correlate with normal function, a 15-minute homecage recording was performed before the mice were sacrificed for ex vivo recordings. For statistical analysis, two-way ANOVAs were performed, and multiple post-hoc comparisons, Tukey correction tests were performed where appropriate.

Results:So far, we have found that during acute deprivation, male mice display a number of spontaneous home cage behaviors indicative of coping with stress, such as increased rearing (non-stressful water vs. alcohol groups,PI number= 0.002) and reduced bubbles (no water stress vs. alcohol groups,PI number= 0.021). Notably, stress seemed to affect water and alcohol stressed mice similarly in the indoor (HC) and outdoor (OF) cage, as the alcohol and water stressed mice spent significantly more time grooming in both arenas (a major effect of house stress in cage,PI number< 0.0001 and open field,PI number= 0.02) and shorter rearing time (main effect of the HC strain,PI number< 0,0001 e OF,PI number= 0.007) and bubbles (main effect of HC stress,PI number<0.001). Preliminary results from Experiment 2 show that during acute withdrawal, female mice show increased excitability of PVNCRH neurons compared to water control mice. In particular, in response to injections with increased current, the alcoholic mice fired more action potentials (main effect of alcohol:PI number< 0.0001). We also found a tendency of male mice to show similar changes in PVNCRH neurons (main effect of alcohol:PI number= 0,092).

Conclusions:We found that a single severe stressor altered the spontaneous behaviors of water and alcohol consuming mice at home. Interestingly, cage-house behavior is generally altered in naïve, unstressed mice, indicating higher coping behaviors. This suggests that even 24 hours after the last alcohol drinking session, the mice show an increased baseline state of stress, which may be further exacerbated by acute stressors. In addition, PVNCRH neurons appear to be more excitable during the same acute withdrawal point.

Keywords:Alcohol withdrawal, stress management, paraventricular nucleus of the hypothalamus, anxiety and stress behavior, neural circuits and animal behavior

Revelation:Nothing to disclose.

Temple University, Philadelphia, Pennsylvania, United States

Bottom:Early life experiences may alter the risk/resistance to disorders associated with changes in motivated behavior. Early stress that is not overwhelming can have a "seeding" effect that promotes later resilience. Our laboratory uses the constrained bed and nest (LBN) model of mild adversity in early life. We found that this model reduced impulsivity and morphine self-administration in male rats but not in females, suggesting a "vaccination" effect against addictive behavior in males. Here, we extend this work to explore how early life experiences alter risky decision-making and explore genetic changes that may contribute to stress inoculation.

Methods:In LBN, pups on postnatal days 2 to 9 and their mothers were exposed to a resource-poor environment and compared to rats housed in standardized housing. When these pups reached adulthood, their risk-taking was compared to rats reared under standard conditions. Here, we predicted that the LBN would reduce men's preference for risky choices in a probability discounting task. When discounting probabilities, rats must choose between two levers: one that always offers a small reward (right lever) and the other that sometimes offers a large reward (dangerous lever). The odds of winning a prize fluctuate throughout the session, and risky leverage is only beneficial when the odds of winning a big prize are high (i.e. 100% or 50%). Given the role of the frontal cingulate cortex (OFC) in impulsive and risky decision-making, we assessed whether LBN-induced changes in gene expression in this region using RNA sequencing (RNA-seq). OFC tissue from adult rats (male controlN= 5; female controlN= 5; male LBNN= 5; female LBNN= 5) were collected and sequenced on an Illumina HiSeq 4000. Fastqc version 0.11.8 was used to assess the quality of the adapter reads, and unpaired reads were removed with Trimmomatic version 0.39. The RRHO test version 2 (Rank-Rank Hypergeometric Overlap) assessed the degree of genetic signature overlap between the sexes. RNA sequence analysis was performed and differentially expressed genes (DEGs) were identified using aPI numbervalue <0.1 and 50% change in expression as cutoff value to determine significance.

Results:We found that LBN exposure reduced risk-taking in a probability discounting task in male and female mice [eat(4, 376) = 2845,PI number= 0.024]. High risk taking is associated with increased drug use, but here we found that LBN reduces risky choices in this task. Since this manipulation produces a phenotype resistant to addiction-related behavior, we were interested in exploring potential molecular substrates that promote stress-induced immunity. RNA sequence in OFC tissue revealed sex-specific changes induced by LBN in gene transcription. RRHO analysis showed little overlap between up-regulated and down-regulated genes between males and females, but we also found a distinction between up-regulated genes in males and down-regulated genes in females due to LBN. We then limited our analysis to genes showing a significant difference between control and LBN and found 161 differentially expressed genes (DEGs) in males and 138 DEGs in females. These genetic changes were largely sex-dependent, with only 18 common genes being altered by the LBN. Analysis of the KEGG pathway showed that "retrograde endocannabinoid signaling" was significantly enriched in men than in women (adjusted forPI number= 0.01), which may lead to LBN-induced behavioral changes in males. The endocannabinoid system is interesting because it is involved in attenuating inhibitory control and promoting risky behavior. Future studies aim to validate major genetic targets, including those related to the endocannabinoid system, with RNAScope.

Conclusions:Taken together, these results suggest that LBN reduces the expression of behaviors associated with increased risk for substance use disorders. Although initially unexpected, these findings add to the growing literature supporting the stress vaccination hypothesis: that some early adversity promotes later immunity. Understanding the mechanisms that promote immunity may lead to better treatments for disorders exacerbated by stress.

Keywords:Stress in early life, risky behavior, gender differences, orbitofrontal cortex

Revelation:Nothing to disclose.

University of California Los Angeles, Los Angeles, California, United States

Bottom:Subjective response (SR) to alcohol is a biobehavioral risk factor for binge drinking and the development of alcohol use disorder (BAD). Identification of SR moderators has been hampered by the small sample sizes often used in alcohol management studies. Several alcohol administration trials were selected in this study to test whether gender, family history of alcohol problems, and impulsiveness (by delay discounting) predict alcohol SR, which includes four domains: agitation, sedation, negative affect, and craving.

Methods:Heavy drinkers and women who are not seeking treatment (N= 250) completed a series of self-report and behavioral scales measuring alcohol use and problems, mood and impulsivity. All participants completed an intravenous alcohol session in which baseline, 20, 40, and 60 mg% SR areas were measured. Due to the nested structure of the data, a series of multi-level models tested whether potential moderators predicted the SR during the alcohol challenge. The nested structure of the data was as follows: repeated measures of SR during an alcohol challenge (Tier 1) nested within the subjects (Tier 2) that were nested in the studies (Tier 3).

Results:We found that male gender independently predicted greater alcohol-induced arousal (B = 0.820, SE = 0.32,PI number= 0.011) and alcohol craving (B = 0.295, SE = 0.10,PI number= 0.005) after checking other moderators. Family history of alcohol problems independently predicted craving while controlling for other moderators (B = 0.995, SE = 0.26,PI number= .0001). Latency discounting did not significantly predict any subjective response domain.

Conclusions:With a large sample size and advanced data analysis methods, this study extends the literature by identifying important mediators of SR in heavy drinkers, namely male gender and family history of alcohol problems. These findings consolidate and extend a growing body of research into who is more likely to report RS characteristics that put them at risk for AUD.

Keywords:Subjective reaction, alcohol, gender differences

Revelation:Nothing to disclose.

National Institutes of Health, Bethesda, Maryland, Stany Zjednoczone

Bottom:A variant of the nicotinic acetylcholine receptor gene CHRNA5 rs16969968 (G>>A) is strongly associated with nicotine use and addiction. A recent preclinical study also demonstrated the involvement of the rs16969968 polymorphism in alcohol consumption and alcohol use disorder (AUD). However, its role in alcohol consumption and sensitivity in humans remains unclear. In this study, we examined the effect of the rs16969968 polymorphism on alcohol consumption and susceptibility in groups of heavy and light drinkers without AUD.

Methods:The study involved 176 healthy AUD non-drinkers who were divided into heavy and light drinking groups based on the results of the Alcohol Use Disorders Identification Test (AUDIT-C) (light drinking: ≤5, heavy drinking: >5). Actual alcohol consumption was assessed using the AUDIT-C subscales and schedule follow-up measures (TLFB). Human alcohol seeking and consumption in the laboratory was assessed using a publicly available intravenous alcohol self-administration (IV-ASA) paradigm implemented in a computer-assisted infusion system (CAIS). IV-ASA measurements included peak and mean breath alcohol concentrations (BrAC) at various time points (30, 60, 90, and 120 min) and total ethanol (in grams) injected during the session. Subjective responses were assessed serially during the IV-ASA session using the Drug Effects Questionnaire (DEQ), the Alcohol Emergency Questionnaire (AUQ) and the Biphasic Alcohol Influence Questionnaire (BAES). In addition, the Alcohol Sensitivity Questionnaire (SRE), the Alcohol Sensitivity Questionnaire (ASQ) and the Alcohol Effects Questionnaire (AEFQ) were used to assess alcohol sensitivity and expected response. Peripheral blood samples were collected from participants for genotyping. Alcohol consumption, subjective responses and related phenotypes were compared between heavy and light drinkers and by rs16969968 genotype. The effect of the rs16969968 polymorphism was tested using the dominant model (GG and AA/AG).

Results:We first compared alcohol consumption and IV-ASA scores between heavy and light drinkers and, as expected, AUDIT-C and AUDIT total scores, as well as TLFB scores, were significantly higher in heavy drinkers than in light drinkers. BrAC levels and ethanol intake during IV-ASA sessions did not differ between the consuming groups. However, in the GG genotype group, heavy drinkers achieved significantly higher BrAC levels at the 60, 90, and 120 minute time points (allPI numbervalues ​​ < 0.05) and more ethanol was given (PI number= 0.045) compared to light drinkers. In the group of AA/AG genotypes, there were no differences in IV-ASA measurements between heavy and light drinkers.

Additionally, heavy drinkers had significantly higher scores on measures of alcohol sensitivity, including all SRE measures (first five, last, heaviest, and total), descending and rising ASQ scores, and social and physical pleasure expectations from the AEFQ. In the AA/AG genotype group, heavy drinkers had higher SRE, ASQ, and AEFQ scores for expected social and physical pleasure than light drinkers. In addition, expectations of sexual improvement were higher in heavy drinkers than in light drinkers. In the GG genotype group, heavy drinkers scored higher on the most recent, most severe, and total SRE scores, ASQ measures, and AEFQ social and physical expectation scores compared to light drinkers.

Conclusions:This study identified a significant effect of the CHRNA5 polymorphism (rs16969968) on increased alcohol consumption and related phenotypes in the laboratory paradigm of human alcohol consumption. The main effect of consumption pattern was observed for several subjective and susceptibility measures across CHRNA5 genotype groups. The AA/AG genotype group had significantly higher expectations for sexual enhancement but had no difference in alcohol consumption, potentially reflecting a protective effect. The GG genotype group showed significantly lower sensitivity and higher alcohol consumption in heavy drinkers.

Keywords:Alcohol, sensitivity, CHRNA5

Revelation:Nothing to disclose.

National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, USA

Bottom:The Neuroclinical Addiction Assessment (ANA) is a clinical framework consisting of three neurofunctional domains believed to underlie substance use disorders: executive functioning (EF), negative emotionality (NE), and motivational dominance (IS). The ANA battery consists of neurocognitive tasks and self-report questionnaires that provide in-depth phenotypic characterization of individuals based on three neurofunctional domains. The goals of the ANA are to better understand the heterogeneity and etiology of substance use disorders, develop individualized treatment for the individual, and identify potential treatment mechanisms. This report focused on the implicit structure underlying ANA batteries and examined differences in these domains between individuals with and without alcohol use disorder (AUD).

Methods:The study included 298 individuals (41.3% women, mean age   42.6 years, 68.5% with a current diagnosis of AUD), representing a wide range of alcohol consumption, who were included in the NIAAA Natural History Protocol. Behavioral tasks and self-report questionnaires assessed the three domains ANA, EF, NE and IS. PE measures included response inhibition (stop sign response task), working memory (digit span - backwards), inference (beads in a jar), task switching (tracing task), mental rotation (dummy), attention (continuous performance task). metacognition (Metacognition Questionnaire) and introspection (Multidimensional Assessment of Receptive Awareness). NE measures included discomfort tolerance (task of gradual visual addition at a pace), ostracism (cyberball), reward motivation (reward effort expenditure task), anhedonia (Snaith-Hamilton pleasure scale), resilience (Snaith-Hamilton-D pleasure scale residence). ), social support (Social Supporting Behavior Inventory), affect (Positive and Negative Affect Scale) and alexithymia (Toronto Alexithymia Scale). IS was measured by approach bias to avoidance (alcohol avoidance approach task), implicit alcohol associations (implicit association with drinking identity task), and alcohol demand (hypothetical purchase task).PI numberand the Barratt Impulsiveness Scale) and the state of craving (Penn Alcohol Craving Scale). Separate factor analyzes were performed in each domain to isolate the underlying factor structures. The interactions of the ANA domains were modeled by structural equation modelling. Group differences between those with and without AUD were determined by assessing structural changes and comparing factor scores.

Results:Five factors appeared in the EF domain: response inhibition, working memory, interaction, rumination, and impulsivity (CFI = 0.93, TLI = 0.92, RMSEA = 0.05). Three factors were identified in the NE domain: negative mood, positive mood, and negative actions (CFI = 0.95, TLI = 0.94, RMSEA = 0.07). Two factors were found in the IS domain: alcohol motivation and alcohol insensitivity (CFI = 0.98, TLI = 0.96, RMSEA = 0.06). We then combined the latent factors from each domain into a single model to investigate cross-domain correlations (CFI = 0.89, TLI = 0.87, RMSEA = 0.06). These cross-domain correlations ranged from undetectable to strong (absolute).Rs = 0.01 - 0.86). Negative mood and impulsivity showed the strongest and strongest correlations with other factors in all domains. Compared to those without AUD, those with AUD showed stronger correlations between factors in the EF and IS domains and between factors in the IS and NE domains. Individuals with AUD differed significantly from those without AUD in all factor scores except response inhibition and positive mood.

Conclusions:Factor analysis of ANA domains revealed significant substructures for AUD. Among the ANA domains, negative mood and impulsivity showed the strongest correlations with other factors, suggesting that they may play a key role in the etiology or progression of AUD. Individuals with AUD showed stronger correlations between EF and IS domains and IS and NE domains. These differences may reflect the pathology of the AUD and thus represent potential targets for therapeutic interventions that may lead to amelioration of other underlying AUD dysfunctions. Future work will focus on identifying relevant domain-associated neural networks and categorizing different AUD phenotypic profiles based on ANA domains.

Keywords:Addiction phenotypes, alcohol and drug use disorders, neurocognitive functions

Revelation:Nothing to disclose.

University of Texas Health Science Center at Houston, Houston, Texas, United States

Bottom:Opioid use disorder (OUD) remains an important public health concern as opioid dependence and overdose deaths increase in the United States. of opioids on particular types of brain cells is unknown. We performed a comprehensive multiomic analysis of cell type-specific gene expression signatures in OUD and generated preliminary postmortem brain single-core gene expression data to verify these signatures.

Methods:We performed next-generation RNA (RNAseq) whole-tissue sequencing (N= 27 opioid users, 14 controls) and proteomics based on liquid chromatography and mass spectrometry (N= 20 opioid users, 12 controls) in Brodmann area 9 (BA9), with further analysis of differential expression and pathway enrichment, and analysis of gene and protein ontologies. Gene expression in the whole tissue was correlated with cell composition by cell type deconvolution. In addition, we performed a preliminary single-nucleotide RNAseq (snRNAseq) in BA9 from a subset of individuals. The cell types of each core sequence were determined by specific cell type markers. All analyzes accounted for covariates including age, sex, PMI, pH and RNA integrity.

Results:We identified 394 differentially expressed coding (DE) and long non-coding (lnc) RNAs, as well as 213 DE proteins in BA9 from individuals with OUD. Changes in RNA and proteins combined in pro-angiogenic gene networks and cytokine signaling pathways. We found cell-type effects in these networks with enrichment in astrocyte, endothelium and microglia related genes. The WGCNA team identified key genes involved in endothelial, astrocyte, microglial and neuronal functions. The snRNAseq project identified cell type-specific clusters that were used to validate gene expression results obtained from whole tissue.

Conclusions:In the brains of opioid users, the network of inflammatory and angiogenic genes is dysregulated, possibly due to damage to endothelial cells, astrocytes and microglia. Single-core RNA sequencing is feasible in our tissue samples and can be used to validate these signatures. To our knowledge, this is the first multi-ohm study examining interference with specific cellular networks in an OUD. This work will help advance our understanding of the molecular effects of opioids on the brain.

Keywords:Opioid abuse, proteomics, single cell RNA sequence

Revelation:Nothing to disclose.

University of Alabama at Birmingham, Birmingham, Alabama, United States

Bottom:The ventral tegmental area (VTA) is a complex area of ​​the brain necessary for reinforcement and reward learning, and whose dysregulation has been implicated in substance use disorders. Although decades of research on VTA function have focused on the role of dopaminergic neurons, recent evidence has also identified critical roles for VTA GABAergic and glutamatergic neurons in reward processing. Interestingly, molecular characterization revealed that subsets of these neurons express genes involved in the transport, synthesis and vesicular packaging of many neurotransmitters, providing evidence for the presence of co-releasing neurons. However, these studies are largely based on low-throughput methods, and the molecular architecture of the VTA has not been fully explored.

Methods:Here, we performed single-stranded RNA sequencing (snRNA-seq) on 21,600 testes of male and female Sprague-Dawley rats to generate a rat VTA transcriptome atlas. Differential gene expression analyzes and Gini coefficient calculations were used to identify putative molecular markers of unique neuronal subtypes. Finally, to investigate which VTA cell types show preferential enrichment of genes associated with GWAS risk, we performed a gene set analysis using MAGMA (Multi-marker Analysis of GenoMic Annotation), a multiple regression model that allows the assessment of the contribution of multiple gene markers to a given phenotype clinical.

Results:We identified 16 transcriptionally distinct cell types, including 7 separable populations of neurons. Additional subgroups revealed several VTA populations that contained gene markers for more than one neurotransmitter system, including a cluster that showed high levels of expression of genes involved in GABA, glutamate, and dopamine synthesis and transport. Statistical comparisons between clusters identified novel gene markers for subclasses of normal and combinatorial dopaminergic neurons. Finally, the MAGMA analysis identified neuronal SNP enrichment from genes involved in schizophrenia and smoking initiation, as well as ADHD risk gene enrichment in VTA glutamatergic neuron populations.

Conclusions:The use of snRNA-seq enabled a comprehensive and unbiased VTA transcript profile, and the resulting atlas is publicly available as a searchable online resource (www.day-lab.org/resources). A study of neuronal subpopulations confirmed the presence of combinatorial neurons and identified new marker genes for co-releasing neurons and classically defined dopaminergic neurons. Gene pool analyzes at the SNP level revealed cell type-specific enrichment of genes associated with GWAS risk, highlighting the contribution of different cell populations to polygenic traits in various clinical disorders.

Keywords:Single cell RNA sequencing, ventral tegmental area (VTA), dopamine

Revelation:Nothing to disclose.

Renaissance School of Medicine, Stony Brook University, South Setauket, New York, United States

Bottom:Maternal sensitivity is critical to parenting behavior and child outcomes, but may be altered in mothers with opioid use disorder (OUD). The incidence of OUD during pregnancy is increasing significantly, with more than 2.5% of pregnant women chronically using opioids each year, and more than 100,000 postpartum women and their families suffering from OUD. Despite buprenorphine (BT) abstinence treatment, mothers with OUD are at high risk of comorbidities, polysubstance use, and a 30% relapse to illicit opioids, which may increase the risk of child abuse or neglect and costly respite care. BT reduces the psychophysiological stress associated with repeated maternal withdrawal cycles, but little is known about how BT/OUD may affect maternal brain function, sensitive parenting, and child outcomes. This is of concern as opioid-induced deficits have been found in preclinical rodent models. Furthermore, we know that human maternal behavior is governed by the maternal nervous system (MBN), which is evolutionarily conserved among mammals to regulate adaptive maternal care and aggressive behavior. We recently reported that human maternal brain responses in the MBN and other cortical networks during face-reflective self-orientation in a functional magnetic resonance imaging (fMRI) task may reflect their maternal sensitivity capacity. Here, we use this task to investigate whether BT/OUD mothers' responses to baby face bouncing are attenuated.

Methods:Of the 22 mothers studied (ages 18 to 40), 6 received BT for OUD and 16 were in the control group without OUD (GC). They underwent the face-to-face reflection task (CFMT) in fMRI, 4 months postpartum. In the CFMT, participants viewed their children's images repeatedly presented in three different task conditions, OBSERVING, REACTING, and RELATING, which were interspersed in random order. In the OBSERVE study, participants were instructed to observe a photograph of a child calmly, without imitating facial expressions or emotions. In REACT, participants were instructed to respond to the child in a normal manner. In JOIN, participants were instructed to empathically imitate (mirror) the child's facial expressions and emotions. Each condition of the own child task was presented in four blocks (16 s/block). In each block, four pictures of the child were presented sequentially for 4 s per picture. Each of these four pictures showed one of four different emotional expressions, happy, sad, ambiguous and neutral, presented in random order. fMRI data were analyzed using statistical parametric mapping software (SPM8).

Results:We explore group differences in Join vs. Observe from the child participants themselves. The Extra Movement Area (SMA) showed significant activation during Join vs. Observe your own child (MNI coordinates [0, 2, 58], 63 voxels, Z=3.53). In addition, the reactivity of this region was reduced in BT/OUD mothers compared to CG (MNI coordinates [2, 8, 60], 230 voxels, Z=3.13). In MBN, the left macula had a similar deficit in BT/OUD mothers compared to CG (MNI coordinates [-16, -4, -6], 23 voxels, Z=4.21).

Conclusions:We show the initial effects of BT/OUD on neural responses during child-centered mirroring. Compared to the comparator group, BT/OUD mothers showed impairments in child-centered mirror-to-face responses in brain regions critical for maternal receptivity, namely SMA related to mirror face and left gyrus, which is important for maternal care response. MBN. More work is needed to relate these deficits in neural responses to actual maternal sensitivity, parental behavior, and child outcomes. However, these preliminary results suggest potential mechanisms in mothers with BT/OUD. More related research is needed on parental interventions that may modulate the same MBN brain circuits affected by BT/OUD, reduce stress, and increase maternal vulnerability to reduce cross-generational mental health risks.

Keywords:Opioid use disorder, maternal brain, maternal vulnerability, emotional empathy, functional magnetic resonance imaging (fMRI)

Revelation:Nothing to disclose.

Brigham Young University, Provo, Utah, USA

Bottom:Research shows that the onset of heavy drinking is a precursor to alcohol-related disorders, and it is widely accepted that central monoamine neurotransmitters modulate changes in alcohol consumption, especially early on. However, more recent research has focused on elucidating the variables that maintain addiction, such as allostatic changes in the CNS that result from chronic alcohol consumption, as well as compensatory processes that reduce the rewarding effects of alcohol and the role of alcohol. Negative buff that causes hunger and relapse. A recent article noted that research on the relationship between central monoamines and alcohol intake is notably absent from recent reviews, and when searching Medline, the authors of this study were unable to find a recent review article that focused primarily on the relationship between central monoamines and alcohol intake alcohol. monoamines and alcohol intake. Despite Cloninger's predictions about the role of monoamines in heavy drinking, few studies have experimentally evaluated the relationship between alcohol-induced monoamine cycling and baseline and variability of alcohol consumption from baseline in nondrinkers. Studies also failed to assess the role of change from baseline and subsequent exposures as predictors of change in alcohol consumption at baseline.

Methods:Using a non-human primate model, this study investigated baseline alcohol-induced monoamine activity and alcohol-induced change in monoamine activity after repeated exposure and their relationship to subsequent alcohol consumption. Rhesus macaques (Macaca mulatta,N= 114) received a standardized intravenous bolus of an alcoholic solution (16.8%, v/v) twice approximately one month apart. One month before and one hour after each alcohol infusion, cerebrospinal fluid was collected and monoamine metabolite concentrations were determined. Approximately six to seven months later, subjects had unrestricted access to an alcoholic solution sweetened with aspartame (8.4%, v/v) for one hour a day, 5 days a week, for five to seven weeks. The alcohol-induced baseline score and monoamine change were used to assess the relationship between monoamines and changes in alcohol consumption.

Results:Overall power analysis results show a statistical power of 0.90 for detecting medium to large effect sizes. The f2 values ​​for the effects ranged from 0.02–0.19, indicating small and large effects. The results showed strong positive correlations between baseline and post-infusion levels of monoamine metabolites in the CSF, indicating a tracer-like response. Lower baseline and post-infusion concentrations of serotonin and dopamine metabolites and smaller changes in serotonin and dopamine metabolites between infusions were associated with higher alcohol consumption. Low baseline and post-infusion norepinephrine metabolite concentrations predicted high alcohol consumption, but unlike other monoamines, a greater change in norepinephrine metabolite concentrations from one infusion to another was associated with higher alcohol consumption.

Conclusions:Studies measuring central monoamine systems before and after alcohol consumption are lacking, limiting our understanding of the role of monoamines in the acquisition of heavy drinking and binge drinking, as well as the acquisition of tolerance to the pharmacological effects of alcohol. The results of this study are important because they suggest that a pre-existing response as well as the initial response of CNS monoamines to alcohol may be important factors in predicting and potentially moderating future alcohol consumption, with the possibility that after alcohol exposure, individuals with low levels of serotonin and dopamine and alcohol-induced low changes in serotonin and dopamine consume more alcohol to compensate for the lower central monoamine function. These findings suggest that individual differences in natural and alcohol-induced monoamine activity, as well as changes between exposures, are stable across individuals and are likely to be important moderators of initial drinking and may play a role in drinking risk. alcohol use. .

Keywords:Adolescent alcohol use, rhesus monkey, norepinephrine, dopamine, serotonin

Revelation:Nothing to disclose.

University of Pennsylvania, Philadelphia, Pennsylvania, United States

Bottom:Alcohol use disorder (ABD) is a chronic, relapsing disorder characterized by compulsive alcohol consumption and negative affective states and withdrawal symptoms when access to alcohol is discontinued. Women are at greater risk than men for the adverse effects of alcohol, including alcohol-related diseases and brain damage caused by alcohol or alcohol withdrawal. Although recent preclinical studies have shown that female rats exhibited a more severe depressive state during alcohol withdrawal than male rats, there is a need to investigate the effects of sex on withdrawal symptoms, liver function, depressed mood, and sleep quality. during withdrawal in people with AUD.

Methods:Here we examined the effect of AUD and gender on sleep quality and depressed mood data collected through screening and natural history inN= 644 AUD participants (193 women) iN= 509 independent healthy controls (264 women). The subgroup also completed a brain MRI for brain volumetric assessment (N= 142 AUD,N= 180 checks). in this groupN= 455 people with AUD (136 women) completed a 3-4 hospital detox treatment program where we investigated gender differences in alcohol withdrawal (CIWA-Ar), need for benzodiazepines to relieve withdrawal symptoms, liver function tests, sleep quality, and depressed mood during treatment.

Results:We found that AUD participants had poorer sleep quality (F1.1153=534.4,PI number< 0.0001) and depressed mood (F1.1153 = 543.4,PI number< 0.0001) compared to healthy volunteers. Women with AUD had poorer sleep (t642=2.8,PI number= 0.005) and depressed mood (t642 = 4.4,PI number< 0.0001) than men participating in AUD, despite having equal amounts of alcohol adjusted for weight between them. In addition, women with AUD admitted to rehab had more severe withdrawal symptoms (F1.454=12.0,PI number= 0.0006), higher use of benzodiazepines (F1.451 = 5.8,PI number= 0.016) and decreased ALT (F1.453 = 6.6,PI number= 0.011), but not AST (F1,454 = 0.6,PI number= 0.80) liver function tests despite equal amounts of alcohol adjusted for body weight before alcohol deprivation. As expected, AUD had the lowest overall GMV (F1.322=19.9,PI number< 0.0001) and larger lateral ventricles (F1.322 = 15.0,PI number= 0,0001) niż grupa kontrolna (skorygowana o objętość wewnątrzczaszkową, wiek, palenie tytoniu i BMI). W grupie AUD ALT i AST były ujemnie skorelowane z GMV i rozmiarem komory (wszystkiePI number< 0.0001), but abstinence scores, sleep quality, or depressed mood were not associated with structural measures of the brain. However, women with AUD had a higher GMV (F1.142 = 50.5,PI number<0.00001) and smaller side chambers (F1.142=4.3,PI number= 0.040) from men with AUD (adjusted for intracranial volume, age, smoking status and BMI). suggesting less susceptibility to the damaging effects of alcohol on brain structure compared to men.

Conclusions:Taken together, these findings suggest that alcohol has a more severe effect on withdrawal syndrome, liver function, sleep quality and depressed mood, but not on brain structural parameters in women than in men. They also suggest that even without cirrhosis, liver dysfunction in AUD may contribute to brain structural changes.

Keywords:Alcohol, alcohol abstinence, gender difference

Revelation:Nothing to disclose.

Virginia Commonwealth University, Richmond, Virginia, United States

Bottom:Disordered use of various medications has been associated with decreased executive functions (EF) such as motor impulsivity or "quick reaction", especially stimulant use disorders. This reduction in self-control is thought to reflect a central EF deficit in earlier neurological development that favored susceptibility to transition from recreational to compulsive substance use or may be a consequence of chronic neurotoxic effects of drugs. However, many measures of motor impulsivity, such as gross commission errors, depend at least in part on central attentional capacity, which is rarely studied.

Methods:As a pilot element of the NIDA Phenotypic Assessment (PhAB) battery as a potential standard assessment for clinical trials, men and women with any of the opioid use disorders (OUD;N= 85), cannabis use disorder (CaUD,N= 48) and cocaine use disorder (CoUD;N= 35) as well as neurotypical controls (N= 82) completed the Attentional Network Task (ANT) flanker task and the SST task (SST).

Results:Orientation ability (using spatial information about where the next target would appear to allow shorter reaction times (RT)) decreased with age, while the conflict (sideslip) effect of inconsistent arrows to increase RT worsened with age. Controlling for age, participants with OUD (PI number< ,05) e CaUD (PI number< 0.01) each showed poor alertness compared to the control group, with these groups not using advanced signaling (time signal) of the approaching flanking target to allow for shorter reaction times (RT) compared to the control group. In addition, CaUD participants showed a trend (PI number< .10) towards greater susceptibility to the flanker effect for RT prolongation compared to the control group. Similarly, when performing SST, OUD and CaUD participants, but not CoUD participants, showed slower RT to target stimuli compared to controls (PI number< 0.05), but there are no differences from controls in the same breakpoint response time in any SUD.

Conclusions:These data suggest that among SUDs, CaUD and OUD may be characterized by only attention deficits in the absence of motor impulsivity.

Keywords:Impulsiveness, attention, cocaine and opioid use disorder, cannabis use disorder

Revelation:Nothing to disclose.

Boston University School of Medicine, Boston, Massachusetts, Stany Zjednoczone

Bottom:Understanding the pharmacogenetics and pharmacokinetics of opioids is critical to therapeutic success, as genetic mutations can dramatically alter therapeutic efficacy and the risk of opioid dependence. Oxycodone (OXY) is a semi-synthetic opioid (the active ingredient in Oxycontin®) and is metabolized in humans by CYP2D6 to the more potent mu opioid receptor agonist oxymorphone (OMOR) and by CYP3A4 to the inactive noroxycodone (NOR). OMOR is further metabolised in humans by UGT2B7 to the inactive oxymorphone 3-glucuronide and by CYP3A4/CYP2D6 to the bioactive noroxymorphone. Since OMOR is an extremely potent mu-opioid receptor agonist and has both analgesic and addictive effects, understanding the regulation of OMOR levels in the brain ([OMOR]) is critical to understanding the neurobehavioral effects of OXY. We observed strong differences in brain [OMOR] between BALB/cJ and BALB/cByJ littermates, with BALB/cJ (J) females showing higher brain [OMOR] at 30 minutes after OXY (1.25 mg/kg, i.p.) compared to BALB/cByJ (ByJ) mice. Behavioral, we also observed increased conditioned place preference (CPP) and locomotor arousal in female J mice compared to ByJ mice, suggesting that increased brain [OMOR] may underlie the increase in OXY-induced behaviors. Since the BALB/c substrains are almost genetically identical, being distinguished only by the 8K variants, the F2 cross with reduced complexity could greatly facilitate the identification of causal genetic factors underlying substrain variation in brain [OMOR] and behavior. The aim of this study was to use the quantitative brain trait locus [OMOR] and gene expression mapping (eQTL) in BALB/c litters to identify potential genetic factors underlying enhanced brain [OMOR] and OXY behaviors.

Methods:We mapped the brain's Quantitative Trait Locus (QTL) [OMOR] and behavior in a 133 BALB/cJ x BALB/cByJ F2 (68F, 65M) reduced complexity cross. BALB/c substrains and F2 mice were trained and tested for condition-dependent CPP for 9 days in a dual chamber apparatus. Mice were scored for baseline preference on day 1, trained on days 2-5 (1.25 mk/kg OXY i.p. or volume-adjusted saline), tested for drug-free CPP on day 8 and conditioned CPP on day 8. Day 9 For OXY/metabolite quantitation, parental littermates and F2 mice previously used as controls in the CPP study received a single injection of OXY (1.25 mg/kg, i.p.) and whole brains were harvested and flash-frozen 30 minutes later. Samples were homogenized and [ACD/metabolite] measured in the brain using combined liquid chromatography, electrospray ionization and tandem mass spectrometry. QTL mapping was performed in eQTL R/qtl and striatal/hippocampal analysis in MatrixEQTL. Analysis of differential mRNA expression in the liver between BALB/c littermates was performed using the scruff package. Whole brain proteomics was performed on native substrates using frozen whole brains harvested immediately after CPP and state-dependent LC-MS and MaxQuant to identify and quantify the peptides.

Results:There was a higher CPP depending on the state (eat(1,156) = 18,8,PI number= 0.014) and brain [OXY] (T(14) = 2,55,PI number= 0.023), [NONE] [T(14) = -1,917,PI number= 0.076] e [MURDER] [T(14) = 2,06,PI number= 0.058] in J women vs. ByJ mouse. QTL analysis identified a single significant genome-wide QTL on chromosome 15 underlying the enlarged brain [OMOR] with the J allele (LOD=6.53,PI number= 0.001; Bayesian range: 32-94 Mb; This explained 29% of the variance) caused by women. Cis-eQTL analysis of the striatum and hippocampus identified Zhx2 as a highly significant (AdjP=7.34E-09) and promising candidate gene located in the middle of the QTL interval (58 Mb) for the brain [OMOR]. Zhx2 encodes a transcription repressor that is highly expressed in the brain and liver, and J mice carry a 6.2 kb murine endogenous retrovirus (MERV) in intron 1 of Zhx2 that dramatically reduces levels of Zhx2 transcript and ZHX2 protein, leading to female dysregulation of pharmacokinetic genes (eg CYP) in the liver. Alignment of short reads of the whole genome sequence confirmed the presence of Zhx2 MERV in J and absence in ByJ. Whole-brain proteomic mass spectroscopy in BALB/c littermates confirmed a reduction in brain ZHX2 (AdjP=2.8E-09), along with an increase in CYP2D11 protein (CYP2D6 homolog) in J mice (AdjP=036). Liver transcriptomics also confirmed reduced expression of Zhx2 (AdjP =1.54E-10) in J mice, along with a plethora of differentially expressed pharmacokinetic genes, including genes encoding CYP and UGT enzymes, which may underlie the distinctive brain mechanism of Zhx2ERV. [OMOR] and OXY-induced behaviors.

Conclusions:Genetic analysis of brain OXY [metabolite] systems and behavior in a cross with reduced complexity between BALB/c substrates combined with a multivariate ohmic approach identified Zhx2 MERV private in J medium as a promising baseline variant of the brain growth quantitative trait [OMOR] and state-dependent reward induced by OXY in J vs. ByJ. We hypothesize that reduced Zhx2 expression leads to altered hepatic expression of one or more key hepato-brain-regulated pharmacokinetic enzymes [OMOR] (e.g., CYP and/or UGT). Future research will use CRISPR/Cas9 gene editing to validate Zhx2 MERV as a quantifiable trait variant and determine the quantification mechanism linking Zhx2 MERV to brain behaviors [OMOR] and OXY.

Keywords:Pharmacogenetics, Oxycodone, Opioid Use Disorders, GWAS, Drug Metabolism

Revelation:Nothing to disclose.

University of California, Los Angeles, Los Angeles, California, United States

Bottom:Risky decision-making, a central feature of many mental disorders, has a complex but largely unknown genetic architecture.

Methods:We examined risk-taking in two independent samples of mostly healthy adults. A sample (N= 1138) included psychiatric patients (53 with schizophrenia, 42 with bipolar disorder, 47 with ADHD). other (N= 911) excluded participants who reported recent treatment or medication for multiple psychiatric disorders but were not screened for ADHD. Corrected mean balloons, a risk-taking index, were recorded during the balloon proportional risk task. DNA was obtained for GWAS analysis on both samples. Polygenic Risk Ratings (PRS) were obtained in each dataset and then compared to a second sample. In addition, PRS were constructed in the pooled sample using a genome-wide MEGA analysis to test the genetic association between BART performance and self-reported risk in the UK Biobank sample and psychiatric phenotypes characterized by impulsivity. ADHD, Bipolar Disorder, Alcohol Use Disorder, Cannabis Use Disorder) at the Psychiatric Genomics Consortium.

Results:PRS for BART performance based on predicted performance of a larger discovery dataset job in a replication sample (R= 0,13,PI number= 0.000012, pFDR = 0.000052), as well as cross-analysis (R= 0,09,PI number= 0.0083, pFDR = 0.04) using a smaller dataset to discover. The exclusion of people with psychiatric diagnoses also yielded significant results. A single significant genome-wide linkage emerged from MEGA-GWAS involving the strong facial relevance gene, IGSF21. IGF21 is known to act on inhibitory synapses in the brain. Common single nucleotide polymorphisms accounted for 27% of the task performance variance. PRS for Cannabis Use Disorders (PI number= 0.00047, pFDR = 0.0053) predicted risky decision making in our sample. Other disorders studied and self-reported risks were not significantly associated with BART exposure.

Conclusions:The results suggest a heritability of risky decisions with shared susceptibility to cannabis use disorders.

Keywords:Polygenetic risk score, risk taking, cannabis use disorder, balloon proportional hazard task

Revelation:Myriad Genetics: Advisory Board (Auto, Tourette Association of America, Advisory Board, Auto

University of Linköping, Linköping, Szwecja

Bottom:Exposure to trauma during childhood or adolescence is associated with increased susceptibility to developing a substance use disorder (SUD) in adulthood. A possible contributing factor is the disruption of the endocannabinoid (eCB) system, a neuromodulatory system involved in the processing of stress and emotions. The eCB system undergoes extensive reorganization during adolescence, and disruptions to this process, such as exposure to trauma, may persist into adulthood. Thus, childhood trauma may affect the functioning of the eCB system, affecting stress and emotional processing, and potentially contributing to the development of SUD later in life.

Methods:Using a quasi-prospective approach, we recently showed that those with documented childhood trauma are at higher risk of developing SUD, even when controlling for potential sources of bias such as recall bias or familial confusion (Capusan et al., 2021 Molecular Psychiatry). To explore the potential contribution of eCB function to this relationship, we recruited individuals (N= 100) with or without a documented history of childhood trauma and/or substance use disorder. All participants provided blood samples at baseline and after exposure to the laboratory stressor for endocannabinoid and cortisol analysis. Emotional processing was assessed by facial electromyography before and after stress exposure.

Results:Those exposed to trauma (trauma only) had higher baseline levels of the eCB ligand anandamide (AEA;PI number= 0.003), which remained high even after exposure to stress (PI number= 0.005). This effect was absent in those who were exposed to trauma and developed SUD. There were no differences in cortisol reactivity to stress.PI number= 0.60), although the LDS groups showed impaired autonomic reactivity to stress (PI number= 0.031). The trauma-only group had lower depression self-report scores than the SUD-only group and the trauma+SUD group (PI number= 0,01).

Conclusions:In people exposed to childhood trauma, increased AEA may act as a protective factor, mitigating the negative effects of exposure to stress. These data support the notion that pharmacological elevation of AEA may serve as a novel therapeutic target for the treatment of trauma-related disorders, supporting ongoing clinical trials targeting this mechanism.

Keywords:Endocannabinoid, stress and trauma, substance use disorders

Revelation:Nothing to disclose.

University of Michigan School of Medicine, Ann Arbor, Michigan, Stany Zjednoczone

Bottom:Activity in the nucleus accumbens (NAc) contributes to the "craving" for food and drugs in response to signals associated with these enhancers. These stimulus-induced bursts contribute to drug addiction and obesity. Blockade of calcium-permeable AMPA receptors (CP-AMPAR) in the NAc prevents the expression of signal-induced food seeking and the "incubation" of cocaine craving. In addition, NAc CP-AMPAR expression and transmission is increased after withdrawal from long-access cocaine self-administration and after ingestion of a high-sugar, high-fat junk diet. These effects persist for more than a month after stopping cocaine use or junk food. However, what initiates and maintains the long-term NAc upregulation of CP-AMPAR is poorly understood. Moreover, the extent to which a junk diet promotes synaptic integration with specific NAc inputs or cell types is unknown.

Methods:Male rats (10–12 per group) were given free access to chow or chow, and whole-cell patch recordings were made by either electrical stimulation or optogenetic stimulation of inputs from the medial prefrontal cortex (mPFC) or basolateral amygdala (BLA). in NAc (pAAV-CamKII-Chronos-GFP). NAc type D1 and D2 MSN recordings were made from D1-Cre+ and A2a-Cre+ mice. Cre+ cells were visualized by viral transduction with tdTomato (pAAV-FLEX-tdTomato; Addgene 28306-AAV1). The contribution of CP-AMPAR to AMPAR transmission was determined using the selective CP-AMPAR antagonist naspm (200 μM). A pharmacological blockade of DREADD-mediated excitatory transmission was used to test the hypothesis that a reduction in glutamate transmission favors CP-AMPAR transmission. Data is analyzed using two-way and one-way ANOVA followed by a Sidak final test or unpaired final testsT- individual tests.

Results:We found that junk food increased CP-AMPAR transmission in mPFC-on-NAc entries but not in BLA-on-NAc entries after short exposure (10 days, unpaired bilateralT- test based on a priori predictions: t11 = 2.4,PI number< 0.05). Preliminary results suggest that the increase in CP-AMPAR transmission occurs in D2 type MSNs (research on D1 MSNs is ongoing). In addition, blocking excitatory transmission facilitated the recruitment of CP-AMPAR in the NAc.

Conclusions:Both mPFC and BLA affect food seeking via interaction with the NAc, but mPFC is more closely related to behavioral flexibility and goal-directed behavior, while BLA is related to emotional motivation. Thus, the preferential effects of unhealthy food on mPFC inputs suggest a possible reduction in behavioral flexibility. In addition, a high-fat diet alters perineural networks and column density in the mPFC, suggesting that changes in NAc transmission may be related to these effects. Finally, preliminary results suggest that increases in CP-AMPAR may be due to decreases in glutamate activity. This effect was independent of prior exposure to the junk diet.

Keywords:Motivation Motivation, glutamatergic synapses, addictions

Revelation:Boehringer-Ingelheim: Konsultant (Auto)

University of Texas, Austin, Texas, United States

Bottom:Exposure to stress contributes to the development of drug and alcohol addiction. Animal models show that stress exacerbates increased alcohol consumption in alcohol-dependent animals. The nucleus tractus solitarius (NTS) is a critical region of the brainstem for the integration and transmission of peripheral signals to modulate the stress response.

Methods:To investigate molecular adaptations in this brain region that may contribute to stress-induced alcohol consumption, we exposed animals to chronic intermittent exposure to ethanol vapor (CIE), forced swimming stress (FSS) or both (CIE+ FSS) followed by transcript profiles NTS at three different time points after the last vapor exposure (0 hrs, 72 hrs and 186 hrs).

Results:Using three different time points, we were able to identify transient, persistent, and compensatory patterns of gene expression associated with each of the behavioral treatments. We identified interferon signaling as a critical genetic network related to the level of alcohol consumption. Using a likelihood ratio test, we identified genes that were differentially expressed over time and between groups. Cluster analysis of these genes to identify unique expression patterns identified a subset of genes that do not normalize in the CIE+FSS group but not in the others. These genes were enriched for cell interactions and cell movement, indicating long-term structural and functional changes in this brain region caused by the unique interaction of alcohol addiction and stress. Specific genes of interest identified in this group include Aqp4, Il16, Reln, Grm4, Gabrd and Gabra6. We also compared changes in gene expression in the NTS with the PFC and found significant overlap between differentially expressed genes between the two brain regions. Overlapping genes in the CIE+FSS cluster were enriched for type I interferon signaling.

Conclusions:Taken together, these results summarize transcription changes over time in the NTS that may be critical to the development of stress-induced increases in alcohol consumption and alcohol dependence.

Keywords:Brain Stem, Core of Loneliness, Acid Stress, Alcohol Addiction, Transcription

Revelation:Nothing to disclose.

National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, USA

Bottom:The speed at which drugs enter the brain affects their rewarding effects, the faster they enter, the stronger the reward, which is why intravenous (IV) injection and smoking are the most addictive ways to administer drugs. However, the rate of increase in AD and changes in brain connectivity related to the rate of drug entry into the human brain have not been studied. To address this issue, we compared DA changes and resting-state functional connectivity patterns associated with intravenous and oral administration of methylphenidate (MP) using a double-blind, balanced, placebo-controlled study in healthy controls. Tested on three different days. We hypothesized that the rate and amount of DA release in the striatum would be greater for IV MP than for oral MP and that the rate of DA increase would be related to changes in HR and subjective MP reward.

Methods:Fifteen healthy participants (36.7 ± 9.5 years, 7 females) underwent a 90-minute simultaneous resting-state PET/fMRI scan to assess dynamic increases in DA, CF, and reward ("getting high") in a 1-minute time resolution , using MP as a challenge in 3 random sessions. In each session, participants received an oral pill (60 mg-MP or placebo) 30 minutes prior to bolus injection (11C-raclopride, 16 mCi) and i.v. (0.25 mg/kg-MP or placebo) 30 minutes after bolus administration. The dynamic increase in DA was calculated from the time-varying differences in the standardized value of the uptake index (SUVr) for the cerebellum between placebo and MP (oral/iv) conditions. fMRI time series (3 mm isotropic, TR=3 seconds, 1800 time points) were re-aligned, warped corrected and spatially normalized in MNI space using human connector design image pre-processing channels and subjected to linear regression. Standard motion and bandpass filtering 0.01 - 0.10 Hz. Removed heart, breathing, and head movement artifacts. Whole-brain HR potential was calculated for each fMRI session using sliding window (long-range HR arrays) and voxel (short-range lFCD). Within-subjects ANOVA was used to test the main effects of elevated AD and MP (iv vs. oral) as well as AD-by-MP interaction effects in edge and voxel statistical analyses, while controlling for multiple comparisons with strict families of approach error (PI number-FWE < 0.05).

Results:Peak "high" scores were stronger for oral and intravenous MP than placebo (PI number<0.001). DA striatal growth over time was smoother and weaker with oral MP than with intravenous (PI number< 0.03, within subjects, one-tailed ANOVA) in which AD increased significantly after iv-PM, in parallel with participants' ratings of "high" in response to MP. The dynamic increase in DA was more sensitive than the traditional static measurement of DA release for detecting differences between drug states. Perceived reward (maximum "high" scores) and increased putamen AD at the time of intravenous injection were correlated during oral administration.R= 0,7;PI number= 0.004), but not during IV-MP. The rate of change of putamen AD in the first 20 minutes after IV injection was greater for IV than for oral MP (PI number< 0.01, two-sidedT-test). The rate of DA change in other regions of the striatum was not significant. Similarly, the decline in mean HR from baseline among IV injection subjects was faster and stronger with IV than with oral MP (PI number< 2E-16), in parallel with the faster increase in DA observed after iv-MP. An MP-sensitive network showing a significant negative correlation in 125 edges between FC power and putamen DA release emerged from long-range array network analysis. Mainly, iv-MP reduced subcortical (53 edges) and motor (49 edges), visual (25 edges) and salience (22 edges) network connectivity compared to placebo (PI number-FWE < 0.05; Bonferroni corrections for 35778 edges). Although HR reductions were greater for iv MP than for oral MP, the association between DA increases and mean MP network connectivity was stronger for iv oral MP (PI number< 2E-08), such that for a given increase in DA, the decrease in HR was greater for oral MP than IV MP, suggesting a greater contribution of additional neurotransmitters to MP IV (i.e., noradrenergic stimulation). The rate of change in HR in the first 20 minutes after intravenous injection did not differ between oral and intravenous PM. Time-varying increases in striatal DA were also associated with time-variable decreases in IFCD in the default mode network (DMN), motor, parietal, temporal, and occipital cortex, and with increases in IFCD in orbitofrontal cortex and cerebellum for IV-MP. but less for oral MP (PI number-FWE < 0.05).

Conclusions:Using simultaneous PET-MRI, we show for the first time in the human brain that DA release is faster with iv-MP than with oral MP. Increased striatal DA was accompanied by increases in self-reported "high" and decreases in HR of subcortical and motor networks that are modulated by AD. The decrease in HR from MP likely reflects a dominant DA stimulation of D2 receptors, which are inhibitory but likely also influenced by the noradrenergic effects of MP. Overall, the results support our working hypotheses that the increase in DA would be faster with IV MP than with oral MP and that dynamic changes in striatal DA are related to dynamic changes in FC.

Keywords:Reward, dopamine receptors (D2, D3), resting-state functional connectivity, simultaneous PET-MR, methylphenidate

Revelation:Nothing to disclose.

National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, USA

Bottom:The negative impact of the COVID-19 pandemic on stress and mental health, both locally and globally, has been well publicized in the media, while more scientific data on the specific effects of stressors associated with the pandemic (e.g. (e.g. social isolation, financial insecurity, Perceived stress, the extent to which an individual perceives life events as stressful, is an important metric to consider in the context of a pandemic as it includes emotional responses and coping with life challenges. stress management and coping are well documented in the literature and are influenced by a variety of factors, including childhood traumas, personality, and history of AUD.This study had three objectives: 1) to explore changes in stress levels related to the pandemic, pandemic and alcohol use disorder (ADD) status; 2) assess whether exposure to childhood trauma is associated with increased perceived stress during the pandemic; 3) examining the relationship of perceived stress with alcohol use during the pandemic and limiting the role of AUD status in this relationship.

Methods:From June 2020 to March 2021, a group of participants who had previously participated in NIAAA clinical trials were invited to participate in a longitudinal study to assess the impact of the pandemic on alcohol consumption and related outcomes. Following consent, participants completed a survey to obtain pre-pandemic baseline data ("In the month before the pandemic [February 2020]...") and impact data ("In the last month..."). Due to ongoing recruitment, basic impact data was collected during various "waves" of the pandemic among participants: Wave 1: 3/11/2020-7/31/2020. Wave 2: 01/08/2020-22/11/2020; Wave 3: from November 23, 2020. The survey included, among others, the Perceived Stress Scale (PSS) and the Alcohol Use Disorders Identification Test (AUDIT). The current analysis also included screening data from when participants were initially enrolled in NIAAA clinical trials, including the PSS, Childhood Trauma Questionnaire (CTQ), and the Five Factor NEO Personality Inventory. Participants in the current study (N= 390; 183 women, 207 men) represented a wide range of alcohol consumption, from non-drinkers to those with severe AUD (219 without AUD, 171 AUD). Perceived stress scores were compared between pre-pandemic and pandemic time points using mixed linear models. Using path analysis, the associations between childhood trauma (as measured by five CTQ subscales, including emotional abuse, physical abuse, sexual abuse, emotional neglect, and physical neglect), perceived levels of pre-pandemic and pandemic stress, and alcohol consumption during the pandemic were analyzed using path analysis. A multigroup pathway model was used to assess the moderating effect of AUD status on these associations.

Results:Overall, perceived stress levels were significantly higher during the pandemic compared to pre-pandemic (PI number< 0.0001). No interaction effect with the pandemic wave was observed, indicating that perceived stress levels were comparable across all three waves. There was a significant interaction between the time point of the pandemic and the AUD status (PI number= 0.03), so perceived stress levels were significantly elevated in non-AUD participants, but not in AUD participants. However, anxiety levels were higher in AUD compared to participants without AUD at both time points. These effects remained significant when controlling for perceived stress levels, CTQ scores, and the NEO neuroticism factor measured at baseline. Pathway analysis found that emotional abuse, sexual abuse, and emotional neglect were positively related to perceived stress levels during the pandemic. Sexual abuse had a direct effect on perceived stress and an indirect effect mediated by neuroticism and pre-pandemic stress levels. all effects of emotional abuse and neglect were indirect. Alcohol consumption during the pandemic (AUDIT-C score) was related to perceived pandemic stress and pre-pandemic alcohol consumption, with the latter showing a stronger effect. Multigroup pathway model analysis showed that a direct effect of sexual abuse on perceived episodic stress was observed only in AUD participants, and that there was a stronger association between episodic stress and alcohol use in AUD participants.

Conclusions:Perceived stress levels were elevated during the pandemic among participants at all points in the waveform where they were initially assessed, although our results suggest that this increase was only true for those without AUD. Exposure to childhood trauma was directly and indirectly related to increased perceived stress during the pandemic through neuroticism and pre-pandemic stress levels. These findings add to existing evidence that exposure to stress early in life can sensitize individuals to increased stress responses later in life. Finally, alcohol consumption during the pandemic, while mainly influenced by pre-pandemic drinking levels, was also associated with perceived pandemic stress, particularly in those with AUD. A better understanding of the trajectory and impact of the COVID-19 pandemic on alcohol and related effects could help design interventions that benefit those exposed to the negative impacts of the pandemic.

Keywords:COVID-19, early life stress, perceived stress, alcohol

Revelation:Nothing to disclose.

University of California - San Diego, La Jolla, California, United States

Bottom:A key question for opioid dependence research remains the question of why accidental drug use increases with problematic use associated with high motivation and compulsiveness, which characterizes substance use disorder in some individuals and not in others. Better characterization of individual differences in propensity to develop addiction-like behaviors could help identify new pharmacological targets, biomarkers, genetic variants and facilitate drug development. However, most studies used small sample sizes (N<8-20), examples of limited self-administration or animal models used with limited individual differences. Such limitations reduce the translational relevance of the results and limit the reproducibility and reproducibility of these studies. To address this issue, we characterized addiction-like behaviors with an advanced model of extended-access oxycodone self-administration in large cohorts of heterogeneous rats (HS) (N= XXX), a unique mouse strain with large individual differences.

Methods:HS rats were allowed to self-administer oxycodone for ~11 weeks for various time periods (2-12 h/day) and were also tested for oxycodone-induced anesthesia (tail), forced use of oxycodone using a progressive response (PR) and withdrawal. allodynia-like behavior (von Frey test) Each animal was also tested for sensitivity to methadone (0.5 mg/kg), naltrexone (3 mg/kg) and buprenorphine (0.5 mg/kg).N= 46–60) was used to minimize cohort-specific effects, and response rates were normalized within cohorts.TTests and ANOVAs were conducted to determine significant effects. With so many animals, small effect sizes (cohenHello= 0.2) can be detected with high power (1-b = 0.95, a = 0.05).

Results:Behavioral characterization showed large inter-individual differences in addiction-like behaviors, with little intra-individual variation. Final oxycodone intake showed a positively skewed distribution. Small differences were observed between the sexes, with women having a higher response rate (FR) than men (PI number< 0.01), but not in a progressive proportion (PI number<0.001). Male and female rats exhibited opioid-induced analgesia and deprivation-induced hyperalgesia/allodynia. There were significantly larger variations between individuals than between genders, with individuals showing resistance, moderate, moderate and severe addictive behaviors. For example, approximately 25% of the animals exhibited a resistant phenotype with a low increase in oxycodone uptake, low opioid-induced analgesia, intolerance to oxycodone-induced analgesia, and low withdrawal-induced hyperalgesia/allodynia (allPI number< 0.01). We found that 65% of the rats decreased their motivation to take oxycodone after at least one treatment (PI number< 0.05). There was considerable individual variation in response to each treatment, with individuals being sensitive to one, two, or all three drugs. Buprenorphine and naltrexone were associated with the highest number of responders, with approximately 8% of patients responding to all three medications.

Conclusions:To our knowledge, this is the most extensive study of intravenous oxycodone self-administration in rats. We identified individuals with resistant, mild, moderate, and severe opioid-dependent behaviors in over 400 heterogeneous rats. Low sensitivity to oxycodone analgesia and lack of tolerance were strong predictors of resistance to the development of addictive-like behaviors, while withdrawal-induced hyperalgesia was only observed in animals with moderate to severe addictive behaviors. Pharmacological tests show individual differences in response to drugs used to treat opioid use disorder in a genetically diverse population of mice. Along the way, a wide variety of biological samples were collected and made available to the community through the Oxycodone Biobank to facilitate the identification of biomarkers of addiction-like behavior and to facilitate reproducibility and reproducibility.

Keywords:Addiction, Opioids, Self-control, MAT, Pain

Revelation:Nothing to disclose.

Oregon Health & Science University, Portland, OR, USA

Bottom:Introduction: The development of alcohol use disorder (ADD) is closely related to the onset of drinking alcohol in adolescence. A better understanding of the effects of alcohol exposure on the adolescent brain in the context of motivated behavior is critical to understanding the etiology and pathophysiology of ASD. Deficits in response inhibition are a critical feature of AUD and are a clinically significant endophenotype of the disorder. However, little is known about the functional and mechanistic consequences of alcohol consumption in adolescence on response inhibition and related cognitive measures in adulthood.

Methods:We used an adolescent moderate voluntary consumption model in conjunction with a newly developed response inhibition task (CRIT) to assess response inhibition, stimulus-response relationships, attention processes, and learning in adolescent or adult male and female rats. This method was combined with a single unit and local field dynamics in two brain regions involved in response inhibition, the orbitofrontal cortex (OFC) and the dorsal striatum (DS). The dependent variables for the behavioral analyzes included the number of correct and premature nudges, the number of nudges and downstrokes during the interval between trials, latency to premature nudge completion, and the ratio of correct to incorrect responses (response inhibition index). Analysis of variance (ANOVA) was performed for all dependent variables with factors of sex, reward and age. The units' firing rate was assessed during the following periods of interest: correct responses, premature responses, reward performance, and inhibition tone presentation. All burn rate data were analyzed by ANOVA. To determine how individual neurons in a network affect the firing rate of other neurons in the same network depending on a behavior or event, we compute peak correlation and evaluate group differences using an ANOVA test. To investigate neural synchrony between the two brain regions, a γ-phase lock index was calculated and then differences between the groups were assessed using an ANOVA test.

Results:We found that adult rats that voluntarily drank ethanol during adolescence were (nonsignificantly) nose poked more frequently between trials, showed more premature responses, and showed reduced response inhibition compared to sucrose-exposed controls. Our analysis of registration data revealed that rats exposed to ethanol have an increased reward response in OFC and an increased response in DS during early response.

Conclusions:Since cortical circuit dysfunction is a critical feature of AUD, these translational results will improve our mechanistic understanding of the brain changes that occur in these circuits as a result of ethanol exposure during adolescence.

Keywords:Adolescent alcohol use, electrophysiology, frontal cortex (OFC), dorsal striatum, response inhibition

Revelation:Nothing to disclose.

SUNY Brooklyn Health Sciences Center, Brooklyn, NY, USA

Bottom:Characterizing whether genetic variants for psychiatric outcomes work through specific or general pathways provides more information about genetic risk and potentially allows us to design more targeted prevention and interventions. We used multivariate methods to separate variants associated with problem alcohol use according to general or pathway-specific pathways and compared the results with standard univariate genetic analysis of problem alcohol use.

Methods:We compared the results of the univariate genome-wide association study (GWAS) of problematic alcohol use with the results from the previous multivariate GWAS of externalizing phenotypes. We identified genetic variants associated with problem alcohol use by broad vulnerability to externalization and those that remained after removing the common variance with externalization. We compared these results for SNP overlap, bioannotations, genetic linkage, and polygenic scores. We have included summary GWAS statistics from the existing GWAS and two US samples: the National Longitudinal Study of Adolescent Health in Adults (Health Supplement) and the Cooperative Study of the Genetics of Alcoholism (COGA). Results included problem alcohol use (ALCP)-O), common risk of externalization (EXT) and specific risk of problem alcohol use (ALCP-S) for GWAS. a pre-registered list of 99 phenotypes available for genetic associations, and criteria for substance use, substance use disorders, and alcohol abuse in polygenic outcome analyses.

Results:The analysis distinguished between SNPs operating under common and specific risk pathways. While ALCP-O has been associated with multiple phenotypes, ALCP-S has been primarily associated with alcohol use and other forms of psychopathology. Polygenic scores for ALCP-O were associated with various other forms of substance use and substance use disorders, polygenic scores for ALCP-S were associated with alcohol phenotypes only. Polygenic scores for ALCP-S and EXT show different patterns of association with alcohol abuse throughout development.

Conclusions:Focusing on the differential effects of general and specific risk may better characterize risk pathways for alcohol use disorders. Multidimensional methods can be a useful tool in the study of many psychiatric conditions. Risk pathway analysis will grow in importance as genetic information is incorporated into clinical practice for psychiatric outcomes.

Keywords:Alcohol and substance use disorders, psychiatric comorbidity, multivariate analysis

Revelation:Nothing to disclose.

California State University, Long Beach, Long Beach, California, United States

Bottom:Ethanol is the drug of choice among teenagers in the United States. Adolescence is a critical period in brain development, making young people particularly vulnerable to the various consequences of ethanol use. Previous literature using the preferred place (CPP) paradigm, a validated animal model for drug reward, revealed the difficulty of reliably demonstrating ethanol preference in adolescent rats, possibly due to sex and dose-related differences. We therefore hypothesized that adolescent male and female rats would exhibit dose-dependent differences in ethanol preference.

Methods:Rats underwent a 10-day CPP procedure to assess ethanol preference during adolescence (experiment 1) and adolescence (experiments 2 and 3). On the first day of conditioning, initial preference for the dual-chamber CPP apparatus was assessed during 15-minute (experiments 1 and 2) or 20-minute (experiment 3) sessions. On days 2–9 of conditioning, rats were conditioned with either saline in the initially preferred chamber or ethanol in the initially non-preferred chamber every other day for 15 minutes. On day 10, after conditioning, preference for the ethanol vapor chamber was assessed using the same procedures as on the first day of the experiment. In experiments 1 and 2, rats were randomly assigned to be injected with ethanol (0.0, 0.5, 1.0 or 2.0 g/kg, IP,N= 7-11) before being placed in the ethanol coupling chamber. In the experiment, 3 rats were randomly assigned to be injected with ethanol (0.0, 0.0156, 0.0313, 0.0625, 0.125, 0.5 or 2.0 g/kg, IP,N=8-11) before placing in the ethanol vaporization chamber. The preference score was calculated by taking the time spent in ethanol vapor during post-conditioning minus the time spent in ethanol vapor during conditioning. The CPP was defined as having a significantly higher preference score compared to the saline controls (group comparisons using Tukey's ANOVA and post hoc, where applicable) and/or a significant increase in time spent on the ethanol side of the pair between conditioning trials and after conditioning (intra-group assessments using scheduled comparisons).

Results:In Experiment 1, male and female rats exhibited ethanol-induced CPP when given the highest dose of ethanol (2.0 g/kg), which was seen as a significant change in preference for the paired ethanol side in males (PI number< 0.05) and a significantly higher preference score compared to saline controls in women (PI number< 0.05). In Experiment 2, gender differences emerged when female rats still preferred the high dose of ethanol (2.0 g/kg) and male rats showed a moderate preference for the ethanol chamber combined with the lowest dose of ethanol (0.5 g/kg). In Experiment 3, male rats showed a strong preference for ethanol when given low doses of ethanol (0.0625, 0.125 g/kg), as evidenced by a significantly higher preference score compared to saline controls (PI number< 0,05).

Conclusions:Overall, gender differences did not emerge during adolescence as the rats showed similar preferences for ethanol regardless of gender. However, during puberty, a change in preference was observed in male rats that exhibited ethanol-induced CPP at low doses of ethanol. In conclusion, ethanol-induced CPP can be reliably demonstrated in adolescent rats, which may help to better understand the brain mechanisms underlying adolescent ethanol use.

Keywords:Alcohol use disorder and drug addiction, adolescence, alcohol, reward, development

Revelation:Nothing to disclose.

University at Buffalo, Buffalo, New York, United States

Bottom:Susceptibility to addiction is influenced by genetic and environmental factors, and is also associated with non-drug-related traits, including response to food-related cues. Using more than 3,200 heterogeneous breeding (HS) mice tested at the University of Buffalo and the University of Michigan, we performed a genome-wide association study (GWAS) to identify regions of the genome that influence the response to food-related stimuli in the air-conditioning example. Pawlowski.

Methods:In this example, the conditioned stimulus (highlighted lever) predicted the delivery of a food pellet in the food store (25 trials per day for 5 days). Lever contacts (cue tracking) and magazine contacts (target tracking) and the last two days were the main metrics analyzed by GWAS, although other metrics such as response error, latency, probability and PavCA index were also obtained. We have identified several loci associated with these agents.

Results:One of the strongest signal-monitoring loci was identified on chromosome 1, which contained a group of amine receptor (Taar) genes. Previous genetic and pharmacological studies of TAAR1 indicate that this receptor is involved in the reinforcing effects of methamphetamine, cocaine and nicotine. To test whether TAAR1 is involved in the development and expression of PavCA phenotypes, we administered the TAAR1 agonist RO5262297 to a subgroup of HS mice tested in this paradigm. RO5262297 restricted the development and expression of tracking cues but not target tracking, and cues and target tracking were also differentially sensitive to the hypothermic effects of this drug.

Conclusions:These studies provide pharmacological evidence for Taar1 as a candidate gene underlying the monitor-monitor phenotype and further suggest that individual differences in Taar1 activity have a similar effect on monitor-monitor and drug reinforcement.

Keywords:Pavlovian conditioning, self-administration, signal monitoring, trace amine-associated receptor 1, methamphetamine

Revelation:Nothing to disclose.

University of Texas School of Pharmacy in El Paso, El Paso, Texas, United States

Bottom:Targeted advertising, the addition of flavorful flavorings, and misconceptions about safety have led to a dramatic increase in recreational e-cigarette use over the last 10 years. Higher concentrations of nicotine, higher rates of use, and unique additives and metabolites lead to different pharmacological properties of nicotine delivered via e-cigarettes and highlight the need for research into the effects of nicotine vapor on the brain and behavior. Preclinical studies investigating the effects of nicotine have primarily used nicotine administration via subcutaneous injections or pumps. However, more recent studies in rodents have begun to investigate the neurochemical and behavioral consequences of nicotine by administering it via vapor inhalation. The purpose of this study was to 1) verify an emerging model of nicotine vapor electronic inhalation in our laboratory, evaluate nicotine addiction after cessation of repeated nicotine vapor exposure in rats, and 2) examine the effects of nicotine vapor nicotine exposure on cost-benefit decision and subunit gene expression of the acetylcholine receptor (AChR) in the striatum and cerebral cortex areas of the rat.

Methods:In Experiment 1, fifty-six adult male Sprague Dawley rats were exposed to 0, 12, or 24 mg/mL nicotine vapor for 9 days. Cotinine levels were assessed on day 6, physical withdrawal symptoms after mecamylamine withdrawal withdrawal on day 7, and anxiety-like behavior after mecamylamine withdrawal withdrawal on exposure day 9. A separate group of twelve rats were implanted with stimulating electrodes in the medial forebrain bundle and trained in intracranial self-stimulation (ICSS). The effect of spontaneous nicotine vapor withdrawal on ICSS thresholds was then assessed. For Experiment 2, twenty-four adult male Sprague Dawley rats were trained in the probability-to-stability reduction task. After training, rats were exposed to 0 or 24 mg/mL nicotine vapor for 10 consecutive days, with discounting task tests immediately after daily exposure. Animal brains were harvested and rt-PCR was used to assess gene expression of the AChR α4 and β2 subunits in the ventral striatum and prefrontal cortex.

Results:Male rats exposed to 24 mg/mL nicotine vapor showed increased cotinine levels, physical withdrawal symptoms, anxiety-like behaviors, and ICSS thresholds compared to 0 mg/mL controls. Additionally, compared with the 0 mg/mL control group, rats exposed to 24 mg/mL nicotine vapor showed an immediate and short-term increase in risky choices, as well as an increase in the expression of genes encoding the AChR β2 subunit, d1 dopamine receptor, and d2 dopamine receptor in medial prefrontal cortex.

Conclusions:The observed increases in ICSS thresholds after cessation of repeated exposure to nicotine vapor extend the existing literature suggesting that e-cigarette use can lead to nicotine dependence and withdrawal symptoms similar to those seen in smokers of traditional cigarettes. The results also provide data demonstrating nicotine vapor-induced changes in AChR subunit gene expression in the cerebral cortex that may underlie changes in cost-benefit decision making observed in smokers. The proposed work helps create a much-needed preclinical rodent model for human e-cigarette use and identifies potential novel mechanisms that drive nicotine's influence on decision-making. Additional information on the effects of nicotine vapor exposure on the brain and behavior will be needed to develop effective government regulations and educational campaigns to reduce recreational use of e-cigarettes.

Keywords:Nicotine vapors, model systems, nicotine addiction, decision making, a4b2 receptors

Revelation:Nothing to disclose.

University of Kentucky, Lexington, Kentucky, United States

Bottom:Due to the alarming increase in the number of women, the disparity in alcohol consumption among women is closing fast. Therefore, it is important to identify potential risk factors for women underlying alcohol use disorder (AUD). Data from our lab and others suggest that poor inhibitory control, both in sober and under the influence of alcohol, is a stronger risk factor in women than in men. We have previously studied the neurobiological factors underlying poor inhibitory control in heavy drinkers and found less brain activity during the inhibitory response in the early follicular phase of the menstrual cycle when sex hormones (estradiol and progesterone) are low. Here, we extend these findings to investigate the neural and hormonal factors underlying susceptibility to the effects of alcohol on brain activity during abstinence in heavy drinkers.

Methods:Heavy drinkers performed a stop task to assess inhibitory control during four sessions of functional MRI. Women were studied while taking intravenous alcohol (60 mg%) and saline infusion during both the early follicular phase (low estradiol and progesterone) and the mid luteal phase (moderate estradiol and high progesterone) of the menstrual cycle. Women also reported subjective sensations of stimulation and sedation at the beginning and during the injection. Blood samples were taken to assess serum estradiol and progesterone levels before and after infusions in all sessions.

Results:Data collection is ongoing and so far 7 women have completed the study. Initial analyzes confirmed low hormone levels in the early follicular phase (mean estradiol in alcohol and saline sessions = 28.5 ng/mL; mean progesterone = 0.44 ng/mL) and moderate to high in the mid-luteal phase (mid (mean estradiol   93 = 6.mL, mean progesterone     13.2 ng/mL). The effects of alcohol on the neural correlates of inhibition varied according to the phase of the menstrual cycle. Specifically, alcohol decreased brain activity during inhibition compared to saline in the right frontal areas, including the right accessory motor area, inferior frontal gyrus, insula, and mid frontal gyrus during the early follicular phase, while alcohol increased brain activity during the mid luteal phase. Moreover, alcohol increased subjective arousal in the early follicular phase, while alcohol increased suppression in the middle luteal phase.

Conclusions:These data suggest that variable estradiol and progesterone levels influence susceptibility to the inhibitory and subjective effects of alcohol in heavy drinkers. Greater inhibition and arousal after alcohol consumption is associated with an increased risk of heavy drinking. Thus, these preliminary findings suggest that the early follicular phase, when hormone levels are low, may be a time of increased risk for heavy drinking in women. Identifying these periods of vulnerability to problem drinking, as well as the related brain mechanisms, may have important implications for the prevention and treatment of AUD in women.

Keywords:Women's health, alcohol, impairment control

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:Substance use disorders represent a massive public health crisis and are among the most difficult to treat diseases in our society. Research into the molecular pathology of addiction continues to focus on the mechanisms by which neural gene regulation is altered in the brain's central reward region, the nucleus accumbens (NAc). Stable chromatin changes are thought to underlie maladaptive transcriptional states in this brain region that persist despite prolonged drug withdrawal. However, there is no direct link between drug-induced epigenetic signals and abnormal gene expression programs leading to relapse. The key challenge is to determine which neuronal subtypes are responsible: the NAc consists mainly of two opposing types of medium spiny neurons (MSNs), dopamine receptor-expressing subtypes D1 and D2, which show dramatic differences in dopamine activity and rewarding effects. Phew. Within these distinct subtypes, we study how chronic cocaine modifies chromatin structure and characterize immediate and persistent changes in gene regulation.

Methods:The NAc mainly comprises (>90%) two functionally distinct MSN subtypes, making the cell type-specific identification of epigenetic alterations crucial. In recent years, the determination of transposase-accessible chromatin by sequencing (ATAC-seq) has become an essential tool in epigenomic research. It is used to assess genome-wide chromatin structure to detect "open" chromatin regions indicating active transcription or gene initiation. Here, we define chromatin accessibility in D1 and D2 MSNs using fluorescence-activated nucleus sorting (FANS) coupled with ATAC-seq and RNA-seq. Combined with objective profiling of histone modifications by mass spectrometry and ChIP-seq, we distinguish between immediate and long-term changes in chromatin and gene expression. In particular, we characterized persistent, circuit-specific changes in chromatin composition with prolonged abstinence (30 days) after chronic cocaine exposure (10d cocaine ip).

Results:We found that chronic cocaine persistently alters chromatin structure in MSN D1, including dramatic depletion of the histone variant H2A.Z - a newly identified memory suppressor - in key neuronal genes related to synaptic plasticity. Genome accessibility is significantly increased in these genes even after prolonged discontinuation, which is associated with aberrant gene expression after drug relapse. The chaperone ANP32E histone promotes H2A.Z clearance, and we show that selective inhibition of the D1 circuit by ANP32E prevents cocaine-induced H2A.Z depletion and effectively blocks cocaine-dependent place preference. In contrast, ANP32E knockdown for D2 enhances cocaine-related reward learning in this animal model.

Conclusions:Our research explores the emerging view of epigenetic adaptation that may promote substance use disorders, providing new insight into genetic facilitation as a key mechanism by which drugs alter brain function and behavior. In particular, our data indicate that cocaine-induced chromatin remodeling involving the H2AZ histone variant underlies a persistent effect on circuit gene regulation in the NAc. Collectively, these findings support epigenetic priming as a critical mechanism and a promising clinical target for drugs to engage in sustained brain function and behavior modification.

Keywords:Epigenetics, chromatin modifications, genetic priming, histone variations

Revelation:EpiVario, Inc.: Fundador (Auto)

National Institutes of Health, Bethesda, Maryland, Stany Zjednoczone

Bottom:The faster an addictive drug enters the brain, the greater its effects will be. Therefore, drugs taken by routes of administration that result in rapid delivery to the brain, such as intravenously (IV), are more satisfying than those taken orally, resulting in slower delivery of the drug to the brain. However, it is not clear how human brain function changes depending on the rate at which the drug is released into the brain, which is related to the mechanisms underlying its rewards. Here, we use a simultaneous PET-fMRI to examine brain activity and dopamine signaling for IV (0.25 mg/kg) versus oral methylphenidate (MP) (60 mg) for doses that achieve comparable brain dopamine transporter blockade, in a double-blind trial, a balanced, placebo-controlled study in healthy adults. We hypothesized that oral and IV MP would decrease and increase striatal activation, respectively, as slow dopamine release would likely stimulate high-affinity inhibitory D2 receptors, whereas fast dopamine release would be sufficient to transiently stimulate low-affinity excitatory receptors. Affinity for the D1 receptor.

Methods:Fifteen healthy subjects (36.7 ± 9.5 years, 7 females) simultaneously underwent a PET/fMRI study of resting 11C-raclopride (16 mCi) on a 3T Siemens Biograph mMR scanner for 90 minutes to assess "high" dopamine changes in striatum in response to PM in 3 different sessions (placebo, oral MP or IV MP). Dynamic PET images were reconstructed using the 3D OSEM algorithm and corrected for scattering and attenuation using a fully convolutional neural network. Changes in dopamine in the striatum were calculated from differences in the standardized value of the uptake index (SUVr) for the cerebellum between the placebo and MP (oral/IV) conditions. fMRI time series (3 mm isotropic, TR=3 seconds, 1800 time points) were realigned, warped corrected and spatially normalized in MNI space using Human Connectome Project image preprocessing channels and linearly regressed. standard motion and bandpass filtering 0.01-0.10 Hz. To model brain activation responses to oral and IV MP, we fitted variable gamma functions to BOLD time series in a general voxel brain linear modeling analysis. in pairsT-tests were used to test the main effects of treatment (oral or intravenous MP vs. placebo) on changes in striatal dopamine and brain activity, controlling for multiple comparisons (pFWE< 0.05). We also used within-subjects repeated-measures ANOVA to test for side effects in the case of "high" scores.

Results:IV MP produced a stronger "high" than oral MP with faster rise and fall, repeating the previous work (PI number< 0.05). Consistent with our hypothesis, IV MP induced a relatively rapid (∼5 min) pattern of increased activation in the striatum, with bilateral clusters centered on the caudate head, whereas oral MP induced a slow and steady pattern of reduced activation in the ventral, ventral, with additional clusters in the ventral /ventral prefrontal cortex and amygdala/hippocampus.

Conclusions:We found that "fast" versus "slow" administration of PM induced positive and negative patterns of striatal activation, respectively, that resembled different temporal patterns of subjective euphoria ("high") for intravenous and oral PM. Thus, the rate rather than the overall magnitude of the dopamine increase may drive different patterns of brain activation that contribute to the rewarding effects of the drug. We hypothesize that the divergent patterns of striatal signaling may reflect mainly excitatory D1 signaling for IV MP versus inhibitory D2 signaling for oral MP.

Keywords:Dopamine, drug abuse, simultaneous PET-MRI

Revelation:Nothing to disclose.

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Bottom:Cognitive flexibility is the act of adjusting behavior in response to changes in reward conditions. Deficits in cognitive flexibility are widespread in mental disorders and are core and prognostic features in disorders such as depression, bipolar disorder, addiction, and schizophrenia. For example, inability to stop drug use despite negative consequences may be due to loss of flexible behavioral control, and the severity of cognitive flexibility deficits in adolescence predicts the onset and severity of mood symptoms in depression and bipolar disorder. The circuits regulating cognitive flexibility have been well studied, highlighting the need for dopamine (DA) release and activation of receptors in the ventral or dorsal cortex and striatum. However, these studies usually measure flexibility after 1–2 sessions after learning the initial discrimination, and few have explored ways to restore flexibility after overtraining. This knowledge gap is important because rewarded behaviors that are repeated eventually become stereotyped and outcome-insensitive habits, coinciding with a shift in relative DA release patterns from the ventral to the dorsolateral striatum. This suggests that the circuitry that regulates cognitive flexibility after learning may be different from restoring flexibility when behavior is well-learned, particularly with regard to AD regulation. Previous studies have suggested a role for the medial septum (MS) and its ability to increase the activity of the DA population in the ventral tegmental area (VTA) and decrease the activity of the DA population in the substantia nigra (SNc) in cognitive flexibility. However, the extent of this role and how it may be affected by education level is unclear.

Methods:To investigate this question, I trained male and female rats to distinguish between the sides of a touchscreen device in 100 trials per day for 1, 10, or 15 days. The day after training, we activated the MS (systemic CNO) or prefrontal cortex to MS (intracortical CNO) pathway with DREADD. Thirty minutes later, we measured their performance in a scenario-switching task in which they were asked to switch from a learned lateralization rule to a visual discrimination rule (e.g., correct). correct). The number of trials and errors made during the transition to the new rule (quantified as 10 consecutive hits) was recorded. A separate group of male and female rats were trained for 15 days and then pharmacologically prevented from multiple sclerosis-induced effects in the VTA, SNc or ​​both immediately after systemic injection of CNO but prior to the test trial. This was done by bilateral injection of scopolamine, which we have previously shown selectively prevents the MS-mediated increase in the activity of the VTA DA population, bicuculin, which selectively prevents the MS-mediated decrease in the activity of the SNc DA population. or both in the ventral hippocampus. Thirty minutes after CNO injection and ten minutes after injection into the ventral hippocampus, the rats performed the same reconfiguration task described above.

Results:Male and female rats that performed the reconfiguration task after 1 day of discrimination training showed similar results and no MS activation effect (gender-matched, tests:PI number= 0.71, errors:PI number= 0.57). However, after 10 days of training, activation of the PS or pre-SC pathway significantly improved kit switching performance in female rats, reducing the number of trials (PI number= 0.0005) and errors (PI number= 0.001) were involved in meeting the criterion of 10 consecutive correct attempts. This effect was not observed in male rats (PI number= 0.18 and 0.11 respectively). After 15 days of training, MS activation improved configuration switching performance in male and female rats to a similar extent, again reducing the number of trials (gender-matched,PI number= 0.041) and errors (mixed gender,PI number= 0.018). These effects were mediated by the MS modulation of VTA and SNc DA activity, as preventing the MS-mediated reduction in activity in the SNc DA population and increasing activity in the DA VTA population abrogated MS activation-induced enhancement in conformational changes in both sexes. Simply preventing the activity of the SNc DA population from declining or increasing the activity of the DA VTA population reduced MS activation-induced amplification of configuration change but did not eliminate it. The difference in the effectiveness of MS activation in male and female rats can be explained by the rate of learning during training. Female rats averaged >92% correct on discrimination training on fewer days (training day 4) than males (training day 6), and more days on this performance criterion predicted poorer control and better performance. Activate MS.

Conclusions:These data suggest that multiple sclerosis activation may be more effective in supporting flexibility as more training is added. They also suggest that multiple sclerosis, through bidirectional regulation of VTA and SNc DA activity, may be a critical relay from the prefrontal cortex to the midbrain, where flexibility is restored when behaviors are well-learned.

Keywords:Cognitive flexibility, dopamine, medial septum, gender differences, top-down control

Revelation:Nothing to disclose.

University of Linköping, Linköping, Szwecja

Bottom:The central amygdala (CEA), a nucleus composed mainly of inhibitory GABA neurons, plays an important role in alcohol dependence behaviors, including alcohol preference, escalation of self-administration induced by withdrawal or addiction, and alcohol versus alcohol choice. awards. We recently discovered that in this structure, PKCδ-mediated signaling is a potential mechanism behind individual differences in susceptibility to binge drinking. To determine the functional role of the CEA-Prkcd chips and their output connections in alcohol-related behavior, we developed and validated a new transgenic Prkcd-Cre Knock-in mouse model.

Methods:The Prkcd-IRES-Cre Knock-in mouse strain was developed by genOway by applying CRISPR nuclease technology to the Wistar genetic background. The presence of the knock-in construct in the offspring was confirmed by PCR and DNA sequencing, and further crosses with wild-type Wistar rats confirmed embryonic transmission. To confirm the specific neuronal expression of Cre and PKCδ and further validate the input and output targets of CeA PKCδ +  projections, we used Cre-dependent adeno-associated viral screening in conjunction with immunohistochemical staining. Forward AAV5-hSyn-DIO-mCherry and reverse viral marker AAVretro-hSyn-DIO-EGFP were bilaterally injected (0.5 μl, 0.25 μl/min) into the CeA of Prkcd-Cre mice (N= 5/group). We then examined the electrophysiological properties of PKCδ+ neurons in sharp slices of the amygdala. Finally, female and male Prkcδ-Cre mice and their wild-type counterparts (N= 10–17/group) were assessed for alcohol self-administration and a range of control behaviors.

Results:In CeA, Cre expression was restricted to PKCδ+ neurons and was detected in approximately 62% of all PKCδ+ neurons. Insertion of the Cre cassette into the Prkcd locus did not affect the overall expression patterns of PKCδ in CeA. PKCδ+  expression was approximately 3.5-fold higher in CeL compared to the central medial amygdala (CeM), consistent with PKCδ expression patterns in CeA in mice. The most prominent targets of the baseline CeL-PKCδ+  projection included CeM, orbitofrontal and insular cortex, BNST, hypothalamus, ventral pallidum, and terminal striatum. In addition, we found direct projections of CeL-PKCδ +  to midbrain dopaminergic nuclei and brainstem projections, particularly to the ventrolateral columns of the periaqueductal grey, which were mainly attributable to the CeL SOM +  subpopulation of neurons. We confirmed retrograde labeling in the insular cortex, BNST, basolateral amygdala, multiple peritonsillar nuclei, and rostromedia tegmental nucleus. However, we did not find input from the nucleus accumbens, previously described as the main input region for CeL PKCδ +  neurons in mice.

Electrophysiological recordings showed similar passive electrophysiological properties in CeL-PKCδ +  and PKCδ− neurons. Optogenetic stimulation (5 ms) induced postsynaptic inhibitory currents in all CeL neurons, which were completely abolished by pretreatment with picrotoxin (100 μM), indicating their GABAergic nature. Furthermore, we found that optogenetic activation of CeL PKCδ+ neurons inhibited the firing activity of PKCδ− neurons, and this effect was abrogated by pretreatment with picrotoxin (100 μM).

Prkcd-Cre rats self-administered similar levels of alcohol enhancers compared to Wistars and there was no effect of genotype in either sexeat(1,81) = 1,1;PI number= NS). Unsupervised grouping identified two subpopulations that responded differently to punished alcohol self-administration in male and female Prkcd-Cre rats, consistent with our recent findings on the bimodal distribution of susceptibility to binge drinking. Prkcd-Cre mice were not impaired when tested on various control behaviors including locomotor activity, anxiety and memory.

Conclusions:Our findings show that Prkcd-Cre Knock-in mice represent an important genetic tool to study the properties and function of PKCδ+ neurons, as well as cell-type-specific manipulations of PKCδ+ neuronal circuits in various mouse behavioral models.

Keywords:Central amygdala, PKCdelta, transgenic mice, alcohol and substance use disorders

Revelation:Nothing to disclose.

South Carolina Medical University, Charleston, South Carolina, United States

Bottom:Hypofrontism refers to the pathological condition of reduced frontal cortex function seen in several neuropsychiatric disorders, including substance use disorders. In abstinent and methamphetamine (METH) addicts, hypofrontality is associated with deficits in working memory and attention, and increased impulsive behavior, which reduces quality of life and increases vulnerability to relapse. However, the mechanisms underlying hypofrontal induced METH remain unclear. In the medial prefrontal cortex (mPFC), the perineural networks (PNNs) contain extracellular matrix (ECM) glycoproteins consisting mainly of parvalbumin-positive fast-spiking interneurons (PV+FSI). PNNs are involved in synaptic plasticity in response to experience, and PNN-related genes are dynamically regulated by neuronal activity.

Methods:Two-month-old Long Evans rats received daily (i.p.) injections of either saline or METH for 14 days (1 mg/kg on days 1 and 14; 5 mg/kg on days 2-13) followed by 7 days of forced cage abstinence in home. In a separate study, LE rats were allowed to self-administer METH (0.02 mg/injection) using a fixed ratio of 1 intravenous operant response for 15 days until stable intake was established. PNNs are labeled using WFA staining together with anti-parvalbumin immunohistochemistry. Using sharp ex vivo sections, whole-cell patch-clamp recordings were made from pyramidal neurons in the deep layers of the prefrontal cortex (V/VI). Inhibitory induced postsynaptic currents (eIPSC) were isolated pharmacologically by adding CNQX and APV. To digest PNN, chondroitinase ABC (chABC) was injected bilaterally into the mPFC. Time order memory (TOM) was assessed with a standardized design involving exploration of 2 identical objects for 5 minutes (phase 1), followed 30 minutes later by exploration of another pair of identical objects (phase 2) and 30 minutes later, the rat is allowed to explore one object from each set for 5 minutes (trial) and the preference ratio is calculated.

Results:We found that repeated unconditional METH administration or conditional METH self-administration induced a significant increase in the proportion of PV+FSI surrounded by PNN in the mPFC, but no change in PV+FSI density. This increase in PNN mPFC was associated with an increase in PV+FSI intrinsic excitability and an increase in the amplitude of evoked and spontaneous IPSCs in deep-layer pyramidal neurons in the mPFC. Moreover, chronic exposure to METH induced deficits in the mPFC-dependent temporal sequence memory (TOM) task, but this deficit was rescued by enzymatic digestion of the mPFC PNN in rats chronically exposed to METH. Similarly, digestion of mPFC PNNs by chABC normalized the amplitude of evoked IPSCs recorded from pyramidal neurons of the mPFC deep layer in METH mice.

Conclusions:Collectively, our data indicate that chronic quota or no METH exposure increases the proportion of PV+FSI surrounded by PNNs, which promotes subfrontal states and cognitive deficits through increased PV+FSI excitability and inhibition of synaptic transmission. In addition, enzymatic digestion of mPFC PNN may reverse METH-induced changes in mPFC activity and behavioral deficits, suggesting a potential therapeutic avenue for treating cognitive symptoms in individuals with methamphetamine use disorder and other disorders with psychopathology that are associated with subfrontal conditions.

Keywords:Perineural networks, hypofrontality, working memory, parvalbumin interneurons, methamphetamine

Revelation:Nothing to disclose.

South Carolina Medical University, Charleston, South Carolina, United States

Bottom:Precortical projections to the nucleus accumbens are critical for cue-induced cocaine seeking, but the importance of the neurons most innervated by this projection and the neuroadaptations that contribute to relapse in these cells remain unknown.

Methods:Male Sprague-Dawley rats were subjected to cocaine or sucrose self-administration, extinction and stimulus-induced reinstatement. Pathway-specific chemogenetics, patch-clamp electrophysiology, in vivo electrochemistry and high-resolution confocal microscopy were used to characterize and elucidate the role of the subset of central neurons in the nucleus accumbens that receive dense input from the prefrontal cortex. induced cocaine and sucrose seeking.

Results:Pathway-specific chemogenetic inhibition of prefrontal cortex projection to the nucleus accumbens suppressed cue-induced cocaine relapse and normalized the real-time induced increase in glutamate release from the accumbens to levels seen in sucrose-seeking animals. In addition, chemogenetic inhibition of descending central neurons of the nucleus accumbens, which received the densest input from the prefrontal cortex, suppressed cocaine but not sucrose seeking. Interestingly, an even smaller subset of these neurons were specifically activated by cocaine and sucrose. We found that accumbens neurons that receive dense prefrontal input also undergo morphological plasticity during peak cocaine seeking in the form of dendritic spine expansion and increased innervation by astroglial processes, particularly in the large heads of the dendritic spine.

Conclusions:We have identified and characterized a unique subpopulation of nucleus accumbens neurons that receive dense cortical feedback. The functional specificity of this subpopulation is underscored by its ability to mediate cue-induced cocaine relapse, but not sucrose seeking. This subset of cells represents a novel addiction treatment target revealed by targeting precursors to explore functional circuitry embedded in a known network.

Keywords:Κοκαΐνη, transsynaptic, astrocytes, prelimbic cortex, nucleus accumbens

Revelation:Nothing to disclose.

Binghamton University, Binghamton, New York, USA

Bottom:Intravenous cocaine self-administration (IVSA) in experimental animals models the voluntary initiation and progression of drug use in humans. This behavior is a complex trait, and much of the individual differences in human and animal cocaine use are determined by genetic variation, although the specific genes and causative alleles remain unknown. IVSA cocaine procedures can be used in experimental animals to accurately and quickly identify relevant genetic effects. Large, genetically diverse mouse populations, including the Hybrid Mouse Diversity Panel (HMDP) and the BXD recombinant inbred panel (a subset of the HMDP population), have been developed for these types of direct genetic approaches. These populations enable high-resolution genome-wide association studies as well as discovery of genetic linkages.

Methods:All topics (N= 86 genetically unique inbred lines,N= 8-12 mice per strain) were surgically catheterized, providing access to the jugular vein. After recovering from surgery, half of the mice received 10 daily sessions of IVSA with cocaine, while the other half had access to saline infusion (as a control). IVSA data for BXD recombinant inbreds (N=  52 strains) were used to map the quantitative trait locus (QTL) and rank candidate locus genes using publicly available RNA expression data obtained from addiction-related brain regions (striatum, nucleus accumbens and ventral tegmental area) (software and data provided by genenetwork .org). Candidates were assessed for cis QTL (eQTL) expression and significant associations of stress level with IVSA cocaine behavior.

Results:We found that IVSA cocaine is essentially heritable in this population, with intake levels ranging from near zero to >25 mg/kg/session. Although IVSA saline has also been found to be heritable, the very modest genetic link between cocaine and IVSA saline indicates that the cocaine enhancer-responsive operant was influenced by much of the unique genetic variation. QTL mapping in BXD strains revealed a suggestive QTL (PI number= 0.063) on chromosome 1 for cocaine but not saline, consumption at baseline. Candidate locus analysis prioritized 3 genes (Pappa2, Nme7 and Kifap3) showing cis-eQTL and a significant genetic association with cocaine use. Phewas expression analysis (systems-genetics.org) of the genenetwork.org database BXD-phenomenon revealed that Kifap3 expression is also genetically correlated with alcohol-dependent saccharin taste aversion, indicating that this gene may modulate the response to cocaine and alcohol.

Conclusions:These data indicate that the HMDP and BXD populations are suitable for advanced genetic approaches in cocaine IVSA analysis. In addition, we identified a genetic locus possibly related to cocaine self-administration as well as novel candidate genes. This project is ongoing and involves full transcriptome RNA sequencing in key regions of the brain for different expression phenotypes (cocaine vs saline IVSA) in all tested strains. Integration of genetic linkage data with expression profiling will help identify candidate genes that modulate cocaine risk.

Keywords:Cocaine self-administration, behavioral genetics, QTL, substance use disorders

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:Opioid use disorder (OUD) is a neuropsychiatric condition that causes significant morbidity and mortality, as well as social, economic, and personal costs to patients and their families. In 2020 alone, approximately 70,000 people lost their lives to opioid overdoses in the United States. Currently available OUD pharmacotherapies are ineffective or intolerable for many patients. Therefore, the development of new interventions, especially those that focus on strategies to promote overall health and resilience to problem opioid use, is of great clinical and societal importance. In recent years, plant-derived dietary polyphenols such as resveratrol have been shown to be effective in promoting behavioral resilience and adaptive neuroplasticity in models of neuropsychiatric diseases. In this study, we investigated the potential of dietary polyphenols in reducing the formation of addictive behaviors and neurobiological changes in a mouse model of OUD.

Methods:To study the effect of dietary polyphenols on behavioral and molecular responses to morphine, 7-week-old male C57BL6/J mice were administered a bioactive dietary polyphenol preparation (BDPP) consisting of grape seed polyphenol extract (1 g/l). , Concord grape juice (1 part juice: 3 parts water) and resveratrol (1 g/L) or drinking water containing sucrose at an appropriate concentration (152 g/L) for 2 weeks, followed by assessment of behavioral response to morphine using a series of paradigms . The effect of polyphenols on the development of motor sensitization was assessed with daily injections of 5 or 15 mg/kg of morphine. A conditioned place preference (CPP) drug-seeking model was used to assess preference for a paired morphine chamber over a range of doses (2.5 - 15 mg/kg administered subcutaneously). To assess the effect of BDPP on morphine preference after withdrawal, an additional group received five daily injections of 5 mg/kg morphine two weeks prior to CPP training. Since polyphenols are known enhancers of sirtuin-1 (Sirt-1) histone deacetylase activity, an additional group received microinjections of the Sirt-1 inhibitor EX527 into the nucleus accumbens (NAc) daily during CPP training. For molecular analyses, mice were sacrificed 24 hours after the CPP assay, and the NAc core was excised for qPCR and RNA sequencing analysis, and blank samples were collected for 16-s sequencing analysis. An additional group of mice were sacrificed 1 hour after acute injection of 5 mg/kg morphine, and the NAc was excised for analysis of acute changes in gene expression.

Results:In the CPP paradigm, we found that at the lower morphine doses of 2.5 mg/kg and 5 mg/kg, control animals developed a strong place preference (PI number< 0,05 ePI number< 0.001, respectively), but BDPP-treated animals did not develop a significant preference for any dose (two-way ANOVA followed by Sidak's post hoc,N= 8-32 per group). Similarly, mice treated with polyphenols did not prefer 5 mg/kg morphine, even after pre-treatment with morphine for one week followed by a two-week abstinence prior to CPP training, while this regimen resulted in a strong preference. in control mice. At the highest dose of 15 mg/kg morphine, BDPP-treated animals developed a preference for (PI number< 0.001), but control mice did not (N= 24 per treatment group). In addition, data from the locomotor sensitization paradigm revealed a significant reduction in locomotion ability after administration of 15 mg/kg morphine in BDPP-treated animals compared to control animals on day 1 and 3 of morphine treatment (two-way ANOVA followed by Tukey's post-hocPI number< 0,0001 ePI number= 0.05 respectively). Taken together, these data suggest that treatment with polyphenols leads to a change in the behavioral dose-response curve of morphine. NAc gene expression data after CPP showed a strong effect of polyphenol treatment on the expression of the nuclear factor κB (NF-κB) subunit of nuclear factor κB, as well as multiple changes in gene networks related to epigenetic regulation. The qPCR data from animals sacrificed 1 h after an acute dose of 5 mg/kg morphine revealed a significant primary effect of BDPP treatment on immediate early genes including EGR4 as well as epigenetic editors including Sirt1 and HDAC1 levels (following two-way ANOVA analysis by post-hoc SidakPI number< 0,05,N= 6-8). To assess the possible mechanistic role of Sirt-1 activity in mediating these effects, BDPP-treated mice and control mice received microinjections of the specific Sirt-1 inhibitor EX527 directly into the NAc while conditioning CPP at a dose of 5 mg/kg morphine (N= 5/4 per group). EX527 treatment did not change the effect of polyphenol on reducing CPP, suggesting that the behavioral effects of polyphenols are not mediated by Sirt-1 activity in the NAc.

Conclusions:Taken together, these data suggest that pretreatment with dietary polyphenols significantly reduces CPP formation at lower doses of morphine, even after a period of abstinence. At a higher dose of morphine, BDPP appears to have the opposite effect of increasing CPP, perhaps by increasing the consolidation of morphine-related reward memory. Molecular analysis to date reveals that BDPP treatment affects the expression of multiple transcription factors and epigenetic editors in the NAc. The inhibition of Sirt-1, which is known to be activated by resveratrol, did not alter the behavioral effects of polyphenol treatment. Together, these studies provide a basis for dietary polyphenols to reduce the formation of addiction-like behaviors in mice, possibly through epigenetic or transcriptional mechanisms.

Keywords:Opioid addiction, resveratrol, epigenetics, immunity, microbiota-gut-brain axis

Revelation:Nothing to disclose.

Duke University Medical Center, Durham, North Carolina, USA

Bottom:Recently, there has been a huge increase in the popularity of electronic cigarettes (e-cigs). While e-cigarettes are likely less harmful than traditional C-cigarettes, the main concern is that long-term use of these products can lead to the development and maintenance of nicotine addiction. As with other drugs, the rate of accumulation in the brain and the size of nicotine are critical to its acute reinforcing effect. Despite recent discoveries that plasma nicotine concentrations following e-cigarette use may be comparable to smoking, the ability of e-cigarettes to produce a rapid, cigarette-like delivery of nicotine to the brain remains poorly studied.

Methods:Brain nicotine uptake was directly assessed in 16 adult dual users (10 females, mean ± SD, 36 ± 11 years) of e-cigarettes and type C cigarettes using 11C nicotine and positron emission tomography (PET). Race was self-reported (White/African American/Other: 56%/6%/38%). PET imaging was performed using the Discovery MI DR PET/CT system (GE Healthcare). Duke University's Institutional Health Systems Review Board and Wake Forest University's Institutional Health Sciences Review Board approved this study, and all subjects provided written informed consent. Each participant underwent two PET scanning sessions in a random order, during which the head was scanned after inhaling vapor (55 ml for 4 seconds) or smoke (35 ml for 2 seconds) containing 11C nicotine. Each standardized puff of vapor was produced with 15 μl of V2 Red e-liquid (1.2% nicotine, 20/80 VG/PG) mixed with 11C nicotine using a V2 EX Blanks rechargeable atomizer connected to a programmable syringe pump. Smoke was generated from a short Capri Magenta cigarette (R.J. Reynolds, USA) by a custom smoke delivery device after application of 11C nicotine. The subject's head was scanned for 15 minutes in a sequence of 249 frames lasting from 1 to 10 seconds each. A whole-body scan was then performed to measure the total absorbed 11C nicotine dose (TAD), which was used to normalize 11C nicotine absorption values ​​between patients and between conditions. Brain nicotine concentration was expressed as %TAD per kg of brain tissue. Final data were analyzed using parT- Test for each of the four kinetic parameters and Holm-Bonferroni correction applied for multiple comparisons.

Results:After inhaling a single puff of e-cigarette vapor or e-cigarette smoke: 1) The mean peak nicotine concentration (Cmax), normalized to the total 11C nicotine delivered dose (TAD), e-cigarette was 23% lower than C-cig ( mean ± SE, 3.7 ± 0.2% TAD vs. 4.7 ± 0.3% TAD). 2) The mean area under the activity time curve from 0 to 15 min was 22% lower for E-cig compared to C-cig (48.5 ± 2.7% TAD*min vs. 62.1 ± 3.1% TAD*min). 3) The mean time to maximum nicotine concentration (Tmax) for E-cig was approximately twice as long as for C-cig (9.0 ± 0.8 min vs. 4.4 ± 0.5 min). and 4) The mean time to half maximum nicotine concentration (T1/2) for the E-cigarette was about two and a half times longer than for the C-cigarette (1.04 ± 0.13 min vs. 0.38 ± 0.06 minutes). all fourPI numberValues ​​are less than 0.0005 after adjustment for 4 comparisons. This lower brain nicotine accumulation after e-cigarette use compared to C-cigarette consumption is, at least in part, due to lower arterial nicotine concentrations resulting from e-cigarette use, which in turn is likely due to lower alveolar nicotine coverage in where nicotine is rapidly absorbed. Indeed, our preliminary results from whole-body imaging showed greater nicotine retention in the upper respiratory tract due to inhalation of e-cigarette vapor compared to C-cigarette smoke. One possible reason for the higher deposition in the upper respiratory tract is the higher pH level typical of e-liquids compared to type C tobacco (pH 7-9 for e-cigarettes and 5-6 for C-cigarettes). The alkaline pH increases the evaporation of the nicotine base from the droplets, thereby increasing its retention in the upper respiratory tract. Nicotine deposition in the upper respiratory tract is expected to be reduced by the use of low pH e-cigarette liquids.

Conclusions:These results suggest that e-cigarettes can rapidly deliver nicotine to the brain, although to a lesser extent than C-cigarettes under current conditions. thus, they can promote smoking cessation and harm reduction.

Keywords:Nicotine addiction, electronic cigarette (e-cigarette), PET imaging, pharmacokinetics

Revelation:Nothing to disclose.

South Carolina Medical University, Charleston, South Carolina, United States

Bottom:Predictive heroin supply cues stimulate heroin seeking by initiating synaptic glutamate release in the nucleus accumbens (NAcore). The intensity of heroin craving is negatively modulated by a signal-induced increase in astrocyte synaptic proximity and increased surface expression of the GLT-1 glutamate transporter. We sought to determine whether the stimulus-induced increases in synaptic proximity of astrocytes and surface GLT-1 were selective for medium spiny neurons expressing the D1 or D2 receptor (D1- or D2-MSN) in the NAcore and how the increase in synaptic proximity of astroglial processes affected synaptic function.

Methods:Rats were trained to self-administer heroin or sucrose before succumbing to extinction and seeking reintroduction. Using confocal microscopy, we quantified the association of astrocytes with D1 or D2-MSN synapses. We then used ezrin-targeted morpholino oligonucleotide to disrupt astrocyte isolation in the NAcore of transgenic mice and used whole-cell patch electrophysiology to study the effect of astrocyte isolation at D1 and D2-MSN synapses.

Results:Heroin cessation, but not sucrose self-administration, decreased GLT-1 and induced astroglial firing at NAcore synapses. Heroin indicates increased synaptic proximity to astroglial processes and GLT-1 surface expression in parallel with heroin search in different NAcore astrocyte populations. Astroglial withdrawal increased the likelihood of release in the D1-MSN in the NAcore and decreased spontaneous excitatory postsynaptic currents in the D2-MSN.

Conclusions:Our data show that increased synaptic isolation from NAcore astroglia does not reduce heroin seeking through GLT-1-dependent mechanisms. Conversely, synaptic isolation from astroglia differentially affects D1 and D2-MSN to suppress relapse behavior.

Keywords:Astrocytes, restoration, heroin self-administration, GLT-1

Revelation:Nothing to disclose.

University of Texas Health Science Center, San Antonio, TX, USA

Bottom:Opioid abuse remains a major public health challenge, despite the availability of effective drugs. Methocinnamox (MCAM), a long-acting mu-opioid receptor antagonist, attenuates the positive, reinforcing and breath-suppressive effects of opioids such as heroin and fentanyl in rhesus macaques, suggesting that it may be an effective treatment for opioid abuse and overdose. In previous studies, MCAM ( ≥ 0.32 mg/kg) on ​​an on-demand or intermittent basis (i.e. once every 12 days) reduced fentanyl self-administration. This study investigated the effect of daily injections of lower doses of MCAM on fentanyl self-administration.

Methods:Five rhesus monkeys (2 females and 3 males) were levered into the i.v. fentanyl infusions (0.032-320 μg/kg/injection) at a fixed ratio of 30. MCAM injected s.c. one hour before each session. The daily dose of MCAM was increased in half-log increments from 0.0032 to 0.1 mg/kg/day under all conditions, and the fentanyl dose-effect curve was redefined for each dose of MCAM treatment. The effects of MCAM were quantified by dividing the ED50 of fentanyl (ascending leg of the dose-effect curve) during each daily MCAM treatment by the ED50 of fentanyl before daily MCAM treatment (ie potency ratio).

Results:Before treatment with MCAM, the number of fentanyl infusions increased and then decreased with increasing dose. the mean (95% confidence interval) ED50 was 0.13 (0.07-0.22) µg/kg/injection. Daily MCAM treatment shifted the fentanyl dose-effect curve to the right, with a mean potency ratio (95% CI) of 1.9 (1.2–2.6), 3.7 (2.5–4.8), 14, 6 (10.1–19.1) and 49.0 (44.3). -53.6) for therapeutic doses of 0.0032, 0.01, 0.032 and 0.1 mg/kg/day of MCAM, respectively.

Conclusions:MCAM attenuated fentanyl self-administration and shifted the fentanyl dose-effect curve to the right by almost 50-fold at the highest daily therapeutic dose of 0.1 mg/kg. These results, together with previous studies showing selective and long-lasting attenuation of opioid self-administration, support the view that MCAM may be a safe and effective treatment for opioid use disorder.

Keywords:Fentanyl, self-administration, methocinnamox, multiple treatments, rhesus monkeys

Revelation:Nothing to disclose.

Laureate Institute for Brain Research, Tulsa, Oklahoma, USA

Bottom:Computer modeling is a promising approach to analyze behavioral processes and dysfunctions in people with substance use disorders (SUDs), but it is not clear how much these processes change during recovery.

Methods:We assessed 1-year follow-up data in a sample of treatment seekers with one or more SUDs (alcohol, cannabis, sedatives, stimulants, hallucinogens and/or opioids).N= 83) previously assessed at baseline in a previous computer modeling study. Regarding healthy controls (HC;N= 48), these participants were considered at the start of the study to exhibit altered learning rates and less accurate choice of actions when performing an exploration-exploration decision task. Here we repeated these analyzes when these patients returned and repeated the task one year later to assess the stability of these differences at baseline. We also tested whether basic modeling measures could predict symptoms during follow-up.

Results:Bayesian analyzes show that: (a) differences between groups in learning rates were stable over time (post-likelihood =   1); (b) relationships between model parameters at baseline and follow-up were significant and ranged from low to moderate (0.25 < < ICCs < 0.54); and (c) learning rates and/or information seeking values ​​at baseline were associated with the 1-year follow-up substance use among stimulant and opioid users (0.36<rs<0.43, 0.002<ps<02).

Conclusions:These findings support the notion that processing dysfunctions involving learning to trade between exploration and exploration are stable during the recovery period. At the same time, individual computational differences at baseline had some predictive value for changes in substance use. Taken together, this computational approach can measure dysfunctional characteristics that may have predictive utility for the severity of substance use.

Keywords:Substance abuse disorder, computer modeling, active reasoning, learning rate, exploration-exploration dilemma

Revelation:Nothing to disclose.

University of Colorado Boulder, Boulder, Colorado, United States

Bottom:Drug-using behaviors typically begin during adolescence, when the brain—especially the prefrontal cortex—is still developing. However, given this critical developmental time, the impact of regular drug use during this time could alter developmental trajectories, resulting in lasting changes in adult brain function in these individuals.

Methods:For a deeper understanding of this process, men (N= 16) and solitary (N= 16) adolescent rats (PND28-42) were given two weeks (14 days) of daily access to 4% v/v. alcohol (controls). The mice reached maturity where they were evaluated in the Pavlovian Conditional Approach (PCA) paradigm in a new context. In this task, a complex set of Pavlovian cues (e.g. left panel light / left lever extended / high pitch) was presented for 10 s and ended with the administration of a sucrose tablet (CS + ). The other stimulus (right panel light/right lever extension/low tone) had no programmed consequences (CS-). The CS+ and CS− stimuli were balanced. Individual differences in adaptive approach during PCA are typically observed in rats that go to the food cup during cues in anticipation of reward (goal tracking, GT), compared to rats that spend the prep period interacting with cues, such as making unnecessary press bar (signal trackers, ST).

Results:Overall, alcohol exposure in adolescents appears to increase the likelihood and intensity of the ST phenotype during early learning (PCA days 1–10), and in particular, shows an increase in stick pressure during cues compared to controls. Electrodes placed in the prefrontal (PL) and submedial (IL) cortex during these PDA behaviors showed a clear distinction between ST and GT phenotypes, particularly in the control group and often with greater phasic activity to the stimuli seen in alcoholics rather than control animals. At the same time, local field potentials collected at the same locations were reduced in ST and GT animals with alcohol relative to controls at the onset of CS+, especially at low and high gamma frequencies. However, this alcohol-related reduction was only observed in ST and non-GT subjects later in the cue and during reward receipt.

Conclusions:Together, these data suggest that voluntary alcohol consumption during development can cause persistent changes in PFC function, even in drug-absent adults. These changes, in turn, may set the stage for more pathological drug involvement through cue-induced craving, approach, and relapse.

Keywords:Electrophysiology, drug abuse, alcohol in adolescence

Revelation:Nothing to disclose.

University of Florida, Gainesville, Florida, United States

Bottom:Neural assemblages in the submellite cortex (IL), as well as their projections to the nucleus accumbens (NAc), have been shown to mediate opiate seeking in well-trained rats. However, it is unclear whether this circuit is recruited during the initial self-administration of oxycodone. Here we tested the need for IL neuron complexes in initial and sustained oxycodone-seeking behavior using the Daun02 inactivation procedure.

Methods:We trained male and female Fos-LacZ transgenic mice (N= 12-13) for self-administration of oxycodone for 3-hour sessions per day until rats met the acquisition criteria (>30 active bar presses, >75% active bar response). We then injected Daun02 to selectively inactivate pools of IL Fos expression associated with initial oxycodone self-administration. We then tested the oxycodone-seeking behavior of the rats 2 days later. We then repeated the experiment using a longer training period to determine the role of IL neuronal assemblies in seeking oxycodone after prolonged training. Here we trained male and female Fos-LacZ transgenic mice (N= 9-10) for self-administration of oxycodone in daily 3-hour sessions on a graduated reminder schedule for 9 days. After 1 week of oxycodone abstinence, we subjected the animals to a 30-minute induction test to reactivate the neural assemblies associated with the withdrawal of oxycodone self-administration and injected Daun02 into the IL. We measured the rats' oxycodone-seeking behavior 2 days later.

Results:We found that inactivation of IL neuronal assemblies reduced oxycodone seeking after initial oxycodone self-administration on test day (t23=2.5,PI number= 0.02). We then found that Daun02 reduced oxycodone-seeking behavior in well-trained rats (t17=2.6,PI number= 0.02). In both experiments, Daun02 infusions decreased Fos expression after testing, indicating Daun02 ablation of the population of Fos-expressing neurons.

Conclusions:These results suggest that IL neural compositions are formed during the initial learning of oxycodone self-administration and are required for initial and sustained oxycodone-seeking behavior.

Keywords:Oxycodone, self-administration, reinforcement learning

Revelation:Nothing to disclose.

University of Cincinnati, Cincinnati, Ohio, United States

Bottom:Clinical evidence shows that stress is a major contributor to substance use disorder (SUD). This is especially problematic because stress is inevitable in everyday life. Therefore, understanding the neurobiological mechanisms underlying the involvement of stress in SUD is critical. A hallmark of SUD is the loss of control over drug intake, which is modulated in rats by conditions that lead to increased patterns of drug self-administration (SA). Repeated daily stress during AS escalates cocaine use in a glucocorticoid-dependent manner. This is likely due to persistent neuroplastic changes that also increase susceptibility to reinstatement of drug-seeking behaviors. This can be explained by changes in stress reactivity, as increased stress reactivity is associated with increased relapse in humans. We hypothesize that repeated stress during AS increases susceptibility to stress-induced return to work and causes lasting changes in stress-related behavior and stress reactivity.

Methods:Male SD rats were trained to SA cocaine (0.5 mg/kg/inf) according to schedule FR 4 in 4 x 30 min SA sessions separated by 5 min drug-free periods. Some rats received intermittent foot shocks in the SA chamber during a 5-minute drug-free period for 14 days. The rats were then tested for increased sensitivity to yohimbine-induced shock recovery (0, 1.25, 2.5 mg/kg, i.p.). Additional groups of rats were tested for changes in stress reactivity and stress-related behaviors after increased stress-induced cocaine consumption. These rats were trained with SA cocaine as described above, and additional saline SA groups were also used, with or without paw exercise. All animals went through a period of extinction followed by yohimbine-induced reintroduction attempts. The rats were then tested for changes in stress-related behaviors (elevated maze, open field, and marble burial), social interaction, and cognition (memory of object location) on different days. After the final behavioral task, the rats were subjected to a 30-minute restraint stress, and tail blood was drawn at 0, 15, 30, 60, and 90 minutes from the onset of stress to measure changes in plasma corticosterone. Rats were sacrificed after 90 minutes and brains were harvested to study stress-induced activation of cFos in stress and reward related brain regions.

Results:Electric shock to the feet delivered daily during self-administration resulted in an emergent escalation of cocaine intake over 14 days that persists in the absence of stress (N= 13-15/group,PI number< 0.05). Rats with increased stress also show increased susceptibility to later reinstatement. Rats with a history of stress during AP show increased recovery to the original cocaine injection (2.5, 5, 10 mg.kg, i.p.;N= 11-14/group,PI number< 0.05), re-introduction of foot shock stress during the 5-minute drug-free period (N= 9–12/group) and injection of the α-2 adrenoceptor antagonist yohimbine (0, 1.25, 2.5 mg/kg, i.p.N= 14-17/group,PI number< 0.05). A variety of changes in stress-related behavior, cognitive and social interactions, and HPA axis responses to constraint stress were observed that depended on the drug history and/or stress of the rat (N= 11-12/group). Analysis of cFos expression in stress and reward related brain regions is currently underway.

Conclusions:Chronic stress induces glucocorticoid-dependent increases in cocaine consumption as a result of persistent neuroadaptations. These persistent neuroadaptations lead to greater vulnerability to reinstatement of drug-seeking behaviors. These stress-induced neuroplastic changes affect behavioral, hormonal, and neural responses to stress, and changes in stress reactivity may be a factor in the enhanced restorative behavior seen in stressed rats. Understanding the unique mechanisms by which stress can lead to drug use has implications for identifying and treating subpopulations of SUD patients in whom stress is a contributing factor.

Keywords:Stress reactivity, cocaine self-administration and reinstatement, c-Fos

Revelation:Nothing to disclose.

Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia, United States

Bottom:Understanding the molecular factors driving the stages of drug addiction may allow the design of pharmacotherapies that block or reverse key events in the progression of drug addiction. Zfp189 is a CREB-regulated gene encoding the nucleus accumbens (NAc) neuronal transcription factor, which has been shown to regulate brain transcription networks in neuropsychiatric disorders. In rodents, NAc gene expression Zfp189 is upregulated after exposure to psychostimulants such as cocaine. To further explore the relationship between ZFP189 and physiological response to different drug classes, we constructed synthetic variants of ZFP189 to exert opposite forms of gene regulation on currently unknown ZFP189 target genes in vivo. These ZFP189 variants were delivered to the murine NAc and the subsequent behaviors and transcriptional response after exposure to psychostimulants (cocaine) or opiates (morphine) were characterized.

Methods:Three variants of ZFP189 were synthesized: ZFP189WT, which is identical to endogenously expressed ZFP189 and contains a transcriptionally repressive N-terminal Krüppel-associated domain (KRAB) and a C-terminal Cys2-His2 DNA-binding domain. ZFP189VPR, in which the endogenous KRAB domain is replaced with the strong transcription activator VP64-p65-Rta (VPR), still retains the endogenous ZFP189 DNA-binding domain. and ZFP189DN in which any transcriptional regulatory domain is completely deleted. Each variant was subcloned into identical expression vectors (p1005) and gene regulation was characterized in conjunction with the ZFP189 response element (RE) luciferase assay in Neuro2a cells (N= 3 in the technical triple jump). The expression vectors were then placed in the herpes simplex virus (HSV) to allow targeted expression in the brain region. Both male and female C57Bl/6J mice aged 8-10 weeks were used in these studies. HSV-ZFP189WT, -ZFP189VPR or -ZFP189DN were delivered bilaterally by stereotactic surgery for NAc (10°; +1.6 AP;  ±1.5 ML; -4.4 DV, 1 µl). For locomotor sensitization, mice received intraperitoneal saline, cocaine (10 mg/kg) or morphine (10 mg/kg) for seven consecutive days, and motility was quantified. Sample sizes range from 10 to 15 mice per group, and statistical analyzes were performed by one-way or two-way ANOVA, including a final test comparing the test groups (HSV-ZFP189WT, -ZFP189VPR) with the control group (HSV-ZFP189DN). After the seventh day testing, we extract RNA from virus-infected NAc tissues, perform ribosomal RNA depletion, prepare libraries, perform RNAseq with the following setup: 2x150 paired-end reads on an Illumina sequencing platform (HiSeq 2500) at a depth of > 30 million reads per sample (N= 5 mice for each condition). Differentially expressed genes (DEGs) were generated using DESeq2 to compare any test group (HSV-ZFP189WT, -ZFP189VPR) to the control group (HSV-ZFP189DN) with adapted WaldPI number-value < 0.05 and absolute log2-fold change > 1 degree is significant.

Results:By co-transfecting the luciferase plasmid ZFP189 RE and our ZFP189 variant repeats, we can observe, relative to the GFP control: ZFP189VPR induces strong activation of target genes (PI number< 0.0001), ZFP189WT induces gene repression (PI number< 0.05) and ZFP189DN exerts no transcriptional regulatory control in this assay (PI number>0.5). By delivering each of these ZFP189 variants with the virus to the murine NAc, we observed that HSV-ZFP189VPR significantly improved the psychomotor response of rodents to daily injections of 10 mg/kg IP cocaine (PI number< 0.0001). Comparing the total daily distance traveled between the virus groups, it can be seen that HSV-ZFP189WT and HSV-ZFP189VPR elicit opposite locomotor behavioral responses to cocaine (PI number< 0.05). When this musculoskeletal sensitization test was repeated with IP injections of saline or morphine, no significant differences emerged for either viral treatment (PI number> 0.05 for all comparisons). In the corresponding RNA sequencing data of these NAcs from these mice, 147 DEGs were identified in cocaine-treated mice, while zero DEGs were identified in saline or morphine-treated mice, indicating that some features of cocaine exposure made ZFP189VPR able to regulate target genes .

Conclusions:These experiments indicate that the transcriptional regulation exerted by ZFP189 in the NAc specifically contributes to the development of cocaine abuse but does not contribute to core behavior or morphine abuse. The synthetic ZFP189VPR was uniquely able to enhance cocaine-induced locomotor sensitization and transcription. The types of NAc cells in which this occurs, the extent to which this represents a cocaine-specific or general psychostimulant effect, and the mechanism by which cocaine allows ZFP189VPR to regulate transcription are areas of future research. This work is evidence that in vivo neurotranscription assays allow scientists to unravel the mechanisms underlying drug-specific disorders.

Keywords:Cocaine and opioid use disorders, artificial transcription factors, RNA sequencing

Revelation:Nothing to disclose.

Rowan University School of Osteopathic Medicine, Stratford, New Jersey, USA

Bottom:Animals must modify their behavior based on updated expected outcomes to successfully navigate changing environments. Cocaine-experienced rats have an impaired ability to update expected scores to suppress behavior toward the predicted reward cue in a Pavlov score devaluation task. We recently showed in naïve animals that the development of prefrontal cortex (PrL) neural coding during learning (Day 1 to Day 10) predicts this ability to suppress approach behavior to a reward cue after effect underestimation (West et al., 2021). Thus, further enhancing neural activity during learning can reinforce specific cue-outcome associations to allow rats to adjust their behavior in response to expected outcomes. In contrast, rats that lost their phase response to PrL during learning showed an inability to stop engaging with cues despite devaluation, suggesting that stiffness is related to loss of PrL binding during learning. In support, the neural activity of PrL during learning is impaired in cocaine-exposed rats, which have an impaired ability to suppress responses after score devaluation, and optogenetic inhibition of PrL during learning recapitulates this behavioral phenotype (West et al., 202). ). Because PrL encodes the online value of the updated score for cues that predict expected outcomes (i.e., extinction devaluation trials) and for cues that predict actual updated outcomes (i.e., post-devaluation trial with delivered outcomes) is unknown.

Methods:To determine whether PrL neural signaling monitors the ability to change behavior, we recorded PrL neural activity to reward predictive cues after training in a Pavlovian task (10 sessions) and after score devaluation. Briefly, male Long-Evans rats (N= 18) presented with 2 cues as conditioned stimuli (CS+, predicting a sugar lozenge or food) and 2 cues not predicting reinforcement (CS−). After 10 sessions, the sugar pellets were combined with LiCl (0.3 M, ip). The food pellets were combined with a saline injection, which remained useless. This happened once. The rats were then tested for extinction to assess their ability to avoid the CS+ associated with the devalued score. The session was then repeated, but with undevalued and devalued (re-learning) scores provided. We examined the percentage of time spent in the chow cup under conditions that predicted no evaluation and overestimation of performance, and calculated a "devaluation index" for each rat (ND-D/ND+D) to correlate with neurophysiological data and two trainings. test.

Results:In both testing sessions after devaluation, rats spent less time in the food cup during CS+ than the devaluation score (13.2% +/- 1.6, extinction, 14.9 +/- 1.7%, relearning up) compared to CS + whi predicts an underestimated effect (19.7 +/- 1.9%, extinction; 20.3 +/- 2.5%, relearning). The PrL recordings revealed different populations of neurons that were either excited or inhibited during signals (classified as "phasic"). The % PrL of phasic neurons for the cue that predicted a devaluation score (D) was significantly lower than the cue that predicted a no-devaluation score (ND) in the extinction test (D: 19.0 +/- 3.9% vs ND: 32, 6 +/- 5.5%). In addition, the % of PrL phasic neurons for the cue that predicted a devalued (but not devalued) score on an extinction test was negatively correlated with the ability to suppress devaluation after a behavioral score (R2 = 0.33,PI number<0.05 and R2=0.03; behavior measured as a devaluation indicator). However, the PrL phase response during the relearning test was not different from the underestimated and non-underestimated cues (D: 32.0 +/- 6.2% vs. ND: 35.6 +/- 6.6%). Moreover, there was no correlation between the percentage of phasic neurons for the cue that predicted underestimation or no score on the relearning test (R2 = 0.01 and 0.02, respectively, behavior measured as an indicator of underestimation).

Conclusions:Increased involvement of PrL neurons during learning correlates with the ability to subsequently avoid a valuable score cue (West et al., 2021) during a testing session (extinction). Here, this reduction in PrL phase response to a signal that predicted an underestimated outcome correlates with the ability to avoid cues based on the expected outcome value. Perhaps the increase in the strength of cue-outcome associations during learning allows for differential and dynamic encoding of PrL neural activity for different cues that predict underestimated and underestimated outcome when tested. When the rats were given an actual score during the re-learning session, PrL neurons reactivated to a cue that predicted both a non-valued and devalued outcome (although most rats avoided the cue that predicted a devalued and negative outcome). ). Future research will investigate how these PrL neural processes are disrupted in male and female rats with a history of cocaine use.

Keywords:Reward devaluation, prefrontal cortex, neurophysiology

Revelation:Nothing to disclose.

University of Illinois at Chicago, Chicago, Illinois, United States

Bottom:Operative self-administration is often used to study alcohol consumption in a preclinical setting. Often, studies using operant paradigms infer the amount of alcohol consumed from the number of reinforcements delivered after the end of the appetitive response (e.g., squeezing a bar). However, recent work in mice suggests that appetite responses do not reliably predict consumption. It is not known if this also applies to mice.

Methods:To remedy this shortcoming, we trained an adult Sprague-Dawley male (N= 16), Wistar (N= 32) e Long-Evans (N= 32) mice on the ethanol lever using a paradigm that accurately measures appetite and drinking behavior. Rats were habituated to ethanol using the standard two-bottle selection procedure in an intermittent home cage. The rats were then trained on a fixed ratio 3 schedule where three presses of the active lever resulted in the presentation of a bright light and 15 seconds of access to a sipper tube fitted with a 20% ethanol lycometer.

Results:During training, three distinct functional phenotypes emerged with different prevalence among rat strains. A significant number of Sprague-Dawley (81.3%) and Wistar (50.0%) rats, but not Long-Evans rats (6.3%), retained their appetitive response but did not consume ethanol (response). In contrast, appetite response was a predictor of drinking(s) in most Long-Evans rats (50.0%) and some Wistar rats (28.1%) but not in Sprague-Dawley (6.3%). There were no significant differences between the strains in ethanol consumption or appetite response in the two phenotypes. Importantly, appetite response was significantly positively associated with intake in heavy drinkers (R= 0,79,PI number< 0.001), but not among responders (R= 0,28,PI number= 0.14). A retrospective analysis of the association between the home cage and alcohol consumption revealed significantly greater 24-hour ethanol consumption in potential drinkers compared with potential responders (PI number<0.001). Withdrawal of chronic ethanol exposure using the Lieber-DeCarli liquid diet procedure resulted in a significant increase in appetite response in both drinkers (PI number< 0.001) and respondents (PI number< 0.05). A similar escalation of ethanol consumption was observed only in heavy drinkers (PI number< 0.001) and non-responders.

Conclusions:Together, these results reveal significant cross-stress differences in the propensity to operatively self-administer ethanol and confirm previous findings in mice requiring the use of appetite measurements to infer consumption.

Keywords:Animal models, Functional behavior, Alcohol consumption

Revelation:Nothing to disclose.

National Institutes of Health, Baltimore, Maryland, United States

Bottom:Growing evidence suggests that the glucagon-like peptide-1 (GLP-1) system is involved in the mechanisms that govern alcohol seeking and consumption. In this sense, the GLP-1 receptor (GLP-1R) has begun to be studied as a potential pharmacotherapeutic target for alcohol use disorder (ABD). We hypothesize that genetic variation in GLP-1R, namely two single nucleotide missense polymorphisms (SNPs) leading to amino acid substitutions in GLP-1R (rs6923761, glycine to serine at position 168; rs1042044, leucine to phenylalanine) will be differentially associated with functional brain connectivity in heavy and low drinkers.

Methods:Potential participants were screened in the National Institute on Alcohol Abuse and Alcoholism (NIAAA) clinical program, and eligible subjects were included in the neuroimaging protocol (N= 181). As part of the screening, the AUD Identification Test (AUDIT) was conducted, in which participants were classified as high-risk alcohol drinkers (AUDIT total score ≥ 8,N= 96.53%) and low risk of alcohol consumption (AUDIT total score < 8,N= 85.47%). Genomic DNA was extracted from whole blood and genotyped using the Illumina OmniExpress Whole Genome BeadChip Array kit. According to the dominant model, participants were divided into two genotype groups based on the presence of a mutant/risk allele for each of the two GLP-1R SNPs of interest. For rs6923761, both groups had the A allele (AA + AG,N= 65) and non-A allele carriers (GG,N= 116), and for rs1042044 allele A carriers (AA + AC,N= 114) and carriers of non-A alleles (CC,N= 67). Brain images were obtained using a Siemens 3T Skyra MRI apparatus. Resting-state functional magnetic resonance imaging (rs-fMRI) lasted 10 minutes, during which participants were instructed to stay awake, lie on their backs in the dark, with their eyes open and without additional stimuli. After pre-processing, independent component analysis was used to analyze the rs-fMRI data. Of the original 75 items, 24 were removed and the remaining 51 were included in the statistical models. Multivariate analyzes of covariance (MANCOVA) were performed separately for each of the two GLP-1R SNPs. The models included the GLP-1R genotype group (A carrier vs. non-A allele carrier), the AUDIT group (low AUDIT vs. high AUDIT) and their interaction (GLP-1R genotype group × AUDIT group) as independent variables and connectivity within and in relation to the lattice of length as dependent variables. Age, sex, years of education, body mass index, smoking status, and ancestry informant scores were also included as covariates.

Results:For rs6923761, three CIs (21, 35, 47) showed a significant effect of the genotype × AUDIT interaction on network connectivity. IC35 and IC47 were mapped to the anterior projecting network and IC21 was mapped to the visuospatial network. Post-hoc analyzes showed that in the group of carrier allele variants (AA+AG), high AUDIT compared to low AUDIT was associated with stronger intra-network connectivity but weaker association or no association. in the protected group (GG). For rs1042044, four CIs (8, 45, 46, 54) showed a significant effect of the genotype × AUDIT interaction on intra-network connectivity. IC8, IC45 and IC54 were mapped to the dorsal pattern function network and IC46 was mapped to the basal ganglia network. Post-hoc analyzes showed that in the group of carrier allele variants (AA + AC) high AUDIT compared to low AUDIT was associated with stronger intra-network connectivity, but in the protected group (GG) the opposite relationship was found. There was no significant effect of the genotype × AUDIT interaction on internet connectivity.

Conclusions:GLP-1R genetic variation was differentially associated with functional brain connectivity in light and heavy drinkers. In particular, the presence of variant alleles was associated with stronger in-network connectivity in heavy and low drinkers. The significant findings in saliency and default function networks are particularly important given their established role in the neurobiology of addictive behavior.

Keywords:Alcohol, GLP-1 receptor, genetic imaging, resting state fMRI

Revelation:Nothing to disclose.

University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Bottom:Recent epidemiological studies suggest increased misuse of gabapentinoids in people with opioid use disorder, and that concomitant use of gabapentinoids (e.g. pregabalin) and opioids increases the risk of opioid-related death. Post-mortem studies have shown that gabapentinoids are present in up to 40 percent of fatal drug overdose cases, mostly those involving opioids. Prescriptions for gabapentinoids are also increasing, with the majority of these prescriptions for off-label indications. Despite these worrying trends, few studies have assessed the potentially harmful interactions between gabapentinoids and opioids. This study examined the effect of pregabalin on the frequency reduction and respiratory depressant effects of heroin and naloxone in reversing heroin-induced respiratory depression in rats.

Methods:Eight male Sprague Dawley rats were trained to respond to sucrose pellets under a schedule of 10-step reinforcement in four 15-minute cycles. Each cycle consisted of a 10-minute decay period and a 5-minute response period during which the rats could acquire up to 5 food pellets. Rats received intraperitoneal (i.p.) saline injections prior to the first cycle followed by sham injections or saline or heroin injections prior to subsequent cycles. Dose-effect curves were determined during sessions using a cumulative dosing procedure in which increasing doses of heroin were administered intraperitoneally. in cycles 2-4, giving cumulative doses of 0.1, 0.32 and 1.0 mg/kg, respectively. Prior to some sessions, pregabalin (0.1–32 mg/kg, i.p.) was administered 15 min before the start of the first cycle. The pressure rate in bar in each cycle was compared under pretreatment conditions. In another preliminary study, three male Sprague Dawley rats were administered intravenous pregabalin (1-10 mg/kg) or saline (IV) prior to increasing heroin doses with ventilation monitored by whole-body plethysmography. Sessions consisted of a 30-minute baseline period followed by a test period in which a cumulative dosing procedure was used in which rats received heroin or saline infusions at 0, 3, and 6 minutes of the treatment period. each heroin injection resulted in a cumulative dose of 0.178, 0.56, and 1.78 mg/kg (intravenously), respectively. Naloxone (0.01 mg/kg) or saline was administered intravenously. 5 minutes after the last heroin shot. The primary outcome of this experiment was minute ventilation—the volume of air ventilated per minute, multiplied by tidal volume and respiratory rate. All procedures were approved by the UT Health Sciences Center San Antonio Institutional Animal Care and Use Committee.

Results:Heroin dose-dependently decreased the speed of lever pressing, with the 1 mg/kg dose completely attenuating the response. The nature of the interaction between pregabalin and heroin differed between individuals, with the heroin dose-effect curve shifted to the left approximately 3-fold in 5 of the 8 pregabalin-treated rats (i.e.). The ability of pregabalin to exert this effect varied between these 5 animals, with an effective dose ranging from 0.32-3.2 mg/kg. Heroin reduced pinpoint ventilation in a dose-dependent manner at a dose of 0.56 mg/kg (e.f.), reducing pinpoint ventilation to 78% of baseline. after i.v. pre-treatment with pregabalin 1 or 10 mg/kg, this effect was enhanced, reducing minute ventilation to 53% and 38% of baseline, respectively. When administered 5 minutes after the highest dose of heroin, naloxone (0.01 mg/kg, IV) restored minute ventilation from 33% to 101% of baseline at 1 minute after administration. This dose of naloxone was significantly less effective in reversing the respiratory depressant effects of heroin in pregabalin 10 mg/kg rats, restoring minute ventilation to only 62% of baseline.

Conclusions:Pregabalin potentiated the depressant and respiratory rate-lowering effects of heroin and significantly reduced the effectiveness of a single dose of naloxone in restoring basal levels of ventilation after a high dose of heroin. These findings have important clinical implications and may help explain recent trends in opioid overdose involving gabapentinoids. Future studies will determine whether this interaction is shared with gabapentin, another gabapentinoid approved for use in humans, and characterize the interactions between pregabalin and other mu-opioid receptor agonists.

Keywords:Pregabalin, Opioids, Breathing

Revelation:Nothing to disclose.

McLean Hospital/Harvard Medical School, Belmont, Massachusetts, Stany Zjednoczone

Bottom:A family history of drug and alcohol use is associated with risk factors for drug use later in life, including early first use of alcohol and drugs, increased impulsivity, impaired executive function, depression, and other mental health conditions. Neighborhood and social factors such as safety concerns, poverty, crime, income, and other environmental disadvantages are also associated with drug use. In addition, individual factors such as coping style, emotion regulation, stress and traumatic life events may play a role. In particular, dysfunctional coping is associated with increased substance use, comorbid mental health conditions, and adverse health outcomes. Here we explore how the family microenvironment, community macroenvironment, and individual characteristics, in particular coping, influence drug use in a community sample.

Methods:Approximately 500 participants from Baltimore and surrounding areas were recruited between 2016 and 2018. The home microenvironment assessments included a children's alcohol screening test and questions about family drug and alcohol use according to the Addiction Severity Index (ASI). Social macro-environmental measures included subjective ratings using the Perceived Neighborhood (PNS) and Neighborhood Conflict (ASI) Scales, as well as objective ratings using the Social Vulnerability Index (SVI) from the Centers for Disease Control and tax code data. Drug use was assessed using the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and/or the ASI. Additional assessments included the Life Events Checklist (LEC), Perceived Stress Scale (PSS), Emotional Regulation Difficulty Scale (DERS), experience issues counseling, and age of first drug use (PhenX Toolkit). Covariates included sex, age, race, ethnicity, marital status, education, number of children and years of living in the neighborhood. The data were examined in three ways: 1) analyzes of variance (ANOVA) to identify group differences in family, community and other characteristics among participants who were lifetime opioid/stimulant drug users, other drug users (marijuana, alcohol) and did not report drug use in life; 2) incremental regression modeling to identify participants as lifetime drug users, lifetime opioid users, lifetime stimulant or other drug users. and 3) stepwise regression modeling to identify participants who used any drug in the last 30 days, used opioids in the last 30 days, used stimulants in the last 30 days. or use of other medications in the last 30 days.

Results:Overall, 154 participants reported no lifetime drug use, 139 reported using opioids/stimulants, and 207 reported using other drugs. Compared to non-drug users, participants who had ever used drugs reported significantly more family conflicts and drug/alcohol use, more unfavorable social environments (PNS, SVI), younger age at first use, higher (worse) PSS, LEC, DERS, and worse coping strategies (3.8 <eat< 74,5; 0,001 <PI number< 0.03). Compared to marijuana/alcohol users, opioid/stimulant users reported more adverse social environments, younger age at first use, higher PSS and LEC scores, and poorer coping strategies (0.001<<PI number< 0.04). Whereas lifelong opioid use was predicted by low neighbor satisfaction, socioeconomic vulnerability, difficulty engaging in perseverance, and coping with a goal (positive reinterpretation, substance use; -0.12<β<0.31, 0.001<PI number< 0.03), lifetime stimulant use was primarily predicted by coping (positive reinterpretation, substance use; -0.13<β <0.23; 0.001<PI number< 0.03). Lifetime use of other drugs was predicted by family alcohol use, family-neighborhood conflict, severe life stress and trauma, and coping (substance use, mood; 0.11<β <0.25; 0.001<PI number< 0.05). Similar results emerged for drug use in the last 30 days: social sensitivity, persistence in pursuing a goal, and coping with intentional opioid use (−0.11<β <0.34; 0.001<PI number< 0.05), while neighborhood conflict, lower tax codes, and confrontation predicted stimulant use (-0.17<β<0.29; 0.001<PI number< 0.04). Other drug use in the last 30 days was predicted by family drug use, family-neighborhood conflict, lower tax code values, lack of emotional awareness and coping (−0.1<β <0.36; 0.001<PI number< 0,04).

Conclusions:Among the measures aggregated here, family microenvironment and social macroenvironment were weaker predictors of drug use than individual factors, especially coping style. Although individual factors generally contributed more to drug use than family or social history, specific predictors varied by drug type. While past family and community experiences cannot be changed, intervention efforts to learn to cope more effectively can help reduce or prevent future drug use.

Keywords:Substance use disorder, opioids, stimulants, treatment

Revelation:Nothing to disclose.

National Institute on Drug Abuse, Baltimore, Maryland, USA

Bottom:The development of non-invasive treatments for brain disorders is at the forefront of neuroscience efforts. New neuromodulation techniques have been developed to alter target cell function under transient and stable control. Widely used technologies such as chemogenetics and optogenetics assume the control of modified/engineered receptors and channels by means of inert exogenous drugs or stimulation (e.g. light). These techniques, however, require surgical delivery of viral vectors and/or implantation of invasive material. Focused ultrasound (FUS) has emerged as a neuromodulation technology that can bypass the invasive procedures described above. However, unlike chemogenetics and optogenetics, it is unknown to what extent local FUS stimulation in a specific brain region can be used to modulate global brain activity as well as the activity of different and molecularly targeted cell types in the brain. specific site of stimulation. The aim of these experiments was to evaluate the effect of specific parameters of FUS stimulation in the CA1 region of the hippocampus of awake, freely moving mice on i) in vivo calcium activity of various cell types in this region and ii) whole brain metabolic activity as assessed by positron emission tomography (PET) and the radioligand [18F]fluorodeoxyglucose (FDG).

Methods:For fiber photometry experiments, mice were transduced using tonsil-associated viral vectors with two different calcium markers, genetically encoded in the CA1 region of the hippocampus. Parvalbumin-positive interneurons in CA1 targeted jRGECO1a, and CaMKII-positive pyramidal neurons targeted GCaMP6. A fiber optic implant was placed in the skull, targeting the hippocampus area to record the cells during ultrasound stimulation. FUS was administered transcranially and the effective parameters were optimized by manipulating the pressure and rate of stimulation. In PET experiments, mice were implanted with a pin on the top of their skull with coordinates pointing to the CA1 region. The pin was used to hold the transducer in place while the FUS was being delivered. Mice were injected with FDG and stimulated with 900 Hz stimulation 10 times for 30 minutes in an open field where the mice were free to move around. After simultaneous FUS stimulation and FDG uptake, mice were anesthetized and scanned for 20 minutes with PET. FDG uptake was assessed using voxel methods to determine objective changes in metabolic activity between unstimulated and unstimulated hemispheres.

Results:Sound pressure fields were investigated for a working transducer attached to the explanted skull with a scanning hydrophone. To optimize the pressure required for effective stimulation of the target region, various pressures (0.26, 0.43, 0.56 and 0.76 Mpa) were tested and calcium signaling was recorded with jRGECO1a and GCaMPP6S. Higher pressures (0.56 and 0.76 MPa) resulted in the most significant increases in calcium signaling in both parvalbumin and pyramidal neurons. Stimulation at 900 Hz initially produced a brief increase in calcium signaling, but quickly resulted in a prolonged depression of calcium signaling that persisted for more than 30 seconds. The results obtained in the fiber photometry experiment were further confirmed similarly with PET. After stimulation at 900 Hz in freely moving, awake mice, FDG uptake analysis showed a significant reduction in the area near the stimulation site in the stimulated hemisphere compared to the opposite unstimulated side. Threshold mapping showed that the FDG signal is most downregulated in the CA1 target region of the hippocampus.

Conclusions:Here we show that focused ultrasound can alter cellular activity in vivo in a way that may facilitate further research in the field of neuromodulation. The invasive techniques used in these experiments (e.g. virus transduction, fiber implantation) were designed to test the feasibility of this new FUS technique. As confirmed by PET experiments using minimal or non-invasive techniques, FUS reliably reduced cellular activity at the target site. This specificity is crucial to elucidate neural circuits with great potential for clinical applications in the treatment of human brain diseases. Future research will explore the long-term effects of chronic stimulation as well as the potential to manipulate drug-related behavior. In addition, FUS can be used as a potential method of delivering systemic compounds to specific areas of the brain without the need for neurosurgery. Such approaches can also be assessed with PET for acute and long-term efficacy.

Hair Sponsor Dr. Michael Lewis

Keywords:Positron emission tomography (PET), focused ultrasound, non-invasive neuromodulation

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:The development of physical dependence and addictive disorders as a result of the misuse of opioid analgesics is a major concern in current pain management. Chronic pain, a debilitating disorder characterized by various sensory and neuropsychiatric symptoms, namely anxiety, depression, sleep disturbances, and increased anxiety, is associated with opioid use disorder (OUD). Very few therapeutic strategies target the physical and emotional symptoms associated with chronic pain, and most available treatments have limited efficacy and tolerability, and have serious side effects. Approximately 20-25% of chronic pain patients are treated with opioids, despite the lack of efficacy and adverse outcomes associated with this treatment strategy. In addition, the increased doses of opioids prescribed for chronic pain lead to a dramatic increase in the number of patients who become addicted to opioids and become addicted. Recent research by our group has revealed that chronic neuropathic pain promotes several genetic adaptations in reward-related brain regions (Descalzi et al., Sci. Signaling, 2017). Here, we use next-generation RNA sequencing and pathway analysis to gain insight into gene expression adaptations induced by persistent oxycodone exposure in the presence and absence of chronic neuropathic pain.

Methods:To assess opioid abuse in chronic pain and pain-free states, we subjected two-month-old C57BL/6 mice to a SNI (serrated trauma nerve) model of neuropathic pain or sham surgery. Mice were assessed for hypersensitivity (mechanical allodynia and thermal hyperalgesia) associated with long-term neuropathic pain every two weeks for 9 weeks, followed by daily subcutaneous injections of oxycodone (30 mg/kg) or saline for two weeks. After two weeks of chronic exposure to oxycodone, the animals went through an extended period of automatic drug withdrawal. At this point, we are monitoring sensory hypersensitivity and emotional behavior to understand the effects of oxycodone withdrawal in terms of chronic pain and pain-free states. As such, we conducted several behavioral tests such as the Novelty Social Recognition Test, the Youth Suppression (NSF) Return, the Marble Burial Test, the Light-Dark Test, and the Motor Activity Test during a 3-week drug withdrawal period to assess related emotional behaviors. drug abstinence. We then sequenced to understand the molecular mechanisms underlying oxycodone withdrawal, subjected a separate group of mice to our oxycodone abuse paradigm, and 21 days after drug withdrawal dissected the medial prefrontal cortex (mPFC), nucleus accumbens (NAc) and ventral VTA region) and performed whole brain tissue RNA sequencing. Using pathway analysis, we identified histone deacetylase 1 (HDAC1) as an upstream regulator in NAc and mPFC. Using a novel HDAC1/2 inhibitor, RCY1305, we administered RCY1305 (3 mg/kg i.p. daily) to mice during oxycodone administration and drug withdrawal for 5 weeks. We then assessed the effects of HDAC1/2 inhibition on oxycodone-induced behavioral changes, such as sensory hypersensitivity and emotional and motor deficits.

Results:Oxycodone administration induced thermal hyperalgesia after five days of daily injection in mice with chronic pain, but not in pain-free mice. During early drug withdrawal, four days after drug withdrawal, both pain and no pain developed significant thermal hyperalgesia compared with saline controls (ANOVA,eat(3248)=57.6). Oxycodone treatment relieves mechanical allodynia in SNI mice and induces significant mechanical allodynia in sham mice during early drug withdrawal (d7) (ANOVA, (3279) = 748.5). We then assessed affective symptoms that were influenced by abstinence states. In the social interaction test, all groups showed a preference for the social goal, with the exception of oxycodone-deprived SNI mice, which showed significant social deficits (ANOVA,eat(3.58) = 1.418). Moreover, all groups except the sham saline controls show significant deficits in recognizing new social goals (ANOVA,eat(3.58) = 0.5616). To assess anxiety-like behaviors, we performed the light-dark box and marble burying test. Oxycodone-withdrawn SNI mice spent significantly less time examining the bright side of the box. In the marble burying test, oxycodone sham mice buried a significant percentage of marbles compared to SNI-oxycodone and its control. Using RNA sequencing, we monitored changes in gene expression in the medial prefrontal cortex, nucleus accumbens and ventromedial tegmental area. Although oxycodone treatment primarily promotes unique transcriptional profiles in brain regions, we observed similar transcriptional effectors and transcription factors that were affected by pain conditions compared to pain-free conditions. Our pathway analysis revealed that histone deacetylase 1 (HDAC1), an epigenetic modifier with a significant role in striatal plasticity, is an upstream regulator of opioid withdrawal in both the NAc and mPFC.

Conclusions:Our findings suggest that chronic pain conditions exacerbate the behavioral and transcriptional signatures of oxycodone withdrawal. Taken together, our studies highlight intracellular pathways, thus providing new avenues for the treatment of oxycodone addiction in painless and neuropathic conditions.

Keywords:Chronic pain, opioid addiction, pharmacotherapy, animal model, abstinence, transcription, negative effect, mesolimbic reward circuit

Revelation:Nothing to disclose.

IRP, NIDA, NIH, Baltimore, Maryland, USA

Bottom:The United States and many other countries are facing a deadly opioid overdose epidemic. Two-thirds of drug overdose deaths in the United States in 2018 involved opioids, and 66% were related to synthetic opioids such as fentanyl. Currently, treatment options for opioid use disorder include substitution therapy (e.g. methadone and buprenorphine) or the non-selective opioid receptor antagonist naltrexone. We previously found that the long-acting κ-opioid receptor (KOR) antagonists 5'GNTI and norBNI reversed the hyperalgesia seen during acute heroin withdrawal in both sexes of rats and that norBNI prevented increased heroin self-administration in male rats. Recently, short-acting κ-opioid receptor antagonists have gained interest as potential therapeutic agents for anhedonia and depression, but little has been done with these compounds in addiction models.

The aim of this preliminary work was to investigate whether KOR antagonism and predynorphin deletion reduce fentanyl self-administration.

Methods:We first trained 64 C57BL/6J mice (32 males and 32 females) and pro-dynorphin knockout (KO) and wild-type (WT) mice (16 female KOs, 18 females WTs, 19 male KOs, and 20 males WTs) to auto-administer vaporized fentanyl (5 mg/mL, 60 W, 1.5 s) and then divided into short access (ShA, 1-hour sessions, independent) and long access (LgA, 6-hour sessions, dependent) groups). We tested the hypothesis that (1) the short-acting KOR antagonist aicaprand and the canonical KOR antagonist norBNI would reduce fentanyl vapor self-administration in dependent but not independent C57BL/6J mice; (2) deletion of the prodynorphin gene (pDyn KO) will prevent fentanyl self-administration from escalating. All animal studies were approved by the local ACUC and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All data will be presented as mean + SEM and analyzed by one-way or two-way ANOVA RM followed by Dunnett's post hoc test.

Results:LgA mice increased fentanyl self-administration while ShA mice did not. Using the within-subject Latin square formula, we tested aikaprand (0, 0.3, 1, 3, 10, and 30 mg/kg, PO, 75 min pre-session) in self-administration of fentanyl. Since we did not detect any differences between the sexes, men and women were analyzed together to increase statistical power. Atikaprant did not reduce fentanyl self-administration in the dependent groups (F5, 75=1.781,PI number>0.05), while the higher dose slightly increased fentanyl self-administration in non-dependent mice (F5, 74 = 4.362,PI number= 0.07). Two groups of mice subjected to scaling and behavioral testing had a 3-week withdrawal phase and then received norBNI (10 mg/kg, IP,N= 8 males and 8 females) or saline (10 ml/kg, IP,N= 8 males and 8 females) prior to the 9-session reprogramming phase. Males and females were analyzed together as no gender differences were detected. A single treatment with norBNI prior to reprogramming significantly reduced mean fentanyl intake in dependent mice but not in independent mice (F1, 60=4.998,PI number= 0,03).

Mice lacking the pro-dynorphin gene (pDyn KO) were tested using the same self-administration protocol. Dependent mice increased fentanyl self-administration (F9, 287 = 6.262,PI number< 0.001) regardless of group, contrary to our hypothesis, female pDyn KO mice tended to increase their intake and intake more fentanyl more quickly than their WT counterparts.

Conclusions:These preliminary data suggest that prolonged κ-opioid receptor antagonism may be necessary to reduce fentanyl self-administration. The effect of chronic treatment with short-acting κ-opioid receptor antagonists on opioid self-administration remains to be investigated. Deletion of the prodynorphin gene may have gender-specific effects that deserve further study.

Keywords:Prodynorphin, kappa opioid receptor antagonist, opioid use disorder

Revelation:Nothing to disclose.

National Institutes of Drug Abuse/National Institutes of Health, Baltimore, Maryland, USA

Bottom:Illicitly produced fentanyl is largely responsible for the current opioid overdose crisis, but several synthetic opioids other than fentanyl have emerged in the non-pharmaceutical (i.e. illicit) drug markets around the world. Little information is available on the pharmacology and toxicology of new synthetic opioids when they first hit the street. U-47700, borphine, and isotonitazene are examples of non-fentanyl synthetic opioids associated with opioid overdose deaths in humans.

Methods:Here, we profile the pharmacology of several synthetic non-fentanyl opioids - including U-47700, borphine and isotonitazene - using in vitro and in vivo methods. In vitro receptor binding assays were performed on rat brain membranes to test the ability of test drugs to displace [3H]DAMGO, [3H]DADLE, or [3H]U-69593 from mu, delta, or kappa receptors. appropriately. In vivo experiments were performed in male Sprague-Dawley rats to evaluate the subcutaneous (sc) effect of the test drugs on analgesia, catalepsy and body temperature. All animal experiments were approved by the Animal Care and Use Committee of the NIDA Intramuscular Research Program.

Results:The results of the [3H]DAMGO binding assays showed that the Ki values ​​of U-47700 (12.0 nM), borphine (37.7 nM) and isotonitazene (14.9 nM) at the mu opioid receptor are weaker than those of morphine (4, 0nM) (N= 3 experiments for each dose-response curve). However, all compounds tested were selective for the mu-opioid receptor at the delta and kappa sites. When administered to male rats in vivo, all compounds produced dose-dependent opioid-like effects, including antinociception, catalepsy and hypothermia.N= 8 rats per drug dose). The results of the hot plate test showed that the ED50 values ​​for U-47700 (0.404 mg/kg, sc), borphine (0.086 mg/kg, sc) and isotoitazene (0.007 mg/kg, sc) are significantly more potent compared to morphine ( 4.163 mg/kg subcutaneous). Isotonitazene was even more potent than fentanyl (0.021 mg/kg, subcutaneous) as an analgesic.

Conclusions:Our results show that synthetic opioids other than fentanyl may be significantly more potent than morphine in vivo, suggesting a serious overdose risk for unsuspecting users. Importantly, the binding affinity of mu-opioids to synthetic opioids in vitro may not predict potency in vivo. Extensive in vitro and in vivo studies are needed to determine the pharmacology and toxicology of new synthetic opioids emerging in underground drug markets.

Keywords:Opioid abuse, analgesia, catalepsy, Mu opioid receptors

Revelation:Nothing to disclose.

UCLA, Los Angeles, California, United States

Bottom:Developing new and more effective treatments for alcohol use disorder (AUD) is a research priority. Identifying the mechanisms of treatment action is an important step towards improving treatment effectiveness, which can be improved by using more reliable and cost-effective assessment methods, such as daily reports. A growing body of research links the immune system to the development and maintenance of AUD. Therefore, there is great interest in new treatments that can restore healthy levels of inflammation and immune signaling to reduce alcohol consumption and speed recovery. Phosphodiesterase (PDE) inhibitors have been tested extensively in preclinical animal models of AUD, as some PDEs are expressed in areas of the brain involved in the reinforcing effects of alcohol. Ibudilast, which is a neuroimmune modulator and selective PDE inhibitor, reduced alcohol consumption and relapse in several animal models of AUD. In addition, our lab tested ibudilast in two AUD clinical samples, where treatment with ibudilast compared to placebo reduced neuronal reactivity to alcohol and decreased binge drinking. In addition, while ibudilast did not significantly alter the subjective response to intravenous alcohol in all subjects, it did reduce the stimulant effects of alcohol in those with high depressive symptoms, warranting further research. Thus, ibudilast is a promising pharmacotherapy for AUD, but its mechanisms of action remain largely unknown in human clinical samples. The current study was designed to test the effects of ibudilast's neuroimmune modulation on subjective alcohol response under naturalistic conditions in a two-week randomized trial that enrolled participants with AUD.

Methods:This study serves as a secondary analysis of a two-week clinical trial (NCT03489850) ibudilast to improve negative mood and reduce alcohol consumption in enrolled men and women who were not presenting for AUD treatment. Fifty-two eligible participants were randomly assigned to ibudilast or the appropriate placebo. Participants completed three in-person visits and morning electronic daily assessments to report drinking, hunger, and mood the previous day. When participants reported drinking alcohol the previous day, they were asked to describe mood and craving before and while drinking, as well as excitement and calmness while drinking. Participants who completed at least one post-randomization daily report (N= 50: ibudilast = 23, placebo = 27) were included in these analyses. Multilevel mixed models with randomized interventions and appropriate covariates (e.g., gender, alcohol intake, depression) examined the effects of drug status (ibudilast vs. placebo) on craving, craving, agitation, and sedation. In addition, multi-level mixed models with random intercepts and inter-level interactions tested whether ibudilast attenuated the impact of same-day and next-day subjective alcohol consumption.

Results:While participants using ibudilast reported lower average levels of agitation and sedation, this between-subject treatment effect was not significant (PI numbers > 0.05). However, analyzes revealed a significant cross-level drug x stimulation (PI number= 0.046) and drugs x sedation (PI number= 0.049) interaction with same-day but not next-day alcohol consumption. Using simple curves to detect these interactions, those treated with ibudilast reported a stronger relationship between arousal (PI number< .001) and attenuation (PI number= 0.001) drinking on the same day as placebo (PI numbers>0.250). Overall, participants in the medication setting reported an increase in alcohol cravings and cravings during the study. In addition, the results revealed a significant drug x time interaction (before consumption vs. during) for alcohol craving, such that ibudilast participants reported a significantly smaller increase in alcohol craving on the same day (PI number= 0.180) than those receiving placebo (PI number< .0001). The same effect was not detected with the urge to drink. However, ibudilast significantly attenuated the effect of the average change in craving (during or before fasting) on ​​alcohol consumption, such that ibudilast (PI number= 0.022), but not placebo (PI number= 0.772) a significant positive relationship was found between the average desire to drink and the number of drinks consumed.

Conclusions:These studies extend our understanding of the mechanisms of action of neuroimmune modulators in AUD clinical samples using ecological references. Although ibudilast did not strongly alter arousal and inhibition in response to alcohol under natural conditions, it appeared to increase participants' awareness of the relationship between subjective response and same-day alcohol consumption. According to previous reports from our lab, ibudilast reduced cravings, thereby reducing the increase in alcohol cravings. These results should be considered in the context of the immunomodulatory and biological effects of ibudilast. Future research should extend these findings to treatment-seeking and higher-severity AUD samples, and explore gender differences.

Keywords:Alcohol use disorder - treatment, subjective response, neuroimmunology

Revelation:Nothing to disclose.

Boston University School of Medicine, Boston, Massachusetts, Stany Zjednoczone

Bottom:The opioid use disorder (OUD) epidemic is a deepening public health crisis, and opioid-related deaths will peak in 2020. Most opioid-related deaths are attributable to prescription opioids, with the semi-synthetic opioid oxycodone (OXY) being one of the most commonly prescribed opioids and is the leading cause of designer drug-related deaths. However, current treatment for OUD is limited and relapse rates remain high, highlighting the critical need to understand the underlying risk factors for OUD, which may lead to more effective OUD treatment. There is a genetic basis for OUD risk, opioid use, and behavioral and physiological responses to opioids, so identifying the genetic basis of OUD-related traits can aid therapeutic development by identifying novel genes, mechanisms, and downstream biological processes that regulate drug responses. Human genome-wide association studies (GWAS) have so far identified several quantitative trait genes for OUD-associated phenotypes. Quantitative mapping of the trait locus in rodent models can overcome the limitations of the human GWAS, effectively elucidating the genetic causes of addiction-related model phenotypes. To identify genetic variants of OUD-related behaviors in mice, we screened 29 mouse strains for lineage-specific differences in behavioral responses to OXY to identify OUD addiction model behaviors and underlying genetic factors that serve to validate genetic manipulations in vivo.

Methods:All mouse strains were tested using our multi-step addiction assessment protocol (MSAAP). Mice were first subjected to conditioned place preference (CPP) at 1.25 mg/kg OXY to assess stress differences in OXY locomotion, OXY environmental preference and state-dependent OXY reward learning. The mice were then injected daily with 40 mg/kg OXY for 1 week, and then the tolerance to the analgesic effect was assessed at 4 mg/kg OXY using an anti-log hot plate test. Mice were re-injected with 40 mg/kg OXY daily for 1 week and then tested for acute OXY withdrawal using the elevated plus maze (EPM) and light-dark test. Data were analyzed using two-way (genotype, treatment) and three-way (genotype, treatment, sex) ANOVA, with post hoc comparisons with Holms-Sidak corrections for major effects or significant interactions. Haplotype association mapping was performed on tissue samples to identify genetic regions associated with different OXY phenotypes.

Sample size (genotype, treatment, sex):

Wild type, saline, male: 14

Wild type, saline, female: 13

Wild type, OXY, male: 11

Wild type, OXY, female: 10

KO, Salina, men: 8

KO, Salina, Feminino: 10

KO, OXY, Male: 10

KO, OXY, Feminino: 10

Results:Using CPP, we found robust stress differences in OXI-induced locomotion, place preference and condition-dependent learning indicative of causal genetics for these phenotypes. In addition, we observed a strong heritability of OXY-induced locomotion (h2 = 0.72). Subsequent haplotype association mapping identified a region on chromosome 17 associated with an OXY-induced translocation containing the candidate gene Cacna1h, which encodes a voltage-gated calcium channel subunit (PI number= 2.9e-8). We then tested Cacna1h knockout (KO) mice in a mixed 129/C57BL/6N background crossed with C57BL/6N for the N2 generation (and then backcrossed) with OXY CPP, in addition to hot plate analgesia, EPM and differential chiaroscuro challenge of tolerance responses and ACID withdrawal between genotypes. We did not observe significant genotypic differences in distance traveled in response to OXY. However, Cacna1h KOs with a prior OXY history walk shorter overall distances during a place preference session (Main genotype effect:eat(1,82) = 5511,PI number= 0.0213; After the OXY treatment:PI number= 0.0219). The effect of Cacna1h KO was specific to OXY CPP-associated locomotion, as there were no genotypic differences in OXY CPP or state-dependent learning, nor significant differences in OXY nociceptive tolerance or spontaneous withdrawal in the elevated plus maze or light maze/dark box.

Conclusions:Our negative findings do not currently support a role for Cacna1h in model behaviors of opioid dependence. However, we only tested one genetic background and it may be useful to test Cacna1h KO on different genetic backgrounds to completely rule out its involvement in behavioral responses to opioids. Additional genetic mapping from our search for MSAAP strains may reveal additional genetic variants that, through in vivo validation, may inform behavioral phenotypes associated with OUD.

Keywords:Mouse genetics, opioid use disorder, haplotype association mapping, addiction phenotypes

Revelation:Nothing to disclose.

Yale University, New Haven, Connecticut, United States

Bottom:There is an urgent need to engage more diverse research participants and researchers to diversify genomics research and the genetics workforce. Joint and coordinated efforts will facilitate the work needed to improve our understanding of the specific genetic vulnerabilities of different and mixed racial populations. These efforts must also include significant input from affected communities so that research and diversification efforts benefit stakeholders. The inclusion of non-European and mixed-ancestry populations in genetic analyzes can facilitate the identification of genetic effects that generalize across populations, the identification of causal variants, and the identification of population-specific genetic variants. In addition, there are well-studied health differences between populations, highlighting the need for fairer genomic research.

Methods:The Latin American Genomics Consortium (LAGC) was established in 2019 to advance the study of psychiatric genetics in Latin American populations, especially between Latin American countries and the United States.

Results:The LAGC has approximately 100 active members representing 8 different countries/regions in Latin America, including Mexico, Argentina, Costa Rica, Brazil, Colombia, Chile, Peru, Puerto Rico and the USA.

Conclusions:This initiative aims not only to address the huge under-representation of non-Europeans in genomics research, but also to facilitate access to training and resources and participation in collaborations around the world.

Keywords:Genomics, Consortium, Latin America

Revelation:Nothing to disclose.

Temple University, Philadelphia, Pennsylvania, United States

Bottom:Experiencing adversity early in life may be a risk factor for the development of many mental disorders, including TUS. However, most people exposed to stress early in life do not develop disorders. Stress that is not overwhelming can have a "vaccinating" effect that promotes the development of immunity later in life. Our lab models adversity in early life with the Bounded Bed and Nest (LBN) model. We have previously found that this manipulation produces a phenotype resistant to addiction-related behavior. Particularly in male rats, the LBN reduces impulsive choice, a behavior partly mediated by the basolateral amygdala (BLA). We also found that this model reduced morphine self-administration in male rats only. This suggests that LBN has a vaccinating effect against addictive behavior in men. Therefore, we sought to outline the potential molecular basis that promotes stress-induced immunity.

Methods:To understand how adversity affects BLA in early life, we used an early life stress model in rodents called LBN, which mimics a low-resource environment by restricting the mother's access to nesting materials during the first week of chick life. Long Evans rats were housed in LBN or Postnatal Day Control (PND) conditions 2 through 9. LBN conditions involved mothers and pups placed in a resource-limited environment where metal mesh blocked access to the bed. and the mothers were given one paper towel to use as nesting material. Control animals were bred under standard laboratory conditions. On postnatal day 10, the LBN animals were returned to standard laboratory conditions. RNA sequencing was performed to determine the effect of LBN on the BLA transcriptional profile in adult mice (male control,N= 4; women control,N= 5; LBN male,N= 5; female pounds,N= 4). BLA tissue from early adult mice was analyzed on an Illumina HiSeq 4000. Fastqc version 0.11.8 was used to assess the quality of reads with adapters and unpaired reads removed with Trimmomatic version 0.39. The Rank-Rank Hypergeometric Overlap (RRHO) test version 2 assessed the degree of gene signature overlap between the sexes. Differentially expressed genes (DEGs) were identified using aPI numbervalue <0.1 and 50% change in expression as cutoff value to determine significance.

Results:We found sex-specific changes in transcription induced by LBN. RRHO analysis revealed different up- and down-regulated genes in males and females due to LBN. There was very little overlap of upregulated genes in men and women, or in men versus women. A large proportion of genes were upregulated by LBN in males and downregulated in females. We narrowed our analysis down to genes showing a significant difference between control and LBN and found 209 DEGs in females and 149 DEGs in males. These changes in gene expression were mainly gender-specific as only 11 genes were modified by LBN in both males and females. Composite LBN DEG heatmaps showed different patterns of up- and down-regulated genes in males and females. Of interest Grin1, the NMDA ionotropic glutamate receptor subunit 1 gene, was downregulated by LBN in females, and pathway analysis showed LBN-induced changes in female glutamate pathways. In men, pathway analysis showed LBN-induced changes in MAPK pathways, and several MAPKs, such as MAPK10, were upregulated in LBN compared to control men.

Conclusions:These analyzes highlight the unique gene expression patterns induced by LBNs in BLA in a sex-specific manner. Future work will focus on validating key targets with RNAscope and manipulating the targets in a cell type-specific manner to directly link changes in gene expression to addiction-related behavior. Taken together, this work advances our understanding of the neurobiological underpinnings of stress vaccination, which may lead to advanced therapeutic techniques.

Keywords:Early life adversity, substance use disorder, basolateral amygdala

Revelation:Nothing to disclose.

Rutgers University - New Brunswick, Piscataway, New Jersey, United States

Bottom:Value-based decision making is an integral concept to understanding addiction, including opioid use disorder (OUD). Despite the availability of the gold standard of treatment (i.e. medication for opioid use disorder or MOUD), people with OUD continue to alternate between states of anxiety-anticipation, drug use, and withdrawal/withdrawal. Changes in normative cognitive processes related to judgment and decision-making, such as risk tolerance and latency, are thought to serve these clinically significant transitions, but there is no empirical support for this association, particularly in patients in everyday physical environments. In addition, psychological and contextual factors such as optimistic beliefs and emotional states can influence the evaluation process to promote the risk of reuse. However, the relationship between these factors and OUD decision preferences over time remains relatively unknown. In the current study, to explore the real interplay of mood, optimism, and decision preferences, we monitored decision-making behaviors in a set of smartphone-powered economic tasks while participants were engaged in their physical environment. We predicted that individuals with OUD, compared to healthy controls, would prefer riskier and more immediate rewards, and that these preferences would especially change during periods of elevated mood and optimistic thinking.

Methods:Nineteen patients enrolled in OUD treatment (58% male, 43.4 ± 13.01 years) were recruited from two university MOUD clinics. Eighteen healthy controls (55% male, 52.8 ± 13.16 years) were recruited from the same geographic area of ​​residence and compared with OUD subjects by gender and race/ethnicity. All subjects participated in a 4-week smartphone instantaneous ecological assessment study (704 person-days, range: 7–30 days/person). The prompts were delivered 4 to 8 times a day and assessed current affective (e.g. positive and negative mood) and cognitive (e.g. optimistic thinking) states. In addition, once a day, subjects performed validated behavioral financial tasks, measuring preferences for delayed and risky real monetary rewards. One decision was selected each day and actually paid at the end of each study week. The impulse choice task examined preferences for monetary rewards that can be delivered with various delays. In a risky choice task, preferences for monetary rewards or losses that can be obtained under various known and unknown (ambiguous) probabilities were examined. Daily decision preferences of patients and controls were predicted by their self-reported emotional state and optimistic thinking. Based on these predictors, two-way interactions (number of observations = 584, linear mixed-effects model with random intercepts and subject deviations) were developed with degrees of freedom computed using the Satterthwaite approach.

Results:Subjects showed dynamic decision preferences with high day-to-day variability for risky and ambiguous choices (SD = 0.31). While there were no significant group differences in overall risk or ambiguous choice between patients and controls, changes in risk preference were associated with mood swings and optimistic thinking. We found that when subjects reported being more optimistic, they preferred riskier deals, especially in the context of unknown/ambiguous losses (b = 0.0009, 95% CI [0.00008, 0.002], t44.74 = 1.71,PI number= 0.03). Subjects also tended to make riskier decisions when they reported feeling better (b = -0.0009, 95% CI [-0.0001, 0.0018], t44.74 = 1.71,PI number= 0.09) with a stronger association between mood and risk observed in patients (b = -0.0019, 95% CI [-0.0001, 0.0018], t89.22 = -2.288,PI number= 0.02). On the other hand, we found that impulsive choice was generally greater in patients compared to controls (t30 = 2.61,PI number= 0.01), but was relatively stable over the course of days and unrelated to mood swings and daily optimistic thinking.

Conclusions:Collectively, our findings suggest a dynamic relationship between mood and optimism in decision-making preferences, particularly for risky choices, potentially identifying a mechanism for detecting and responding to specific cognitive and contextual changes associated with risky clinical situations in OUD (e.g., reuse/recursion).

Keywords:Opioid addiction, value-based decision-making, momentary ecological appraisal, optimism, mood

Revelation:Nothing to disclose.

University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States

Bottom:Circadian clocks regulate neural mechanisms related to rewards, and disruptions in these clocks increase substance use. Drug abuse, in turn, disrupts the timing mechanisms of circadian clocks in reward circuitry, thereby contributing to substance use disorder (SUD). Adolescence is a time sensitive to both circadian rhythm disruptions and the development of SUD because it is a transitional stage accompanied by more reward-motivated, impulsive, and sensation-seeking behaviors. The interplay between circadian clocks and reward pathways is a critical aspect of SUD development during adolescence, but the molecular mechanisms underlying this relationship are not fully understood. Therefore, as part of the Adolescent Reward, Rhythm, and Sleep Center, our primary goal is to understand how genetically abnormal rhythms and environmental arrhythmias contribute to susceptibility to substance abuse in adolescence.

Methods:To understand the relationship between natural changes in circadian characteristics and drug addiction, we conducted several analyzes measuring circadian rhythms, addiction-related behaviors, including the 5-choice serial response time task (5CSRTT) and intravenous self-administration using heterogeneous stocks (HS) in adolescent litter rats (P28-48). We also used longer-period, lower-amplitude primary mouse skin fibroblasts to test potential therapeutic compounds for their ability to modulate molecular rhythmicity in vitro. To address the effects of environmental circadian disruptions such as jet lag on reward functions, 5CSRTT and electrophysiological recording were performed on neuronal tissues in rats with chronic interruptions in their light-dark cycle (12 hours every 3 days). In addition, we collected the prefrontal cortex and nucleus accumbens (NAc) after an acute sleep interruption to measure changes in gene expression using RNA sequencing.

Results:We found that HS mice showed high circadian phenotypic diversity compared to conventional mouse crosses, possibly due to their genetic heterogeneity. This shows that this model is a powerful tool for studying complex traits such as circadian rhythms and addiction-related behaviors. We observed a large variation in circadian period and daily sleep percentage in these rats (23.76 - 24.18 hours and 32 - 56%, respectively). Our rat fibroblast cultures showed a duration similar to that observed in behavioral rhythms. In addition, our preliminary tests showed that previously identified compounds that increase indices in mouse cell cultures have similar effects on molecular indices in mice. Interestingly, mice with chronic interruptions in the light-dark cycle took longer to develop 5CSRTT compared to mice with stable light-dark cycles. In addition, NAc electrophysiological recordings showed that circadian misalignment altered synaptic AMPAR levels and membrane excitability in medium spiny neurons.

Conclusions:Our preliminary data show that genetic diversity contributes to phenotypic variation in circadian rhythms, and that circadian mismatch in adolescence increases addictive behavior in HS rats. In addition, our transcription study will provide more useful information to elucidate the mechanisms underlying the interplay between circadian rhythms and drugs during adolescence.

Keywords:Puberty, circadian rhythms, substance use disorders, inbred rats

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:Substance use disorders are debilitating neuropsychiatric diseases that lead to significant morbidity and mortality. Despite significant advances in understanding the intracellular signaling cascades and neural signaling networks associated with long-term drug use, there are currently no drugs approved by the FDA for the treatment of psychostimulant use disorders. This has led to the development of work exploring regional factors as potential targets for translational research. A growing literature has characterized an important cross-talk between the brain and the bacterial population in the gut - the gut microbiome. This signaling may influence both normal brain function and pathogenesis in animal models of neurodegenerative diseases as well as neuropsychiatric diseases such as autism, depression and addiction. While the complex mechanisms of this gut-brain communication require further study, strong evidence suggests that the gut microbiome produces numerous neuroactive metabolites that can signal to and regulate brain function. The strongest evidence exists for a class of bacterial-derived metabolites known as short-chain fatty acids (SCFAs), which have been shown to modulate molecular, cellular and behavioral effects in animal models of neuropsychiatric diseases. Recent work from our lab has shown that animals whose microbiomes have been depleted in antibiotics show a greater preference for cocaine, and that this effect can be reversed by supplementation with SCFA metabolites. In this study, we used our established models of microbiome depletion and metabolite supplementation to analyze the effects of gut microbiome signaling on cocaine-seeking behavior in a translational model of drug relapse behavior. Behavioral studies are combined with state-of-the-art transcriptomics and metabolomics profiles to help determine the mechanisms underlying these gut-brain effects.

Methods:Gut bacteria and their metabolites were depleted in Sprague-Dawley rats by adding non-absorbable broad-spectrum antibiotics (neomycin, bacitracin, vancomycin) to the drinking water of their cages and compared to water-treated controls. To investigate the SCFA-specific mechanistic contribution, the three major gut-produced SCFAs (butyrate, acetate, propionate) were restored to normal physiological concentrations by adding drinking water in the home cage. Adult male rats were first trained to self-administer cocaine (FR1 0.8 mg/kg/inf) and then cocaine-seeking behavior was tested using: (1) a threshold test during sessions to assess cocaine motivation at various doses (N= 10 H2O,N= 7 Abx) or (2) 21 days of abstinence followed by a cue-triggered cocaine-seeking task to model relapse behavior (N= 6-7/group). After all behavioral testing, nucleus accumbens tissue was isolated and processed for RNA sequencing. Transcriptomes were compared by differential gene expression analysis using the DeSeq2 analysis package. Functional pathways changed between groups were determined using the Ingenuity Pathway Analysis and G: Profiler software packages. All experimental protocols in animal studies were approved by the Institutional Animal Care and Use Committee at Mount Sinai and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Results:The depletion of the microbiome by antibiotics did not affect the acquisition of cocaine in the FR1 regimen. However, when animals that were already consistently self-administering cocaine were subjected to a dose-response threshold task during the session, those with a depleted microbiome showed significantly increased motivation for a low dose of cocaine (two-way ANOVA, main effect by Abx:PI number< 0.01). Similarly, microbiome depletion increased cue-induced cocaine seeking after prolonged abstinence in a relapse model (T-test, effect Abx:PI number< 0.01). To determine the molecular drivers of gut–brain signaling in this model, we supplemented with SCFAs in animals with and without a depleted microbiome. SCFA supplementation completely reversed the behavioral effects of microbiome depletion on signal-induced cocaine seeking after abstinence. RNA sequence analysis further showed that microbiome-depleted animals showed significant changes in gene expression in networks known to affect synaptic plasticity compared to SCFA-treated individuals and water-treated controls. Taken together, our findings suggest that the microbiome and its metabolic by-products are critical regulators of cocaine-seeking behavior and synaptic plasticity.

Conclusions:Animals lacking a complex gut microbiome show significantly increased cocaine-seeking behavior and altered expression of synaptic plasticity genes. In the absence of a normal microbiome, supplementation with bacterial SCFA metabolites reverses the behavioral and molecular changes associated with microbiome depletion. Molecular analyzes revealed marked changes in activity-dependent gene expression in mice treated with antibiotics compared to controls. These findings suggest that gut bacteria may serve as homeostatic regulators of gene expression in the brain through their metabolites and suggest that the microbiome may be a potential target for translational research.

Keywords:Cocaine self-administration, cocaine-seeking behavior, cocaine use disorder, metabolites, gut-brain axis

Revelation:Nothing to disclose.

National Institutes of Health, Bethesda, Maryland, Stany Zjednoczone

Bottom:Black people are at higher risk for alcohol use disorder and high-risk alcohol use disorder (AUD). Also, they are less likely than Caucasians to seek and complete AUD treatment. AUD has a biphasic effect on the hypothalamic-pituitary-adrenal (HPA) axis, reflecting a shift between over-responsiveness to stress (during alcohol withdrawal) and decreased responsiveness (during early withdrawal). Independent of AUD, there are racial differences in HPA axis function: black individuals without AUD, compared to white individuals without AUD, show flatter daily cortisol gradients and a reduced response of the HPA axis to a psychosocial stressor. These differences are related to socioeconomic status (SES), early life adversity, including trauma, stress in adulthood, and psychopathology. Although related to altered HPA axis function, racial minority status has not been well studied in the context of AUD-related HPA axis dysregulation. Here, our aim was to investigate race self-identification as a potential contributing factor to dysregulation of the HPA axis in individuals with ASD during alcohol abstinence.

Methods:We compared ACTH and morning plasma cortisol levels in 449 subjects (282 men, 167 women), namely: inpatients seeking treatment for alcohol addiction (69 non-Hispanic blacks, 70 non-Hispanic whites); not seeking AUD treatment and actively drinking without clinically significant symptoms of alcohol withdrawal (65 non-Hispanic blacks, 13 non-Hispanic whites); and controls without a current AUD (127 non-Hispanic blacks, 105 non-Hispanic whites). ACTH and cortisol were collected: (a) in abstainers with AUD as a morning value on day 2 of admission to the NIAAA Hospital Ward; (b) in active AUD drinkers and control AUD non-drinkers during the screening visit at the NIAAA Clinic from 11:00 to 12:00 ACTH and cortisol were measured using FDA-approved chemiluminescent assays performed on an IMMULITE® 2000 XPi (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). ANOWA,TTests and/or chi-square tests were performed to compare the variables between our three subsamples and between blacks and whites respectively in each subsample. Multiple linear regression was used to examine the principal effects and interactions on the HPA axis of race reactivity, gender, age, body mass index (BMI), SES (assessed using a combination of social health determinants, e.g., income, relationship status, and current urban/rural address ), childhood and adult stress (as measured by the Childhood Trauma Questionnaire [CTQ], Early Life Stress Questionnaire [ELSQ], and Perceived Stress Scale [PSS], respectively), depression (as measured by the Montgomery-Lsberg Depression Rating Scale [MADRS] ) and anxiety (as measured by the Situational Anxiety Inventory [STAI-T]).

Results:Post-withdrawal AUD subjects showed HPA axis overactivity with significantly higher ACTH and cortisol levels than active AUD drinkers and non-AUD control subjects (PI numbers = 0.001). ACTH levels were significantly higher in black people compared to those with AUD. white people (PI number= 0.02) and black without AUD vs. White (PI number= 0.004), while black and white individuals with AUD who were active drinkers showed comparable levels of cortisol and ACTH. Cortisol levels were significantly lower in black people compared to those with AUD. white people (PI number= 0.009), while in the other two groups they showed no significant racial differences. Regression analyzes showed that black race, AUD abstinence status, male gender, younger age, and higher BMI predicted higher ACTH levels (R square 0.285). Caucasian race, AUD abstinence status, higher BMI, and higher MADRS scores predicted higher cortisol levels (R-square     0.175). There were no significant intervariate effects except for a weak interaction between black scores and the STAI-T score, which predicted higher cortisol levels. Collinearity was low because all variable factors of inflation were within the acceptable range (i.e. <4).

Conclusions:Black people with AUD show a significantly lower adrenal response to withdrawal, possibly due to the blunted HPA axis stress response seen in previous studies in black people compared to controls. discharge. However, they also show a much greater pituitary response to withdrawal. One hypothesis is that the latter observation may be related to CRH positive feedback in the ultrashort loop to enhance ACTH release under stress. This HPA axis dysregulation occurs independently of SES, childhood adversity, and adult stress, which did not contribute to differential allostatic load in the black and white individuals in our sample.

Keywords:Alcohol use disorder, racial differences, hypothalamic-pituitary-adrenal axis

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:Cannabidiol (CBD) has gained much interest in recent years as a potential treatment for a range of disorders. However, while the number of CBD-based products and publications is growing exponentially, little is still known about its pharmacokinetic (PK) properties, especially in studies that have important implications for the clinical use of this cannabinoid. Therefore, we intend to combine the available evidence from many studies to begin evaluating the factors affecting CBD pharmacokinetics.

Methods:A systematic review and meta-analysis was performed (PROSPERO ID: 269857). In this first phase, we focused on serum/plasma pharmacokinetic parameters of single-dose pure CBD products in healthy adults, excluding studies of CBD-THC combinations or any concomitant medications, patient populations, multiple doses of CBD, or urine/sputum testing. Following PRISMA guidelines, we systematically interviewed references from Medline, Embase, Web of Science Core Collection and gray literature from inception to June 2021. Data extracted on demographics and sample size, study design, CBD composition and supplier, session duration PK, measured metabolites, any reported pharmacokinetic parameters, especially Cmax, AUC0-t and Tmax. Data from various studies were transformed and log-transformed as required for each PK parameter. Post-regression random effects models were used for the analysis, considering each PK parameter as a dependent effect and CBD dose, CBD supplier, route of administration, cannabis withdrawal status, fasted or fed state, and PK session duration (especially for AUC0 - t ), as predictive. We include all predictors in each model. Quality assessment of included studies is ongoing using the NHLBI's before-after (before-after) uncontrolled quality assessment tool.

Results:Fourteen studies were included, with 39 different treatment arms as a unit of analysis. Five treatment groups were excluded from the final analysis: two did not report fasting/sedation, three did not report an average CBD dose. The analytical sample size included 480 participants. In individual treatment arms, the dose of CBD ranged from 10 to 6000 mg, Cmax [geometric mean (CV%), ng/ml] from 0.43 (112.77) to 1628 (51.4), AUC0-t [geometric mean (CV% ), h * ng/ml] from 1.469 (120.49) to 8347 (34.1) and Tmax [number mean (SD), h] from 0.05 (0.034) to 10.45 (7.057) . In post-regression, GW Pharmaceuticals (Epidiolex), as supplier, was associated with a higher Cmax (PI number< 0,001), AUC0-t (PI number< 0,001) e Tmax (PI number= 0.002) compared to non-GW suppliers. A higher dose of CBD was not significantly associated with Cmax in the entire trial (PI number= 0.054), but was significantly associated with higher Cmax in the subgroup analysis of the GW Pharmaceuticals evaluation (PI number= 0,021), a nie GW Pharmaceuticals (PI number= 0.035) treatment arms separately. CBD dose was not significantly related to AUC0-t or Tmax. Nutritional status was significantly associated with a higher Cmax (PI number= 0.020) compared to the fast state, but not significant for AUC0-t or Tmax. PK session duration was not a significant predictor of AUC0-t. The percentage of inter-study variability that could be explained by models (R2) was 86% for Cmax, 77% for AUC0-t and 89% for Tmax.

Conclusions:Factors such as CBD supplier/chemistry and fasted/fed state are important determinants of CBD pharmacokinetic parameters to the point that significantly different doses from different CBD suppliers/chemicals are required to achieve similar pharmacokinetic parameters and efficacy. Given the significant development of research on CBD, future pharmacokinetic studies must include some consistent and standardized formats that will allow for comparable and synthetic information.

Keywords:Cannabidiol, pharmacokinetics, meta-analysis, new drug development

Revelation:Nothing to disclose.

Vanderbilt University, Nashville, Tennessee, Stany Zjednoczone

Bottom:Substance use disorder (SUD) is a chronic neuropsychiatric disorder characterized by a drive to use and pursue drugs such as cocaine. Defining the neural mechanisms underlying cocaine-related reward and motivation, and how they can be modulated to control cocaine-related behavior, will be critical to the development of evidence-based therapeutic pathways for the treatment of SUD. While previous work has focused on the role of entire brain regions or genetically defined populations of cells, recent data show that stimuli activate only a small percentage of neurons in the nucleus accumbens (NAc), a key brain region in valence-based decision making. . Here, we combine behavioral tasks, optogenetic manipulations, and genetic mouse strains that allow us to label transcriptionally active neurons in a time-specific manner to identify, manipulate, and characterize a functional pool of cocaine-activated neurons and determine their role in drug intake.

Methods:Using transgenic mice (Arc-CreERT2) that allow time-specific labeling of transcriptionally active neurons, we expressed Cre-dependent viruses such as canaldopsin and diphtheria toxin in cocaine-activated neurons in the NAc. We combine this approach with functional tasks to determine the necessity and sufficiency of these cocaine-activated neurons to control reinforcement learning for drug and non-drug stimuli. We also used electrophysiology techniques to study the physiological properties of these neurons. Through these experiments, we defined the role of cocaine-activated neurons in the NAc in encoding cocaine-related information and in guiding learning and motivated behavior to determine how cocaine works in the NAc and leads to the development of substance use disorder.

Results:We found that the neuronal populations activated by cocaine in the NAc core are physiologically different from those activated during saline injection, demonstrating that cocaine selects a group of neurons with unique physiological properties. Furthermore, we have shown that neurons activated in the NAc are sufficient to drive reinforcement on their own, but are unable to modulate learning or performance on reinforcement tasks for physical rewards or negative reinforcements. Damage to these cocaine-activated neurons does not affect cocaine self-administration or modulate behavioral responses to other types of operant learning, suggesting that this set of neurons initially activated by cocaine may not play a critical role in the development of substance use disorder. Finally, we show that a subset of neurons recruited to chronic cocaine are also able to direct reinforcement, but do not modulate other reinforcement behaviors, and are also not required for self-administration of cocaine.

Conclusions:Together, we discovered that upon cocaine exposure, transcriptionally active neurons in the NAc core are physiologically unique and sufficient to drive reinforcement. However, despite these unique physiological properties, these neurons recruited by cocaine do not alter the behavior associated with other major reinforcers. This suggests that the neurons in the NAc may be intrinsically reinforcing, but there is another set of neurons that drives the development of drug addiction, or that there is plasticity in this initial set of neurons that cannot be fully reproduced under these experimental conditions. drug-using and drug-seeking behaviors. Furthermore, damage to the set of neurons recruited by chronic cocaine exposure is insufficient to modulate the acquisition or performance of cocaine self-administration. These data are extremely important as they shed new light on the underlying mechanisms underlying cocaine use disorder and raise the question of the role played by bands in the NAc and how these groups of neurons may be involved in the dysregulated behaviors seen in SUD.

Keywords:Cocaine use disorder, nucleus accumbens, cocaine self-administration, operant behavior, optogenetics

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:Protein translation is a critical component of brain function, and abnormal translational processes are associated with mental illness. Transfer (t)RNAs, cloverleaf-shaped non-coding RNAs that play a key role in ribosomal protein synthesis, have been poorly studied in the brain. Nsun2, a mammalian cytosine tRNA methyltransferase, is highly expressed in the brain and is associated with neurodevelopmental defects in humans and mice. Previous work has shown that loss of cytosine tRNA methylation causes deficits in protein synthesis in non-neuronal cells, but the role of tRNA methylation in adult neuronal function has not been explored.

Methods:We used conditional knockdown and transgenic Nsun2 overexpression in the prefrontal cortex (PFC) of male and female mice to manipulate postnatal tRNA methylation levels. We use targeted RNA bisulfite sequencing and tRNA sequencing to assess tRNA methylation/expression, LS-MS/MS to measure protein expression, electrophysiological recordings to assess synaptic transmission, and behavioral testing for complex behaviors including learning behaviors, desperate behaviors, and behavioral related to addictions.

Results:The results showed that complex behaviors related to emotions, cognition and addictions are particularly sensitive to bidirectional changes in Nsun2 in prefrontal cortex (PFC) neurons. In addition, the Nsun2-deficient cortex was selectively deficient in multiple glycine tRNAs, leading to codon-specific changes in the proteomic landscape, with deficits in glycine-rich neuronal proteins associated with impaired neurotransmission and altered behavioral phenotypes.

Conclusions:tRNA methylation is a key process regulating plasticity and synaptic behavior through proteomic changes in the mature cerebral cortex. These data suggest that, in addition to glycinergic receptors, there is another mechanism by which glycine may critically regulate brain function and complex behavior, suggesting the potential for new therapeutic avenues in psychiatry.

Keywords:Epigenetics, non-coding RNA, opioid addiction

Revelation:Nothing to disclose.

University of Texas Health Science Center at Houston, Houston, Texas, United States

Bottom:Drug overdoses have steadily increased in recent years, with the majority of cases attributable to opioid abuse. Opioid dependence is associated with impaired risk decision making. However, it is still unclear how the brain processes risk in an attempt to use opioids to change motivated behavior in conflict situations. The prefrontal area of ​​the medial prefrontal cortex (PL) has been linked to executive functions and behavioral responses to rewarding and aversive stimuli. Therefore, we hypothesized that repeated exposure to opioids suppresses PL activity and increases risk behavior.

Methods:To test our hypothesis, we developed an approach-avoidance conflict model involving the presentation of a fear-inducing predator's innate scent. Using an opioid-based place preference (CPP) model, we injected saline into adult male Long-Evans rats (N= 36) or morphine (N= 24) and were exposed to the less preferred side of the chamber every other day for a period of 10 days. After 72 hours of forced abstinence, the rats were given a 10-minute preference test to assess the time the rats spent on each side of the chamber. Immediately after the preference test, we added a predator scent (cat saliva) to the previously paired drug site to create a motivational conflict, and re-exposed the animals to the chamber for a 10-minute conflict test.

Results:During the preference test, morphine-treated rats showed a greater preference for the drug pair compared to the saline group (repeated measures ANOVA followed by Tukey's post-hoc test,PI number<0.001). During the conflict test, the saline group showed greater cat smell avoidance (PI number<0.001). In contrast, the morphine group continued to move toward the paired drugs despite the presence of cat odor (PI number= 0.45), showing that morphine conditioning increases risky behavior. Single-unit recordings from PL neurons revealed a significant number of cells with suppressed firing rates after acute administration of morphine (22%, 19/86) but not saline (6%, 5/64; Fisher's exact test,PI number= 0.011). On the last day of conditioning, administration of morphine did not cause changes in the frequency of LP firing compared to saline (PI number= 0.397), suggesting that PL neurons adapt to the effects of repeated morphine over time. Different populations of PL neurons were excited during the crash test (N= 28 neurons,PI number= 0.011) or inhibited (N= 14 neurons,PI number= 0.0061) when salt rats entered the cat side with a scent pair compared to entering the same side during the preference test. Interestingly, this change in neural activity was not observed in the morphine group during the conflict test.

Conclusions:Taken together, our results show that opioid-induced CPP is associated with reduced inhibition of PL activity after repeated administration of morphine. In addition, after repeated exposure to opioids, PL neurons did not respond to threatening stimuli, and this pattern of neuronal activity emerged with increased risk behavior during approach-avoidance conflict.

All experimental protocols in animal studies have been approved by the Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals

Keywords:Prelimbic cortex, recordings, morphine, approach avoidance, reward retention

Revelation:Nothing to disclose.

University of California San Diego, San Diego, California, United States

Bottom:The rates of opioid use disorder (OUD) represent an urgent health crisis. Much evidence suggests that the risk of OUD is hereditary. As a surrogate (or replacement) for OUD, we investigate the genetic basis of over-the-counter opioid use. We hypothesize that opioid abuse may be an inherited risk factor for OUD.

Methods:To test this hypothesis, we conducted a genome-wide association study (GWAS) of problematic opioid use (POU; "always take prescription opioids off-prescription") in 132,113 participants in the 23andMe study of European descent (Ncases =27,805).

Results:Our GWAS identified two significant genome-wide loci (rs3791033, an intronic variant of KDM4A; rs640561, a transgenic variant near LRRIQ3). POU showed a positive genetic association with opioid dependence and OUD as measured by the highest available GWAS (rg=0.58–0.80). We also identified numerous additional genetic associations with POU, including alcohol dependence (rg=0.74), initiation of smoking (rg=0.63), use of pain medication (rg=0.49), major depressive disorder (rg=0.49) .44), chronic pain (rg = 0.44). = 0.44) = 0.42), insomnia (rg = 0.39) and loneliness (rg = 0.28). Although POU was genetically positively associated with risk-taking (rg   =   0.38), adjusting for POU for risk-taking did not fundamentally change the magnitude or direction of these genetic associations, suggesting that POU simply does not reflect a general tendency toward dangerous behavior. Finally, we performed association effects and laboratory analyzes that revealed additional POU-associated phenotypes such as respiratory failure, insomnia, ischemic heart disease, and metabolic and blood-related biomarkers.

Conclusions:We concluded that opioid abuse can be measured across population cohorts and provides a cost-effective complementary strategy for understanding the genetic basis of OUD.

Keywords:Prescription opioids, opioid addiction, GWAS, human genetics

Revelation:Nothing to disclose.

Center for Alcohol Research in Epigenetics, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL, USA.

Bottom:Heavy drinking during adolescence alters normal neurodevelopmental pathways, increasing an individual's risk of developing alcohol use disorder (AUD) and comorbid anxiety in adulthood. Non-coding RNAs, including microRNAs (miRNAs) and enhancer RNAs (eRNAs), collectively regulate neuronal gene expression, and their expression may be altered by early exposure to alcohol. Here, we used a miRNA microarray to identify miR-222 as a candidate that, as a result of alcohol exposure during adolescence, could regulate gene expression in the amygdala to induce behavioral phenotypes of anxiety and increased alcohol consumption in adulthood.

Methods:Adolescent male Sprague-Dawley rats were exposed to 2 g/kg ethanol (EIA) or intermittent saline (AIS) on a 2-day on/off schedule by intraperitoneal (i.p.) injection on postnatal days (PND) 28-41. When the mice reached maturity, we collected brain tissue and performed an unbiased miRNA microarray. In other cohorts of adult EIA/AIS mice, we injected miR-222 mimic, Fos LNA, and miR-222 antagonist into the central nucleus of the amygdala (CeA) and assessed their subsequent behavioral phenotypes. Brain tissue was harvested to measure Fos mRNA and eRNA expression. cFOS protein levels in the tonsils were measured by gold immunostaining. 5 to 9 mice were assigned to each group. Statistical analyzes were performed using the Studenta programT-test, two-way ANOVA and repeated measures ANOVA with post hoc comparison using Tukey's test.

Results:miRNA microarray data showed that miR-222 expression was significantly higher in the amygdala of EIA mice compared to AIS controls, which was confirmed by qPCR. Target gene analysis of miR-222 revealed a highly reliable binding site in the Fos 3'-UTR, and downregulation of Fos mRNA and cFos protein was observed in the adult EIA amygdala. The expression of Fos eRNA-2, a highly conserved enhancer of regulated activity, was also downregulated in the amygdala of adult EIA mice compared to AIS mice. Injection of the mimetic miR-222 or nucleic acid inhibitor blocked by eRNA-2 Fos into the CeA of alcohol-naïve animals resulted in increased anxiety-like behavior and decreased Fos mRNA expression in the amygdala. Interestingly, injection of miR-222 antagomir directly into the CeA of adult EIA mice reversed EIA-induced increases in anxiety-like behaviors and voluntary alcohol consumption, and normalized Fos mRNA and eRNA expression in the amygdala.

Conclusions:Adolescent alcohol exposure disrupts the epigenetic circuitry in the amygdala, increasing miR-222 targeting of Fos mRNA and reducing transcription of the conserved Fos enhancer, which increases individual risk of developing psychopathology in adulthood (Supported by NIAAA UO171AA-049 (NADIA Project), grant SCP P50AA022538 and the Department of Veterans Affairs Senior Career Scientist award)

Keywords:MicroRNA, teen alcohol, amygdala, anxiety, c-Fos

Revelation:Nothing to disclose.

University of Michigan Health System, Ann Arbor, Michigan, United States

Bottom:Opioid use disorder (OUD) is serious, common, and costly. Reliable indicators of who will take opioids as prescribed and who will develop OUD remain unclear. Much evidence links childhood trauma to the development of OUD later in life, but the underlying mechanisms have not been identified. Hippocampal function and contextual learning and memory (CDLM) are affected by childhood traumas, and CDLM deficits have been observed in OUD. We propose that CDLM processes mediate the link between childhood trauma and OUD.

Methods:In this pilot study, we examined the associations between childhood trauma, CDLM scores, and opioid abuse behaviors in 23 adults who were receiving prescription opioids. Participants completed questionnaires and tasks in a virtual format. Childhood trauma was assessed retrospectively using the Childhood Trauma Questionnaire, and opioid abuse was assessed using the Current Opioid Abuse Measure. CDLM was assessed using a mnemonic similarity task and a contextual discrimination task.

Results:Childhood trauma was significantly associated with poorer CDLM scores,R(17) = -0,510,PI number= 0.036. Childhood trauma was also associated, at a trend level, with increased opioid abuse,R(17) = 0,458,PI number= 0.065. Increased opioid abuse was associated with poorer CDLM outcomes,R(21) = -0,447,PI number= 0,042.

Conclusions:These preliminary results in a small sample of adults taking prescription opioids support further research into the associations between childhood trauma, CDLM, and opioid abuse. Future work will explore these factors as predictors of OUD in a larger study of patients receiving prescription opioids. Our goal is to develop neurocognitive approaches to identify and protect at-risk youth, perhaps by enhancing hippocampus-dependent CDLM pathways prior to initiation of substance use. We hope to ultimately reduce the public health consequences of opioid and other substance abuse later in life.

Keywords:Childhood trauma, context, memory, hippocampus, opioid use disorder

Revelation:Nothing to disclose.

Yale School of Medicine, New Haven, Connecticut, Stany Zjednoczone

Bottom:Cannabis use is the most widely used illicit drug in the world,1 and rates of daily or near-daily use in the United States have nearly doubled in recent years. Although treatment outcomes for cannabis use (CU) vary widely between individuals, little is known about the brain predictors of CU treatment outcomes. Here, for the first time, we extend our previous work—demonstrating robust, substance-specific neuroindicators of opioid and cocaine treatment outcomes—to identify a UC treatment outcome neuroindicator using a fully data-driven approach.

Methods:Fifty-eight people with cannabis use disorders participated in neuroimaging at the start of the study. Reward neuroimaging data and cognitive tasks were used to compute functional connectivity matrices (two per participant) and fed into connectome-based predictive models (CPM) to identify predictive networks of baseline addiction severity and subsequent abstinence from cannabis biologically. confirmed within three months. CPM was performed using a 10-fold cross-validation with 100 replicates, and a permutation test was used to determine statistical significance. The generalizability of the model to other substances was tested on an external sample of 53 multi-substance users, and cannabis network strength was compared to a group of control participants (N= 46).

Results:CPM reward and cognitive task data successfully identified networks predicting cannabis withdrawal during treatment (reward:R= 0,39,PI number= 0.004; cognitive:R= 0,35,PI number= 0.005). Combining reward data and cognitive tasks also successfully predicted marijuana withdrawal, more accurately than either task alone (R= 0,44,PI number= 0.002). Out-of-sample analyzes showed that the identified cannabis withdrawal network did not generalize to predict withdrawal from other substances, consistent with previous work showing substance-specific neuromarkers of withdrawal. The CPMs from the combined task data also predicted baseline addiction severity as measured by the Addiction Severity Index (R= 0,43,PI number<0.001). Consistent with other CPM work, the networks identified for both analyzes were complex and included cortical and subcortical connections. Despite this complexity, the spatial extent of the abstinence-gravity network combined comprised only 758 edges (584 abstinences, 174 severity) or less than 2.2% of possible connections. The spatial overlap of the network was minimal and covered 9 edges. Finally, healthy controls showed an intermediate potency of the cannabis network over responders and non-responders.

Conclusions:These data indicate dissociated neural networks of cannabis severity versus post-treatment outcome. Contrary to classical views, these findings add to the growing body of evidence implicating unique, substance-specific predictors of addiction treatment outcomes.

Keywords:Cannabis use disorder, biomarkers, neuroindicators, treatment prediction

Revelation:Nothing to disclose.

Hunter College/CUNY, New York, New York, United States

Bottom:Alcohol use disorder (AUD) is a huge burden for people with the disorder, those helping them with treatment, and society. A complex disorder involving behavioral, genetic, and neurochemical mechanisms, AUD interacts with basic motivational and nutritional systems that are altered by the acute experience with ethanol (EtOH). Lack of food increases EtOH intake, and chronic EtOH consumption adversely affects nutrient absorption and metabolism. Endogenous opioid systems also play an important role in the processes of EtOH intake and reward, as well as in those processes related to food intake. We found that the underlying motivational system during early EtOH experiences can influence alcohol self-administration and progression to AUD. This study assesses the role of food deprivation and opioid mechanisms in the motivation to consume EtOH.

Methods:Rodents received their first exposure to EtOH or saline under two incentive conditions: A) Rats (female Sprague Dawley,N= 13) were deprived of food or given food and water ad libitum for 24 hours. before accessing 2 cc 3% EtOH three times. These sessions were separated by three days when the animals were allowed ad lib. food and water. All animals received two bottles with a choice between EtOH or water, during which EtOH concentrations were increased every three days (1, 3, 5, 7 and 10%). B) C57BL/6 mice (C57 males,N= 20) when naloxone HCl (5 mg/kg) or saline was administered three times immediately after exposure to EtOH (2 cc 3%) (no food was given prior to EtOH ingestion). Following this exposure to EtOH, the animals were given a choice of two bottles between 10% EtOH and water for 1 hour.

Results:A) Food deprivation combined with EtOH intake resulted in an increase in EtOH consumption over controls when acquiring a choice of 2 bottles by increasing the concentration (T= 3,45,PI number< 0.05) B) Pre-exposure of paired naloxone reduced EtOH consumption compared to unpaired consumption during a selection of 2 vials of EtOH (eat(3, 21) = 13,64,PI number< 0,05).

Conclusions:EtOH exposure under conditions of food scarcity increased voluntary self-administration of EtOH. The increased drive associated with food deprivation may have increased stimulus arousal and EtOH incentive value, leading to increased preference. On the other hand, combining naloxone with initial EtOH exposure reduced voluntary self-administration. Opioid receptor blockade may have reduced the EtOH incentive value, leading to decreased preference. These data suggest that the reinforcing effects of EtOH are mediated by motivational factors that engage drive and opioid mechanisms, which may lead to improved AUD handling.

Keywords:Alcohol preference, food deprivation, naloxone, stimulus enhancement, stimulus inhibition

Revelation:Nothing to disclose.

University of Kentucky, Lexington, Kentucky, United States

Bottom:The claustrum (CLA) has long been hypothesized to be involved in various brain functions, including attention and sensorimotor integration. CLA is also the most densely innervated structure in the brain and has the highest expression of the serotonin 2A receptor (5HT2AR). CLA, as well as 5HT and 5HT2AR, have been separately suggested to play a role in substance use and cognitive flexibility, the ability to adapt behavioral strategies to environmental changes. Here, we investigate the effect of serotonin 2A receptor signaling on neuronal excitability in the claustrum in the context of cocaine-induced deficits in cognitive flexibility, a set-shift task.

Methods:Electrophysiological cross-sectional records were obtained from back-labelled CLA neurons projecting into the anterior cingulate cortex (ACC). Spontaneous excitatory (sEPSC) and inhibitory postsynaptic currents (sIPSC) were analyzed along with measurements of cellular excitability including resting membrane potential, membrane capacity and action potential firing rate. Recordings were made after assessing behavior in a set-switching task with and without cocaine self-administration history (extended access, 6 h/day).

Results:Retrovirus injected into the ACC labeled a large population of CLA neurons with a label closely following the anatomical contour of the CLA. The use of 5HT resulted in a significant reduction in the frequency and amplitude of sEPSCs, accompanied by membrane hyperpolarization and decreased firing of CLA neurons projecting from the ACC. In contrast, the strong increase in sIPSC frequency and amplitude induced by 5HT is accompanied by sIPSC bursts. Blockade of 5HT2A receptors with ketanserin abrogated the synaptic effects of 5HT, indicating a regulatory role of 5HT2AR in signaling across the cerebral cortex. Our behavioral experiments showed that both cocaine and the serotonin-releasing agent, MMAI, had profound negative effects on cognitive flexibility.

Conclusions:These findings provide the first physiological evidence that a large population of CLA-ACC neurons is under the strong inhibitory control of 5HT and 5HT2AR. The overall inhibitory effect on neuronal production appears to be driven by a synergistic bi-directional regulation of inhibitory and excitatory synapses by 5HT2A receptors. Ongoing experiments are investigating whether these effects are responsible for cocaine-induced cognitive deficits.

Keywords:Cloister, electrophysiology, serotonin, cocaine, cognitive flexibility

Revelation:Nothing to disclose.

Icahn School of Medicine in Mount Sinai, New York, New York, United States

Bottom:A key feature of drug addiction is the maladaptation of the systems governing the evaluation and projection of reward. Emerging evidence from animal models suggests that the lateral habenula (Hb), the epithelial center of corticolimbic communication, is a key neural substrate of reward function affected by psychostimulant addiction. In particular, while the prefrontal cortex (PFC) provides top-down control of predictive drug responses, neural adaptations in the encoding of negative reinforcement from Hb lateralization are hypothesized to induce aversive states characteristic of withdrawal, possibly accelerating relapse and compulsion. However, direct translational evidence for a role for this circuit in human drug addiction remains substantiated. We wanted to fill this gap by using non-invasive pathway reconstruction to map the anatomical PFC-Hb junctions in a population of healthy individuals and cocaine addicts.

Methods:We performed diffusion MRI tractography on 31 subjects with cocaine use disorder [CUD, including 16 urine positive (CUD + ) and 15 urine negative (CUD−) for cocaine on the day of testing as a measure of recent use] and 28 healthy controls (CTL). Clinical interviews were conducted to assess measures of life expectancy and recent drug use. PFC-Hb projections were divided into fiber bundle components (subsegments): medulla striatum (SM) and anterior branch of the internal capsule (ALIC). Since ALIC contains multiple parallel fiber tracts, we constructed additional tracts between the PFC and the ventral cingulate region as well as the anterior thalamus as control regions to target the medial supralateral bundle of anterior and anterior thalamic radiation, respectively, and evaluate the specificity of the results for Hb. White matter microstructure was analyzed by diffusion tensor imaging to obtain mean fractional anisotropy (FA), an index representing the orientational coherence of fibers in the sections of interest, as well as mean total diffusivity (MD) and overall average diffusivity in axial (AD) and radial (RD) ). Differences in DTI measures were tested by ANOVA 3 (group: CTL, CUD + , CUD-) × 2 (subunit: SM, ALIC) × 2 (side: left, right), and the test statistics were found to be significant inPI number< 0.05 with Bonferroni correction for multiple comparisons.

Results:FA was generally reduced across the range of PFC-Hb in CUD compared to CTL (main group effectPI number<0.001). This was characterized by a significant Group × Subsection interaction effect (PI number< .001), where CUD + < CTL = CUD- in SM and CUD- < CTL = CUD + in ALIC. Importantly, right SM FA was negatively correlated with duration of cocaine abstinence (r2=0.21) and preference for viewing drug images over food (r2=0.21; moderated by the CUD group, r2=0.20). In addition, there was a significant main group effect for DM, where CUD->CTL=CUD++. No significant group effects or interactions were observed for AD or RD. In addition, there were no significant differences between the groups in DTI measurements in the ventral control region or anterior thalamic orbit, supporting the specificity of the results for Hb.

Conclusions:White matter abnormalities in PFC-Hb projections have been observed in cocaine addicts, and their severity has been associated with objective (urine toxicology) and self-reported (duration of abstinence) measures of current drug use. Distinct subregions of these regions may be associated with the severity of recent drug use (in MS) or predisposing or long-term factors (in ALIC). These results support further questions about the role of Hb in addiction and cognitive impairment related to drug seeking and relapse. Further analyzes will include functional magnetic resonance imaging during the drug selection task to assess the role of Hb in drug-related responses.

Keywords:Diffusion tensor imaging (DTI), side lobe, PFC, cocaine addiction

Revelation:Nothing to disclose.

Perelman School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania, United States

Bottom:Opioid use disorder (OUD) affects more than 2 million Americans. Clarifying the genetic risk of OUD could improve prevention, diagnosis and treatment. Despite an estimated twin heritability of ~50%, the largest genome-wide association study (GWAS) of OUD to date, with 10,544 cases of mostly MVP, revealed only one major genome-wide (GWS) locus, OPRM1, which was restricted to European Americans (EA).

Methods:We expanded our sample size using the latest version of the MVP data and a less stringent phenotypic definition to identify new loci. We performed a crossover meta-analysis of OUD in MVP with 31,480 cases (phenotype definition requiring code 1+ICD9/10 for OUD) and 394,484 controls exposed to opioids (any outpatient opioid prescription). For comparison, we performed GWAS using two other features: strict definition (codes 1+ for inpatients or 2+ for outpatients;Ncases = 23,552) and opioid dependence (addiction code 1 + ICD9/10).Ncases = 28190).

Results:Ten loci exceeded genome-wide significance in the (less rigorous) OUD cross-origin meta-analysis, 9 of which are novel. Three additional loci were identified in ancestry-specific analyzes (1 in African Americans (AA;N= 88502; 1 w EA (N= 302,596); 1 em Latin America (LA;N= 34,865). With the exception of a previously identified exon variant in OPRM1, which was still the highest genome-wide SNP (PI number= 9.72x10-11), intronic variants have been identified in RABEPK, FBXW4, NCAM1 and KCNN1.

Conclusions:Many of these genes have previous associations with other diseases, including mental disorders such as bipolar disorder and schizophrenia. Genetic association analyzes identified significant positive genetic associations with schizophrenia, ever vs. never, and cross disorder. Gene-based association analyzes identified 3 genes in AE: OPRM1, DRD2 and FTO. With a sample size three times that of the largest previous GWAS, we identified 12 new loci for OUD, including lineage-specific loci. These findings will increase our understanding of the biological pathways associated with OUD, which will underpin preventive, diagnostic and therapeutic efforts.

Keywords:Opioid use disorder, a genome-wide association study, New Therapeutics

Disclosures:Dicerna Pharmaceuticals: advisory board (own)

Sophrosyne Pharmaceuticals: Consultant (independent) of the Alcohol Clinical Trials Initiative of the American Society for Clinical Psychopharmacology, supported for the last three years by AbbVie, Alkermes, Dicerna, Ethypharm, Indivior, Lilly, Lundsukabor, Pfizer, Amygdala Neurosciences: , Other financial or in-kind support ( own)

PCT Patent Application No. 15/878640 entitled: Patent (own)

Mayo Clinic College of Medicine and Science, Rochester, Minnesota, USA

Bottom:The outer globus pallidus (GPe) is the center of integration and the gateway to behavioral flexibility in reward-related behaviors. However, it is not known whether GPe-enriched astrocytes drive behavioral flexibility.

Methods:We used two operant behavior tasks with effort or time-based reward performance (10 ul of a 20% sucrose solution) to generate habitual and goal-directed action choices. We then used in vivo calcium imaging to investigate the temporal dynamics of GPe astrocytes during target learning and habit learning. Using a machine learning approach, we test whether GPe astrocyte dynamics can predict task-like behavior in mice. Finally, we determined whether promoting calcium signaling in GPe astrocytes adapted from habitual learning attenuated habitual reward-seeking behaviors.

Results:GPe astrocytes were generally silenced during routine learning compared to goal-directed learning. On the time scale of action events, GPe astrocyte activity increased immediately after the end of the rewarding behavior before the next action. However, during normal learning, this increase was not noticeable during goal-directed learning. In addition, support vector machine (SVM) analysis showed that GPe astrocyte dynamics predicted whether the mice performed goal-directed or habitual behaviors. Interestingly, the chemogenetic activation of GPe astrocytes, which attenuates the firing of GPe neurons, exhibited habituation-directed behavior. In addition, brief and repeated attentional stimuli summarized the effects of GPe chemogenetic activation on intervening habitual reward-seeking behaviors with increased GPe astrocyte activity.

Conclusions:Our findings reveal new knowledge that activation of astrocyte GPe attenuates habitual behavior and improves behavioral flexibility, which may be a potential therapeutic target for decision-making disorders such as obsessive-compulsive disorder and addiction.

Keywords:Astrocytes, Globus Pallidus, Habits, Goal-directed behavior, Calcium image

Revelation:Peptron: Advisory Board (independent)

New York University School of Medicine and NKI, Hewlett, New York, USA

Bottom:There is evidence from animal and in vitro studies that THC can affect cannabinoid receptors (1 and 2) in the brain and periphery and influence immune responses, and limited evidence from animal studies that it can affect methylation processes related to epigenetics . THC consumption has also been reported to cause schizophrenia in respectable individuals. This is less direct work on these effects in people who smoke cannabis, the study of these types of chemical biomarkers in peripheral blood cells. This study also assessed whether smoking marijuana in humans induced changes in cannabinoid receptors, biomarkers of the DNA methylation cycle, and mRNA expression of immune-related genes.

Methods:23 people took part in an experiment in which they smoked cannabis cigarettes with one of two doses of marijuana (5.3% or 13.4% THC) or placebo (0.02%) and their driving skills were assessed. Blood samples were collected at the beginning of the study and several times after smoking. Plasma and WBC (PMC) were separated and stored at -80°C for further analysis. Samples were analyzed for mRNA content for cannabinoid receptors 1 (CBR1) and 2 (CBR2), methylating and demethylating enzymes (DNMT, TET), glucocorticoid receptor (NRC3) and immune markers (IL1B, TNFa) by qPCR using TaqMan probes. The results were correlated with total blood THC levels and baseline TCOOH levels. Statistical analysis used analysis of variance and covariance andT-tests or non-parametric equivalents for those values ​​that were not normally distributed.

Results:There were no differences in the baseline characteristics of the subjects, except that the higher THC dose group was older than the low dose and placebo groups, and the low THC group had higher baseline CBR2 mRNA levels. Both the 5.9 and 13.4 THC groups showed elevated THC levels over the next 2 hours and then declined toward baseline. the 13.4 THC dose still showed higher THC levels than placebo after four hours. However, there were no significant differences in THC levels between doses of 5.9 and 13.4 at any time point. At the 4-hour post-drug time point, the 13.4% THC group had a higher CBR2 (PI number= .021(eDNMT3A)PI number= 0.027), placebo mRNA levels and DNMT1 mRNA levels trended in the same direction (PI number= 0.056). The group with the highest 13.4 THC content had significantly higher CBR2 mRNA levels than the 5.9 group at various time points after drug administration, and tended to differ between the 5.9 and 13.4 THC groups for other mRNAs. TET3 mRNA levels were higher in the 13.4 THC group 55 minutes after taking the drug. When the high and low THC groups were combined, none of the differences in mRNA levels compared to placebo remained statistically significant. Changes in plasma THC levels were not associated with changes in mRNA levels.

Conclusions:During this study, CBR2 levels increased in human PMCs in the high-dose THC group, but were not accompanied by changes in immune markers. Changes in DNMT and TET mRNA suggest potential epigenetic effects of THC in human PMCs. An increase in DNMT methylation enzymes has been linked to some pathophysiological processes in schizophrenia and therefore should be further explored as one possible mechanism linking cannabis use as a schizophrenic trigger.

Keywords:THC, Dnmt, Cannabinoid Receptors, Schizophrenia (SCZ)

Revelation:Nothing to disclose.

University of Pennsylvania, Philadelphia, Pennsylvania, United States

Bottom:Craving and relapse are the main symptoms of addiction that occur during withdrawal. How do the pathological effects of drug use persist and increase when the drug signal is lost? We recently discovered that a transcription factor, nuclear receptor subfamily 41 (Nr4a1/Nurr77), regulates persistent gene expression in the nucleus accumbens (NAc) during withdrawal after self-administration of cocaine (SA) in mice. We found that CRISPR activation of Nr4a1 expression in the nucleus accumbens (NAc) during withdrawal reduces cocaine rewarding behavior, suggesting that Nr4a1 acts homeostatically to reverse the effects of cocaine. We have recently begun to investigate the cell-type-specific nature of this regulation, providing evidence for a cell-type-specific role for Nr4a1. For example, recent studies indicate a specific direct function of the (Drd1 + ) Nr4a1 pathway, given its activation of Drd1-specific pERK signaling. Our goal is to define a specific population of striatal neurons in which Nr4a1 regulates persistent transcription to moderate reward behavior.

Methods:All experiments are performed in C57B/L6 mice or transgenic mice aged 8 to 12 weeks. We used "isolation of specific neuronal cell types" (INTACT) in conjunction with "sub-target cleavage and nuclease release" (CUT and RUN) to determine Nr4a1 occupation, chromatin landscape and Nr4a1 target gene expression in specific cell types. To determine the type of NAc cells responsible for the regulation of cocaine-seeking behavior by Nr4a1, we activated or inhibited Nr4a1 (CRISPRa/i) in one of two subtypes of medium spiny neurons (MSNs) and measured their time-dependent place preference. and exit/re-entry.

Results:All experiments were performed one to three times, and duplicate data were observed in duplicate or otherwise reported experiments. Main effects and interaction effects were considered significant forPI number< 0.05. We recorded chromatin and gene expression profiles in MSNs of the direct and indirect pathways. A2a- or Drd1-Cre? LS1L-Sun1-GFP mouse striatum (N= 3) was INTACT followed by H3K27me3 or H3K4me3 CUT and RUN. Spearman correlations were calculated between the A2a+ repeats and the published H3K27me3 ChIP-Seq datasets of NAc and CamK2a+ whole excitatory forebrain neurons. The A2a+H3K27me3 peaks were more closely correlated with NAc MSN mass (rs=0.79) than with forebrain excitatory neurons (rs=0.38). To determine the accuracy of the CnRs in each cell type, K27me3+ from A2a+ MSNs was superimposed on the published lists of cell type-specific genes (PMID: 31171808). K27me3 in A2a+MSN was enriched in a greater proportion of Drd1-specific genes (2.05%) than A2a-specific genes (0.17%), consistent with the repressive role of H3K27me3 in gene expression. To regulate Nr4a1 expression in specific MSN subtypes, we designed Cre-dependent dCas9 expression plasmids for expression in A2a or Drd1-Cre mice; LSL-Sun1-GFP. NAc was transfected stereotactically and monomerically (JetPEI) with non-targeted control (NT) or Nr4a1-sgRNA and dCas9-VP64. Testes were isolated using INTACT and gene expression was quantified by qRT-PCR. Nr4a1 CRISPRa activated Nr4a1 and Cartpt expression specifically in Cre+ intermediate MSN (A2a + ) compared to NT (2-way ANOVA, influence of sgRNA: Nr4a1eat(1, 10) = 12,05PI number= 0.0060). To confirm the specificity of our approach, we measured the expression of A2a and Drd1. Cre+ nuclei isolated from A2a-Cre mice were enriched in A2a and depleted in Drd1 relative to Cre- nuclei (A2a 2-way ANOVA, Effect of Cre,eat(1, 8) = 14,23PI number= 0.0054; Drd1 2-way ANOVA, effect of Cre,eat(1, 8) = 107,2PI number< 0.0001). Activation of CRISPR Nr4a1 in A2a+MSN significantly reduced cocaine-dependent place preference (CPP) (N= 3–6 na grupę. Dwukierunkowa ANOVA Efekt leku:eat(1, 7) = 0,005593,PI number= 0.9425; Action of sgRNA:eat(1, 7) = 21,65,PI number= 0,0023).

Conclusions:Using SA cocaine epigenetic editing, INTACT/CUT and RUN+RNA-Seq and CRISPR, we found that activation of Nr4a1 in direct pathway MSNs is sufficient to moderate and even reverse cocaine rewarding behavior.

Keywords:Cocaine self-administration, epigenetics, CRISPR

Revelation:Nothing to disclose.

Rutgers University, Piscataway, New Jersey, United States

Bottom:There is ample evidence that social environments, including partner-mediated social interactions, influence ethanol consumption in humans and animals. Differences in gene expression are also thought to play a role in modulating individual levels of ethanol consumption, although much less is known about the involvement of specific genes in the context of ethanol consumption related to social interactions.

Methods:CD-1 (drinking) mice had continuous access to the two-bottle free-choice (ethanol vs. water) paradigm when housed in a home cage in the presence of a nearby same-sex or opposite-sex spouse. Since the amount of ethanol consumed varied significantly between different individuals, we compared mice with high ethanol consumption (heavy drinkers) with mice with low ethanol consumption (little drinkers) and analyzed the steady-state mRNA levels of the Drd4 dopamine receptor gene, with a sex-match between Bebedor and Cagemate as the independent variable.

Results:Drd4 dopamine receptor gene expression varied significantly depending on the effect of steaming on ethanol consumption. Differences in gene expression levels were more pronounced in pots mated with cagemates of the opposite sex.

Conclusions:These findings suggest that voluntary ethanol consumption may be regulated by social interaction with sexual association as an influencing factor and that the dopamine neurotransmitter system may play a role in underlying regulatory mechanisms.

Keywords:Social interaction, sexual mating, ethanol consumption, gene expression

Revelation:Nothing to disclose.

UCSD School of Medicine, La Jolla, California, United States

Bottom:Cocaine addiction is a serious public health problem. Despite decades of intense research, there is no effective treatment. Both preclinical and clinical studies of drug addiction strongly suggest that the nucleus accumbens (NAcc) is a viable target for deep brain stimulation (DBS). Although previous studies have shown that DBS from NAcc reduces cocaine seeking and recall, the effect of DBS on cocaine intake in cocaine-addicted animals has yet to be studied.

Methods:rats (N= 14) were conditioned to cocaine, allowing them to self-administer cocaine during a long access session (6 hours, 0.5 mg/kg/injection). The effect of high-frequency NAcc envelope DBS on cocaine consumption was then examined. In addition, cocaine-induced locomotor activity, excitability-like behavior during cocaine withdrawal, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit levels 1 and 2 (GluR1/GluA1 and GluR2/GluA2) after examining the DBS.

Results:Contrary to our expectations, DBS from the NAcc shell caused a slight increase in cocaine self-administration and cocaine-induced locomotor activity. In addition, after 18 hours of abstinence from cocaine, we found that DBS reduced irritability-like behavior. We also found that DBS caused a strong increase in GluR1 levels in cytosomes and synaptosomes, but not GluR2, especially in the central nucleus of the amygdala, but not in other areas of the brain.

Cocaine self-administration escalation (N= 14) was analyzed using one-way repeated measures analysis of variance (ANOVA) or paired Student analysisT-test (two-sided). The effects of DBS treatment and sham on the escalation of cocaine self-administration were analyzed using paired or two-way repeated measures Student's ANOVAT-test (two-tailed) using average consumption in two sessions OFF/ON. The effect of DBS on irritability-like behaviors was analyzed withT-test (two-sided). Cocaine-induced locomotor activity was analyzed by a two-way repeated measures ANOVA. Differences in GluR1 and GluR2 protein levels were analyzed using a two-way ANOVA followed by a Bonferroni post hoc multiple comparison test. Western blot data were analyzed using a two-way ANOVA followed by a Bonferroni post hoc multiple comparison test. His valuesPI number< 0.05 was considered statistically significant.

Conclusions:These preclinical results with cocaine addicted animals do not support the high frequency of NAcc shell DBS as a therapeutic approach to treat cocaine addiction in active cocaine users. However, the reduction in irritability-like behavior during cocaine withdrawal, coupled with previous findings showing that NAcc cortex DBS reduces cocaine-seeking reinstatement in abstinent animals, warrants further investigation of DBS as a treatment for emotional situations. Negatives and cravings during abstinence.

Keywords:Cocaine addiction, DBS, withdrawal, neuromodulation, amygdala

Revelation:Nothing to disclose.

McLean Hospital, Belmont, Massachusetts, United States

Bottom:Reactivity to smoking cues leads to motivation to smoke and is associated with poorer treatment outcomes (Ferguson 2009). Many studies have been conducted evaluating the neurobiological and physiological basis of stimulus reactivity and its relation to treatment outcome. For example, we have previously shown that increased insula reactivity to smoking cues predicts vulnerability to relapse (Janes et al., 2010; 2017), while others have reported a similar association between relapse and greater cortisol responses elicited by drug cues. (Fatsea 2011). The present work aims to fill the gap by assessing the relationship between stimulus-related increases in cortisol and brain reactivity to smoking-related stimuli in nicotine addicts.

Methods:Our sample was from a larger project investigating changes in the brain after trying to quit smoking. Participants were included if they had smoked daily in the past 6 months and were excluded if they were diagnosed with a psychiatric or neurological disorder. The final sample included 27 nicotine addicted participants (9 females and 18 males, mean age [SD] 27.0[5.9]) who had smoked for an average of 10.2[6.7] years, with a mean of 11.4[ 6.1] cigarettes per day. Participants underwent a 90-minute scanning session that included anatomical, resting, and functional neuroimaging of an event-related stimulus reactivity paradigm that included smoking cues, neutral cues, target images, and a fixation cross. Salivary cortisol levels were obtained immediately before and after the scanning session.

FSL was used to model BOLD responses to smoking and neutral cues. At group level, whole-brain BOLD activation for smoking with contrast > neutral cues was correlated with salivary cortisol change (post-sweep minus pre-sweep) using a mixed-effects model (FLAME 1). The cluster level cutoff was set to z=3.1,PI number= 0.01. Ad hoc analyzes used linear regression to determine whether time of day was related to baseline cortisol levels or change in cortisol.

Results:Increases in BOLD activation in the right anterior insula and right dorsolateral prefrontal cortex (DLPFC) when viewing smoking cues compared to neutral cues were positively correlated with increases in salivary cortisol before scanning. On average, we did not observe a statistically significant increase in salivary cortisol response when comparing post- and pre-scanning levels. The time of day of the scan had no effect on baseline cortisol levels (R= 0,23,PI number= 0.27) or change in cortisol level (R= -0,013,PI number= 0,95).

Conclusions:The present findings show that the increase in cortisol after smoke stimulus exposure is associated with smoke stimulus-induced brain activation in the right insula and DLPFC. This discovery advances the field in two ways. First, by combining two previous independent studies showing that both stimulus-induced increases in insula activity (Janes et al., 2010; 2017) and cortisol (Fatseas 2011) are associated with poorer outcomes. This paper goes a step further by proposing a link between insula-mediated interoceptive processing of smoking cues and a measurable physiological response. Taken together, these findings suggest that these normal neurobiological signals work together to facilitate the motivation to smoke.

Keywords:Substance abuse, cortisol, island, DLPFC, stimulus reactivity

Revelation:Nothing to disclose.

University of Texas Galveston Medical Branch, Galveston, Texas, United States

Bottom:Relapse is a dynamic and fundamental obstacle to recovery from substance use disorders. Relapse is often accelerated by exposure to drug-related cues and has been linked to impulsive behavior, particularly in cocaine use disorder. Motor impulsivity is characterized by impulsive action or an inability to stop a premature reaction. Here, we tested the hypothesis that phenotypic levels of motor impulsivity can predict drug-seeking behavior after long-term abstinence from cocaine.

Methods:Naive male Sprague-Dawley rats (N= 48) were trained for stability in a 1-choice reaction time task (1-CSRT) and phenotyped as high impulsive (HI) or low impulsive (LI). Rats were then trained in self-administration of cocaine (SA) (0.75 mg/kg/inf) until stabilized in a fixed-ratio 5-reinforcement regimen, followed by repeated fixed task performance of 1-CSRT. On a day corresponding to 30 days of SA cocaine abstinence, the rats were subjected to a drug-seeking test session in which lever presses were reinforced with a distinct signaling complex previously associated with drug injection.

Results:SA cocaine acquisition and observed cumulative levels of cocaine consumption did not differ for HI vs. LI. Rats identified as HI or LI maintained their baseline locomotor impulsivity phenotype during 30 days of abstinence from SA cocaine, with HI rats showing increased barometric pressure for cocaine-related cues compared to LI rats.

Conclusions:These data suggest that prior levels of motor impulsivity are not major factors influencing cocaine use in current settings, but motor impulsivity is a predictor of cocaine seeking during long-term abstinence. Importantly, these results demonstrate the efficacy of motor impulsivity endophenotype in predicting relapse-like behavior. Identifying motor impulsivity can provide more accurate and individualized diagnoses and/or treatment for patients, which can improve treatment outcomes, especially in relapse prevention.

Keywords:Cocaine, impulsivity, LDS-like phenotypes

Revelation:VidaLibreBio, Inc: Contributed research (own)

Mayo Clinic, Rochester, Minnesota, United States

Bottom:Sex and gender differences are related to differences in alcohol consumption and risk of developing alcohol dependence (AD). Depressive and anxiety disorders are also common among alcohol users and contribute to AD risk, progression, and outcome in a gender-specific manner. However, the biological mechanisms underlying these complex relationships remain poorly understood, mainly due to the limited number of samples to study such relationships. Here, we present analyzes of associations between sex hormone levels (testosterone and estrogen) and phenotypes associated with AD and depression in a large community sample taken from the UK Biobank. We also consider the role of sex hormone binding globulin (SHBG) and albumin levels in these phenotypes.

Methods:AD cases (N= 2,900) was defined as having any ICD-9 or ICD-10 AD code, alcohol withdrawal syndrome, or alcohol-related mental health problems. controls (N= 448,918) had none of these ICD9 or ICD-10 codes or codes related to alcohol-induced organ damage. We also excluded people with an AUDIT score of 8 or higher. A "narrowly defined" depressive phenotype has been identified based on an ICD-9 or ICD-10 diagnosis of major depressive disorder. In addition, the defined "broad depression" phenotype by Howard et al. (2018), i.e. self-description of seeking help from general practitioners or psychiatrists in the case of neuroses, anxiety and tension or depression was included in the analyses. Total testosterone (TT), total estradiol (TE2), SHBG and albumin levels were measured by immunochemical assays performed on a Beckman Coulter Uicel Dxl 800 instrument. Free and bioavailable testosterone was estimated based on putative binding to SHBG and/or albumin.

For each hormone/protein, we compared sex hormone levels between AD and non-AD patients using linear regression. We also assessed whether these associations were moderated by depression phenotypes, including interaction conditions. All analyzes were broken down by gender and adjusted for age and BMI.

Results:The incidence of major depression was higher in women than in men and almost twice as high in AD compared to the control group in men (67.5% vs. 35.0%) and women (82.8% vs. 46.2% ). The frequency of ICD-10 depression was even higher (8-fold and 6-fold) among AD cases compared to controls (67.1% vs. 8.2% in men and 89.7% vs. 14.4% in women). Interestingly, in the case of AD, the incidence of major depression and ICD depression was similar (men: 67.5% vs. 67.1%; women: 82.8% vs. 89.7%), while in the control group the incidence of CDI was significantly lower than for generalized depression (men: 8.2% vs. 35.0%, women: 14.4% vs. 46.2%). Due to the limited number of people with ICD depression, hormone correlation analyzes were performed only for the broad depression phenotype. Men with AD had significantly higher TT (PI number< 0,001) e TE2 (PI number< 0.001), but lower levels of bioavailable testosterone (BioT;PI number< 0,001) estradiol (BioE2;PI number< 0.001) levels than control males. Overall, in women, those with AD had significantly higher levels of TT, free testosterone (FT) and BioT (PI number< 0,001,PI number= 0,003, npPI number= 0.014, respectively), but lower TE2 (PI number= 0.010) levels than women in the control group. In both sexes, higher levels of SHBG (PI number< 0.05) and lower albumin (PI number< 0.001) has been found in people with AD. All these differences were statistically significant after adjusting for age and BMI.

FT, BioT and albumin levels were significantly affected by the interaction effect between AD and major depression in men. Men with comorbid AD and major depression had significantly higher FT and BioT levels but lower SHBG levels than men with only AD, only major depression, or none (AD*BDep: β=10.38,PI number= 0.002 e β = 328.23,PI number= 0.001, respectively). TT levels were negatively associated with major depression (β=-0.115,PI number< 0,001).

In contrast, no significant interaction effects were found on female hormone or protein levels, but AD and major depression had significant independent effects on testosterone and estradiol levels. Specifically, major depression was negatively associated with levels of TE2, free estradiol (FE2), and BioE2 (β=-17.59,PI number< 0.001; β = -0.274,PI number= 0.005; β=-8.89,PI number< 0.001 respectively). TT and FT levels were associated with major depression in the opposite direction (β = −0.008,PI number< 0.001 i β = 0.184PI number= 0.005, respectively).

Conclusions:We found significant sex-specific associations between circulating levels of testosterone and estradiol and DA in men and women. SHBG levels were much higher and albumin levels much lower in those with AD. Moreover, the associations between testosterone levels and AD were moderated by major depression in men, where those with AD and major depression had the highest levels of FT and BioT. We found that in women, major depression (ie, treatment-seeking depression/anxiety) was associated with lower levels of TE2, FE2, and BioE2, independent of AD. Taken together, these findings support the importance of estradiol in modulating depression/anxiety in women but not in men, and the importance of testosterone in modulating depression/anxiety in men with AD but not women with AD. Future studies are needed to replicate these findings and uncover the biological mechanisms underlying these associations.

Keywords:Sex hormones, alcohol addiction, depression

Revelation:Nothing to disclose.

University of California, San Diego, San Diego, California, United States

Bottom:Anxiety (e.g. generalized anxiety disorder, social phobia, phobias) is the most common mental disorder in adolescence, experienced by over 30% of young people and often coexists with alcohol use. Most of the research examining neural traits in adolescents with trait anxiety is small, and there is a lack of larger studies explaining the relationship between anxiety and alcohol consumption in humans. The National Consortium on Alcohol and Neurodevelopment in Adolescence (NCANDA) is an ideal resource for studying these neural features. It was designed as a comprehensive study (N= 831), which recruited adolescents aged 12 to 21 and followed them for 10 years, taking annual measurements of substance use, psychiatric function and brain structure using magnetic resonance imaging (MRI). Some young people in the trial progress to anxiety and/or heavy drinking after healthy baseline levels. Data from this study will help explain the longitudinal relationship between stress and binge drinking in adolescence.

Methods:We used implicit parallel process curve modeling to examine trends in the direction between adolescent anxiety and heavy drinking using annual measures of anxiety from the Achenbach Empirical Assessment System (ASEBA) and Routine Drinking and Drug Diaries (CDDR) of monthly binge drinking alcohol consumption (4+ drinks for women/5+ drinks for men on one occasion; also known as heavy episodic drinking or HED) from baseline to 5 years of follow-up (ages 12-21). Parallel Process Latent Growth Curve Modeling (PPGM) is a multidimensional extension of latent growth models that simultaneously examines levels and changes over time in multiple measures. This approach made it possible to estimate the average stress and trajectory of HED development. the trajectories of each person in the sample and assess the correlations between these change processes. We assessed model fit using the adjusted χ2 difference test, where a non-significant result indicated adequate model fit. We supplement this test with a number of alternative fit indices, including comparative fit index (CFI), Tucker-Lewis index (TLI), root mean square error of approximation (RMSEA), and root mean square residual. normalized (SMR). In our final integrated model, we included race as a covariate to control for the confounding effects of majority racial identity. All analyzes were performed inRusing lava.

Results:In the NCANDA study from baseline to 5 years, we found no significant linear and/or quadratic relationship between raw ASEBA anxiety scores and monthly CDDR binge drinking.

Conclusions:Our paper presents a novel study of the interaction between raw ASEBA anxiety scores and monthly CDDR binge drinking in the NCANDA study. This is in line with other studies that have found no clear link between stress and substance use. This may underline the importance of primary treatment of excessive alcohol consumption prior to treatment of comorbid anxiety disorders.

Keywords:Binge drinking in adolescence, anxiety in adolescence, NCANDA, a longitudinal study

Revelation:Nothing to disclose.

Rutgers University, Piscataway, New Jersey, United States

Bottom:The orexin (hypocretin) receptor system has emerged as a promising target for new therapies to treat cocaine use disorder. Suvorexant, a dual orexin receptor antagonist, has been approved by the FDA for the treatment of insomnia and has a strong safety/tolerance profile, making it easily reusable. While there is some evidence in rats to suggest that suvorexant may reduce cocaine-related behaviors, this has not been tested in a cocaine self-administration model that promotes a polyaddiction-like phenotype. Additionally, the dose-response profile of suvorexant when administered to rats by a clinically significant (oral) route has not been studied, nor is it known if there are sedative effects or other adverse effects at the doses required to reduce cocaine-seeking behavior. Thus, here we tested the efficacy of suvorexant (orally administered) in the expression of addiction phenotypes in rats with a history of intermittent cocaine self-administration. We also investigated any off-target effects of suvorexant using tests of general activity and psychomotor alertness.

Methods:macho (N= 16) and female free bike riders (N= 18) Long Evans rats were assessed for initial financial need for cocaine using an intra-session threshold procedure. The rats were then given intermittent daily access to cocaine (5 minutes of access every 30 minutes for 6 hours) for 2 weeks before reassessing economic demand and compulsive (punished) response to cocaine. The effect of suvorexant (0, 3, 10, 30 mg/kg, oral gavage) on seeking and penalized response was evaluated. The potential sedative effects of suvorexant were assessed on motor reactivity and psychomotor alertness during surgical tasks.

Results:In both male and female rats, intermittent access to cocaine was associated with a significant decrease in the elasticity of demand for cocaine (increased motivation). In male rats, this enhanced motivational profile was significantly reduced by suvorexant (10.30 mg/kg), which was enforced (punished) by responding to cocaine. At the minimum effective dose of suvorexant (10 mg/kg), no sedative effect was observed in the locomotor test in male rats. The effect of suvorexant on the psychomotor alertness task is under investigation. Data are also available on efficacy and sedative potential in female rats.

Conclusions:In addicted male rats, suvorexant was effective in reducing key addictive endophenotypes at non-sedating doses, supporting a potential repurpose of this compound for the treatment of cocaine use disorder. The study is investigating the effect of a lower effective dose on cognitive/psychomotor performance and the effectiveness of suvorexant in reducing addictive behavior in women. These studies may also be informative for the repurposing of other FDA-approved dual orexin receptor antagonists for the treatment of substance use disorders, including opioid use disorders.

Keywords:Dual orexin receptor antagonist, cocaine, addiction, insomnia, neuropeptides

Revelation:Nothing to disclose.

Boston University School of Medicine, Boston, Massachusetts, Stany Zjednoczone

Bottom:The prevalence rates of opioid use disorder (OUD) have increased dramatically, accompanied by an increase in overdose deaths. In addition, most patients treated for OUD recurrence within the first year of treatment. Although OUD has been extensively studied in preclinical models, understanding of the molecular changes that occur in the brains of chronic opioid users diagnosed with OUD remains limited. Corticostriate circuits in the brain are heavily involved in opioid reward, craving, and relapse, including the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc). To address this limitation, we used proteomics to investigate protein changes in DLPFC and NAc homogenates and synaptosomes from post-mortem brains in OUD subjects.

Methods:Both DLPFC and NAc were collected from the brains of postmortem healthy comparison subjects (N= 20) and osoby z OUD (N= 20). Between the unaffected cohorts and the OUD cohorts, subjects were matched for postmortem interval, RNA integrity, age, sex, and other criteria. In the OUD cohort, those diagnosed with OUD were active opioid users at the time of death. Directed mass spectrometry was used to measure protein levels in DLPFC and NAc homogenates and synaptosome preparations. Experimenters were blinded to diagnosis regarding sample preparation and proteomics experiments. Protein expression analyzes between healthy subjects and OUD were performed using regression modeling (Limma) with covariates (eg, age, sex, autopsy). Differential protein expression (DE) analyzes used fold shift (FC greater than plus/minus, 1.2) andPI number-courage (PI number< 0.05) thresholds. For DE proteins in OUD, we performed pathway enrichment analyses, gene cluster enrichment analyzes (GSEA), and protein-protein interaction (PPI) and transcription factor (TF) analyses. Integration analyzes between transcriptomic (RNA-seq) and proteomic data in brain regions from the same individuals were also used to determine isoform- and peptide-specific enrichment in DLPFC and NAc.

Results:Between healthy and OUD subjects, we identified 61 DE proteins (10 decreased and 51 increased in OUD) in DLPFC homogenates. Enrichment analyzes in the DLPFC of people with OUD included pathways related to metabolism, synaptic vesicle signaling, and glucocorticoids (PI number< 0.05). In NAc homogenates, we identified 29 DE proteins in OUD patients (22 decreased and 7 increased). NAc enrichment analyzes identified pathways related to the cytoskeleton, immunity, nuclear receptor signaling and dopamine signaling (PI number< 0.05). In synaptosomes, GSEA identified innate immunity, RNA-binding pathways and oxidative phosphorylation pathways enriched in the DLPFCs of OUD patients (143 synaptic DE proteins), while pathways related to glial cell activation, metabolism and lactate signaling were enriched in synapses. NAc (58 DE synaptic proteins). PPI and TF analyzes predicted the involvement of HIF, PPAR, and SP1 protein families, along with RNA-binding proteins, in protein interaction networks in the DLPFC and NAc of people with OUD (PI number< 0.01). Using integrated approaches to transcriptomic and proteomic datasets from the same individuals, we also found isoform- and peptide-specific changes in DLPFC and NAc associated with OUD.

Conclusions:Our findings are beginning to reveal molecular changes in cortical circuits in the brains of people with OUD. Taken together, we suggest further links between brain immune system, cellular metabolism and synaptic function in chronic opioid and OUD use in the human brain.

Keywords:Opiate use disorder, opiates, opiate addiction, human brain autopsy, proteomics

Revelation:Nothing to disclose.

Yale University School of Medicine, New Haven, Connecticut, Stany Zjednoczone

Bottom:Alcohol abuse is the leading cause of disability in the United States, and female drinkers are at greater risk than male drinkers for many of the consequences of alcohol consumption [NIAAA 2017 Women and Alcohol]. Alcohol has been shown to interact with several neurotransmitter systems to produce pharmacological effects. The endogenous kappa opioid receptor (KOR) system is strongly involved in the positive and negative reinforcing effects of alcohol, negative affect and stress [Bruchas et al. 2010 Brain Res]. Dysregulation of the KOR system by chronic alcohol consumption contributes to individual differences in alcohol-related behavior [Kosterlitz et al. 1989 NIDA Res Monogr] and KOR blockade reduces alcohol consumption in dependent rats but not in independent rats [Walker and Koob 2008 NPP]. Many studies show that women are more likely to drink to regulate negative emotions/stress, while men are more likely to drink for positive reinforcement [Logrip et al. Alcohol 2018]. These differences can be attributed to neuroadaptations in the KOR system, in particular the coupling of projections from the amygdala-frontal cortex, which are critical for the regulation of negative emotions/stress [Tejada et al. 2015 nuclear power plant]. Currently approved drugs for alcohol use disorder (AUD) have relatively low efficacy, were originally developed on male samples, and do not address gender targets. We previously observed that individuals with AUD had lower levels of available KOR in the corticolimbic system than healthy individuals (HC) (Vijay et al. 2018 NPP). We also observed that healthy men had higher levels of available KOR than healthy women [Vijay et al. 2016 AJNMMI], suggesting that AUD men and women should be compared to their HC counterparts. The aim of this study is to examine the KOR system in women and men with AUD compared to their HC counterparts. Based on preclinical and human behavioral studies and our previous work referenced above, we hypothesized that AUD males would have a lower KOR than HC males in the striatum, amygdala, and frontal cortex, and that AUD females vs. Women with HC will have a lower KOR in the amygdala but show no differences in the frontal and striatum.

Methods:Fifty-two AUD patients (19 females) and 25 CH patients (9 females) underwent positron emission tomography (PET) with [11C]LY2795050, a selective, high-affinity KOR antagonist radiotracer with kinetically favorable in vivo KOR imaging [Kim et al. others 2013 JNM]. Partial volume correction was applied to all PET data from AUD and HC patients to correct for possible atrophy. Marker distribution volume (VT) was calculated regionally as a measure of NRA availability. VT AUD vs. HCs were compared for 3 a priori areas of interest based on their behavioral involvement - striatum (reinforcement), amygdala (negative affect) and frontal cortex (negative affect/stress regulation). independent samplesT-tests were used to compare VT values ​​for men with AUD vs. HC men and women with AUD vs. Women HC.

Results:We found tentative evidence of AUD-related gender differences in NRA availability. KOR availability was significantly lower in AUD males compared to HC males in the striatum (PI number= 0.038, d CohenHello= 0.374) and amygdala (PI number= 0.013, CohenHello= 0.597) and tends in the same direction in the frontal cortex (PI number= 0.069, CohenHello= 0.420). AUD females had less KOR availability than HC females in the amygdala (PI number= 0.008, CohenHello= 0.319), but showed no differences in the availability of KORs in the striatum (PI number= 0.973, CohenHello= 0.302) and frontal cortex (PI number= 0.787, CohenHello= 0,370).

Conclusions:Consistent with our hypotheses, KOR availability was lower in AUD men than in CH men in the striatum and amygdala, and lower, but not statistically significant, in the frontal cortex. Women with AUD vs. HC showed lower KOR availability in the amygdala, but no differences in the frontal and striatal cortex. These data suggest that men and women with AUD have different patterns of KOR neuroadaptation compared to HC. In women, the pattern of amygdala-frontal KOR detachment may explain why women drink to regulate negative emotions. These findings point to possible neurobiological underpinnings of gender differences in alcohol use behavior and possible differences in the clinical efficacy of drugs targeting opioid receptors. We are now investigating image-based stress biomarkers in an expanded sample of more women.

Keywords:Alcohol Use Disorder Gender Differences Kappa Opioid Receptor Positron Emission Tomography (PET) Corticolimbic

Revelation:Nothing to disclose.

Addiction and Mental Health Center, University of Toronto, Toronto, Canada

Bottom:Relapse rates of alcohol use disorder (AUD) are high and current drug treatments are limited. In this sense, astrocytes have been identified as a new therapeutic target for AUD. Monoamine oxidase-B (MAO-B) is a putative marker of astroglial levels, however, in vivo studies evaluating MAO-B in AUD are lacking. We aimed to determine whether MAO-B levels, inferred from the binding of the positron emission tomography (PET) radiotracer [C-11]SL25.1188, would be elevated in the living brain in AUD during early withdrawal and subsequent abstinence from alcohol, as suggested by some preclinical and post-mortem data.

Methods:In this pilot study, treatment-seeking patients diagnosed with AUD DSM received a PET scan with the radiotracer MAO-B [C-11] SL25.1188 after 3 to 7 days of abstinence (N= 15; M/K 10/5; 49 ± 10 years) with a subgroup of four participants who completed a second study after 2–4 weeks of monitored abstinence from alcohol. Participants were not asked to refrain from smoking cigarettes, if any. healthy controls (N= 14; M/K 8/6; 42 ± 13 years) passed the exam. [C-11]SL25.1188 The total volume of distribution (TV) in the cortical and subcortical areas of the brain was compared between AUD and controls, and between early and late alcohol withdrawal.

Results:Repeated ANOVA (rm-) revealed that [C-11]SL25.1188 VT was not significantly different between controls and AUD in early abstinence (eat(1,27) = 0,22, -4%,PI number= 0.63). However, people with AUD who smoked cigarettes (N= 5) had, as expected from the literature, a significant reduction in [C-11]SL25.1188 VT compared to the control group (eat(1,17) = 19,5, -36%,PI number< 0.001) and non-smokers with AUD (eat(1,13) = 24,6, -43%,PI number<0.001). With regard to smoking, rm-ANCOVA tended towards a marginal effect of abstinence time (eat(1,2) = 10,4,PI number= 0.09, +3%) and marginal time x interaction with smoker (eat(1,2) = 11,4,PI number= 0.08, smoker (N= 1) +28%, non-smoker (N= 3) -6%). [C-11]SL25.1188 VT did not differ between non-smoker controls and non-smokers AUD (eat(1,20) = 0,95, +6%,PI number= 0,34).

Conclusions:Our exploratory study is limited by the small sample size. To the extent that astroglial status can be inferred from lead SL by PET, our data suggest that brain astroglial status may be normal in AUD with early and slightly longer abstinence, however smoking effects may occur. We also replicated the previous finding of low brain MAO-B binding in current cigarette smokers (Fowler et al., 1996). Strong inhibition of MAO-B in AUD smoking can alter the reinforcing properties of alcohol. Continued recruitment of AUD participants in our ongoing study may provide findings that will modify these initial conclusions. Conforms to US NIH NIAAA 026680 and CIHR GSD-159264 of Canada.

Keywords:Monoamine Oxidase B, Positron Emission Tomography (PET), Alcohol Use Disorder

Revelation:Nothing to disclose.

Washington State University, Pullman, Washington, USA

Bottom:During chronic cocaine use, associations are made between the motivational effects of cocaine and the environmental stimuli in which these effects occur. Re-exposure to environmental cues predicting cocaine leads to the recovery and destabilization of relevant cocaine-related memories. As a result, cocaine's volatile contextual memories must be re-stabilized in long-term memory stores through a protein synthesis-dependent process of memory reconsolidation to be maintained over time. Importantly, memory recall intervention attenuates cocaine-related memories and reduces cocaine-seeking behavior in preclinical models of drug relapse.

The dorsal hippocampus (DH) is a brain region that plays a key role in cocaine-induced memory consolidation. Inhibition of nerve conduction or Src family tyrosine kinase activity in HD immediately after retrieval-induced memory destabilization interferes with subsequent cocaine-seeking behavior. Inhibition of protein synthesis in DH does not alter memory reconsolidation, but nerve conduction in DH is required for reconsolidation of protein synthesis-dependent memory in the basolateral amygdala. We hypothesize that DH maintains or accesses unstable cocaine-related memories prior to their re-stabilization in the basolateral amygdala during recall. In this case, the functional contribution of DH is likely limited to the early stages of cocaine memory reconsolidation. We assessed the time-dependent involvement of DH in cocaine memory reconsolidation by time-precise optogenetic manipulations.

Methods:Rats received bilateral intra-DH injections of AAV5-hSyn-eNpHR3.0-eYFP-WPRE-PA (eNpHR3.0, 5.5 x 1012 vg/ml) or AAV5-hSyn-eYFP (eYFP, 3.5 x 1012 vg/ml) ) μl/ml. hemisphere), permanent fiber optic implants targeting HD, and cervical catheter implantation. Rats were trained to press a lever for intravenous cocaine injections on a schedule with a fixed ratio of 1. Training sessions were held in a different environmental context for 2 hours per day for 10 days. The rats then underwent extinction training sessions in a different context for 2 hours a day for 7 days. Twenty-four hours after the last extinction training, the rats were re-exposed to an environment previously paired with cocaine for 15 minutes to induce destabilization of cocaine-related memories or remain caged. Thereafter, rats in each group received either laser light stimulation (light on, 532 nM, 10 mW, 5 s off, duration 1 h) or no laser light stimulation (photo off) immediately after or 1 h after recovery from session memory or home cage (8 groups in total.N= 6-8/group). The following day, daily blanking training was continued until the bar pressing fell to the predetermined blanking criterion (~2 days). During this period, the potential impact of optogenetic manipulations on extinction memory strength was assessed on the basis of non-reinforced vertical presses in the context of extinction. Finally, the rats were re-exposed to combined cocaine to assess the effect of optogenetic manipulations on cocaine contextual memory strength. The effectiveness of the optogenetic constructs was verified in a separate cohort of mice. Brain tissue was harvested from these rats immediately after a 15-minute session of memory retrieval and manipulation of turning lights on and off (N= 6-7/group). C-Fos immunoreactivity was quantified in GAD67 and CaMKII immunoreactive cell populations in the dorsal region of maize ammonia (dCA3), just ventral to the optic fiber tip.

Results:There were no pre-existing differences between experimental groups in lever response during self-administration, blanking, memory recall, and post-treatment blanking sessions. Inhibition of DH neurons expressing eNpHR3.0 immediately (PI number= 0.02), but not one hour (PI number=   0.48), after memory retrieval, moderated cocaine-seeking behavior in the context of cocaine in the pair, but not extinction, in the test. In contrast, inhibition of DH neurons expressing eNpHR3.0 without memory retrieval (i.e., after cage conditioning) did not change cocaine-seeking behavior (PI number= 0.64). Similarly, light-ON manipulation of DH neurons expressing eYFP immediately after memory retrieval (PI number= 0.49) did not change cocaine-seeking behavior during the test. Light-ON manipulation in the group expressing eNpHR3.0 decreased c-Fos expression in dCA3 compared to light-OFF manipulation (PI number= 0.04), while the group expressing eYFP was unaffected. These results in the eNpHR3.0 expressing group were observed in GAD67-(PI number< 0.0001) e CaMKII-immunoreactive (PI number= 0.05) dCA3 cell populations.

Conclusions:Optogenetic inhibition of DH reduced memory strength in a cocaine context, as measured by the magnitude of cocaine-seeking behavior in a context previously paired with cocaine, in a time-dependent manner when applied during the first but not the second; cocaine memory consolidation time. DH involvement was required for cocaine-induced memory recovery, and behavioral changes did not reflect non-specific effects of viral expression or laser light exposure on cellular health. Collectively, these findings suggest that the critical contribution of DH is limited to early memory reconsolidation, consistent with the idea that DH may support the retention of unstable cocaine context memories before their re-stabilization in long-term memory stores.

Keywords:Cocaine self-administration, memory recovery, dorsal hippocampus, optogenetics

Revelation:Nothing to disclose.

Harvard Medical School McLean Hospital, Belmont, Massachusetts, Stany Zjednoczone

Bottom:Alcohol use disorder (AUD) is characterized by persistent drinking despite negative consequences. People with ASD have difficulty abstaining from alcohol in part because alcohol-related stimuli attract attention and lead to use (Cox et al., 2006). For example, individuals with AUD show greater cue-induced behavioral disruption when consuming Stroop alcohol (Lusher et al., 2004). However, it is unclear whether this alcohol-induced increase in disturbance is due to interference from the relevant drug-related stimuli or is due to a more general deficit in cognitive control. The current study addresses this gap by assessing brain reactivity using the classic and alcohol Stroop (i.e., color word) that assesses alcohol-related and non-alcohol-related disturbances. During both tasks, we measured brain reactivity in the dorsal and anterior cingulate gyrus (rACC and dACC) for their involvement in emotional and cognitive disruption, respectively (Mohanty et al., 2007). We also correlated brain reactivity in both ACC regions with AUD severity to determine whether brain reactivity during alcohol or classic Stroop is more strongly related to addiction severity.

Methods:46 participants (32 male, 13 female, 1 transgender person; mean [SD]     40.8 [11.0] age) were enrolled in an ongoing double-blind, randomized, controlled trial of an anticonvulsant drug for AUD. The current analysis used data from the baseline pre-treatment data collection period. Participants completed the Alcohol Dependence Scale (ADS) and functional MRI. During the fMRI scan, participants performed a modified Stroop task that included both Stroop alcohol and the classic Stroop color word. The fMRI data were analyzed in FSL, and the weights for the rACC and dACC regions of interest were extracted for the contrast of alcohol Stroop (alcohol > neutral) and classic Stroop (inconsistent > consistent) of interest. AUD severity was correlated with rACC and dACC activity for each contrast.

Results:Participants showed behavioral disruptions with both alcohol and classic Stroop, with significantly slower reaction times with alcohol trials compared to neutral trials (T= 7,85,PI number< 0.001) from inconsistent and congruent trials (T= 8,78,PI number<0.001). There was significantly greater rACC activity during an alcoholic Stroop (alcohol > neutral contrast) compared to a classic Stroop (i.e. inconsistent > congruent contrast) (T= 2,80,PI number= 0.01). dACC activity was not significantly different between alcohol and classic Stroop (T= -0,08,PI number= 0.94). The severity of AUD was associated with increased dACC activity during classic Stroop (R= 0,31,PI number= 0.03), but not Stroop alcohol (R= - 0,05,PI number= 0.75). Stroop performance (i.e., reaction time and accuracy) did not correlate with rACC or dACC activity or AUD severity (allPI numbers > 0,05).

Conclusions:In a sample of adults with AUD, both alcohol and classic Stroop caused behavioral disruption. We found that rACC exhibited enhanced Stroop alcohol activity over classic Stroop while dACC did not. According to current findings, increased rACC activation is associated with emotional disruption during Stroop emotional paradigms (Mohanty et al., 2007). More generally, the rACC is a key node of the default mode network that supports self-referential processing (Qin and Northoff, 2011), and activation of this network is inversely correlated with cognitive task performance (Frida et al., 2005). The current findings suggest that when individuals with AUD complete an alcoholic Stroop, regions involved in self-referential processing show increased activation, which may contribute to cue-related emotional disruption. In addition, we found that AUD severity was associated with dACC activity during classic Stroop. Together, these findings indicate that although alcohol-related words activated self-referential processing regions in adults with AUD, only participants with more severe AUD required greater recruitment of cognitive control regions to meet the cognitive demands of Stroop tasks. Our findings have an impact on the development of treatment. In particular, while treatments that reduce the impact of alcohol conditions may be beneficial for people with a wide range of AUD severity, treatments that also improve executive function may be especially beneficial for people with more severe AUD.

Keywords:Alcohol and substance use disorders, functional magnetic resonance imaging (fMRI), anterior cingulate cortex (ACC)

Revelation:Nothing to disclose.

School of Life Sciences, Arizona State University, Tempe, Arizona, United States

Bottom:The human immunodeficiency virus (HIV) and cocaine use disorder (CUD) remain common public health issues in the United States and around the world that disproportionately affect minority men. Exposure to HIV and its protein products (e.g. HIV envelope glycoprotein gp120) increases susceptibility to the reinforcing effects of psychostimulants, increases drug-induced dopamine transmission in the brain, induces chronic neuroinflammation in the brain, and facilitates the search for behavioral drugs. On the other hand, psychostimulants are known to increase viral replication and proliferation, highlighting the synergistic nature of this comorbidity. One obstacle to combating comorbid HIV and CUD is the increased motivation (or "craving") of cocaine, which is usually exacerbated during prolonged abstinence, an effect known as the "craving incubation effect". Importantly, the combined effect of HIV and chronic cocaine self-administration on relapse-like behavior and neuritis after long-term abstinence is unclear. We addressed these critical knowledge gaps by assessing cocaine-seeking behavior and neuritis after prolonged abstinence from cocaine self-administration and systemic gp120 exposure. Importantly, few studies have attempted to elucidate whether HIV affects the therapeutic efficacy of drugs that are otherwise effective in reducing cocaine-seeking behavior. Thus, another objective of this study was to investigate whether partial stimulation of the dopamine D3 receptor agonist (D3R), which we have shown to inhibit cocaine (but not sucrose) self-administration, reduces the signal-induced seeking of cocaine in gp120 exposed animals.

Methods:We investigated whether the D3R partial agonist MC-25-41 can suppress cue-induced cocaine seeking after a period of prolonged abstinence and whether systemic exposure to HIV gp120 modulates this effect. male rats (N= 6–7/group) received surgical implantation of intracranial guide catheters and jugular catheters for the first time (cocaine rats only). The rats were then trained to self-administer sucrose (45 mg/granule) or intravenous cocaine (0.75 mg/kg/injection) 2 hours a day, first on a fixed ratio (FR) -1 and then a variable -ratio reinforcement schedule. (VR)-2, VR-3 and VR-5 enhancement programs. After ≥12 self-administration sessions, rats entered a 5-day or 21-day abstinence period where they received intracerebroventricular (icv) microinjections of gp120 (50 ng/1 μl injection) or vehicle (1 μl PBS) once daily. day for days 1-5 of abstinence. Mice with a 5-day withdrawal were sacrificed to collect fresh brain tissue. 21-day-old abstinent rats received a 1-hour stimulus reactivity test in which they received an injection of MC-25-41 (10 mg/kg, i.p.) or vehicle (20% cyclodextrin + 3% 1M HCL in saline, 1 ml/kg , i.p.) 10 min before the test. Immediately after the test, the mice were sacrificed to collect fresh brain tissue for further protein analysis. All animal procedures were approved by the Institutional Animal Care and Use Committee at Arizona State University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Results:Treatment with MC-25-41, relative to vehicle treatment, significantly reduced signal-induced cocaine seeking in the icv. PBS (PI number< 0.05). However, MC-25-41 did not reduce signal-induced cocaine seeking in gp120-exposed rats (PI number> 0.05). Importantly, gp120 by itself did not suppress or enhance cue-induced cocaine seeking among vehicle-treated rats (PI number> 0.05). Studies are underway to analyze protein expression of a wide range of neuroimmune substrates (e.g. cytokines) in the nucleus accumbens (NAc) and prefrontal cortex (PFC). Interactions between type of reward (cocaine vs. sucrose), icv. type of treatment (gp120 vs. PBS), i.e.PI number. The type of treatment (MC-25-41 vs. vehicle) and duration of abstinence (5 days vs. 21 days) on expression levels of these neuroimmune substrates will be investigated.

Conclusions:The present findings support our previous research showing that a D3R partial agonist is effective in suppressing cocaine-seeking behavior. However, mice exposed to gp120 did not respond to D3R partial agonist treatment. Taken together, these findings suggest that targeting D3R may not be an effective therapeutic strategy for people with HIV/CUD comorbidities. This study highlights the need for future studies attempting to elucidate the therapeutic value of new drugs targeting CUD in animal models of HIV/CUD comorbidity.

Keywords:Cocaine seeking, HIV, dopamine D3 receptors, withdrawal, neuritis

Revelation:Nothing to disclose.

Linköping University, Clinical and Experimental Medicine, Linköping, Sweden

Bottom:It has been suggested that individual differences in subjective reward and the stimulating effects of alcohol are a risk factor for alcohol use disorder. Alcohol intoxication is also associated with risk-taking behavior, which may be due in part to reduced sensitivity to punishment. However, few studies have directly assessed the effects of alcohol consumption on human reward and punishment learning or have examined whether alcohol-induced behavioral changes in reinforcement learning correspond to the self-reported subjective effects of alcohol. In the current study, we examined the effects of acute alcohol consumption on reinforcement learning and sensitization using a probabilistic reverse learning task and assessed whether reward and punishment learning in this task is related to subjective feelings of alcohol intoxication.

Methods:Socially healthy drinkers (students.N= 32) performed a probabilistic reverse learning (PRLT) task under alcohol (dose 0.6 g/kg) and placebo in balanced order. Behavioral data was analyzed using a reinforcement learning model to provide individually tailored parameters for the rate and sensitivity of reinforcement learning (i.e. rewards and punishments). Self-report questionnaires were used to assess the subjective effects of mood and alcohol.

Results:Although overall behavioral performance on PRLT did not differ between alcohol and placebo (e.g., "correct" response rates), computer modeling analysis revealed that alcohol consumption was associated with greater sensitivity to reward and punishment compared to sham drugs. In addition, the punishment learning rate was higher with alcohol consumption compared to placebo, while the reward learning rate was lower with alcohol consumption compared with placebo. Neither the rate of reinforcement learning nor the sensitivity parameters were significantly correlated with self-reported (or other) alcohol rewards.

Conclusions:Alcohol consumption significantly alters reinforcement-based decision-making, but does so differently for reward or punishment. The implications and future directions for understanding alcohol-induced changes in reinforcement learning as a mechanism for alcohol abuse will be discussed.

Keywords:Alcohol, probabilistic learning reversal, reward sensitivity

Revelation:Nothing to disclose.

University of Wyoming, Laramie, Wyoming, United States

Bottom:Reward-based positive reinforcement is an evolutionary strategy common to all species. However, with drug addiction, reward seeking becomes maladaptive and threatens survival. Although drugs and natural rewards such as sucrose involve overlapping brain nuclei, we have recently shown that drug abuse and natural rewards are associated with distinct sets of neurons, defined as a distinct number of simultaneously activated neurons. Accumulating evidence shows that drug exposure strongly and unambiguously influences gene expression across the transcriptome. However, beyond the identification of a small number of plasticity-related genes in methamphetamine-seeking neuronal assemblies in rodents, little is known about how drug exposure alters mRNA expression profiles, particularly in drug-associated assemblies. To fill this knowledge gap, we plan to study gene expression in specific sets of rewards and differences in neurons activated during the search for different types of rewards. We hypothesize that a distinct number of key genes, possibly activity-dependent, are differentially expressed in each specific set of rewards and in cells responsive to all kinds of rewards.

Methods:Using inducible cFosiCreERxAi14 reporter mice, we demonstrated that we can fluorescently label different sets of reward-specific neurons in the nucleus accumbens (NAcore), the center of reward processing. To study gene expression in these different pools, we used the same approach to induce fluorescent labeling of 3 different pools: cocaine, sucrose, or overlapping pools. We then isolated NAcore neurons from mouse brains, sorted the labeled clusters using the FACSMelodyTM cell sorter, extracted the RNA and performed RNA sequencing to compare gene expression in the 3 clusters with unlabeled cells without clusters. Differences in gene expression profiles between 4 cell groups were compared and used to identify cell types and generate cell groups based on transcriptional profiles.

Results:The first step of the proposed experiments was to establish a tissue harvesting protocol that would allow the sorting of labeled cells into different sets, i.e. small percentages of 1% or less NAcore cells, using FACS technology without mRNA destruction and sequencing difficult. The presentation will include a detailed description of the protocol with key findings and practical tips on how to increase the reproducibility and yield of good quality mRNA. We then identified differentially regulated genes in each labeled set of neurons, sets of cocaine, sucrose and overlap, compared with unset cells.

Conclusions:Differential profiling of the expression of genes uniquely associated with drug seeking offers the possibility of therapeutically targeting maladaptive drug seeking without affecting the underlying, biologically adaptive seeking of physical rewards.

Keywords:Cocaine addiction, reward, RNAseq

Revelation:Nothing to disclose.

Howard University, White Plains, Maryland, United States

Bottom:The Minority AIDS Network Effort (HUMANE) program at Howard University addresses significant service gaps by providing peer support, case management, and HIV/hepatitis prevention services in three different locations—outpatient mental health, primary care, and infectious clinical disease. With the goal of improving the health and well-being of people with severe mental illness (SMI) or co-occurring disorders (COD), who are living with or at risk of HIV or hepatitis, one of the goals of the program is to provide Integrated Dual Disorder Management (IDDT). The IDDT model is an evidence-based practice that improves the quality of life for people with comorbid serious mental illness and substance use disorders by combining substance abuse and mental health services.

Methods:Center for Mental Health Services (CMHS) National Outcome Measures (NOM) Client-level measures for discretionary programs providing face-to-face services were used during program enrollment and six-month reassessment. A sample of 138 participants (ages 18–75) enrolled in the program and were recruited by a peer support specialist trained to perform the weekly IDDT. Satisfaction with the treatment of services was measured by consumer perceptions of the service (CPC).

Results:Of the 138 participants, 76 are women and 132 are African American. Among the registered, 37 (26.81%) have a diagnosis of recurrent MDD, 22 (15.94%) - bipolar disorder, 10 (7.25%) - schizophrenia, 12 (8.70%) - anxiety disorders, 21 (15 .21%) – opiate use disorders, 13 (9.42%) with alcohol use disorders. At study entry, 125 (90.58%) participants reported having been tested for HIV and 30 (21.74%) were HIV positive. 23 HIV+ participants said they had always used antiretroviral therapy (ART). 118 (85.51%) participants reported that they had been tested for HBV at registration and 2 of them had HBV+ and were covered by the treatment service. Similarly, 120 (86.96%) participants were tested for HCV at enrollment and 8 of them were HCV+ , all of whom were related to treatment services. At 6-month reassessment, 98.81% of participants reported having been tested for HIV. Similarly, 98.81% of participants reported being tested for HBV and HCV at the 6 month reassessment.

Of the 101 participants who had to be reassessed after 6 months, eighty-four completed the NOM questionnaire. All IDDT interventions were conducted by a trained peer support specialist. Participants were generally satisfied with the IDDT evidence-based intervention received. During the re-evaluation, 77 (91.67%) of participants agreed that they had received the necessary information and 73 (86.90%) agreed that they were encouraged to use consumer-led programs. All participants agreed that they enjoyed the service they received from the programme. Twenty-four (28.57%) strongly agreed that if they had other options, they would continue to use the service and recommend it to others.

Conclusions:Participants' perception of expert mutual support groups using the evidence-based IDDT model in this addiction/SMI service program was generally positive. When reassessed after 6 months, there was an overall improvement in the overall health of the participants. While this study provides support for the use of evidence-based intervention for dual diagnosis, more work is needed to implement the use of a peer support specialist in a comprehensive MCS/addiction services program.

Keywords:Substance use disorder, serious mental illness, evidence-based approach

Revelation:Nothing to disclose.

Indiana University School of Medicine, Indianapolis, Indiana, United States

Bottom:Adolescent overexposure to alcohol (ABAE) is a common problem in our society. A recent trend in teen/young adult binge drinking is that the baseline rate of binge drinking has decreased and there has been a sharper increase in the overall rate of binge drinking in the teen/young adult transition. Heavy drinking during adolescence induces numerous changes in adolescent/adult phenotypes that have multiple deleterious consequences. We have recently published several findings that ABAE induces persistent changes in the alpha-7 nicotinic acetylcholine (a7) receptor and that pretreatment with multiple negative allosteric modulators (NAMs) a7 prevents ABAE-induced increases in alcohol consumption in adults. The purpose of this experiment is exactly the opposite. The current study determined whether pre-treatment with an α7 positive allosteric modulator would enhance the effects of moderate adolescent alcohol exposure (AMAE) in promoting adult alcohol consumption.

Methods:Male and female Wistar rats were pre-treated (2 hours) with 7 PAM PNU (0, 3 or 5 mg/kg) prior to exposure to ABAE (2 g/kg - half the standard dose or water) in our standard ABAE example. Adult drinking used voluntary beer consumption to stimulate consumption of pharmacologically relevant levels of alcohol (>100 mg% BEC). In males, on postnatal day 75, rats were exposed to 24-hour drinking of beer of choice for 6 weeks, beer deprivation for 2 weeks, then reinstatement of beer for 2 weeks (ADE relapse). In females, on postnatal day 75, rats were given 1 hour of functional access to beer. Daily caregiver access to beer was maintained for 10 weeks, followed by 1 week of extinction training, 2 weeks of the home cage period, 4 days of the beer-seeking test, 2 weeks of the home cage period, and finally 2 weeks of beer re-exposure (relapse, ADE trial). , during).

Results:All male rats bingeed on beer with no effect on AMAE or PNU (PI numbervalues ​​> 0.54). In the 6th week of access to beer, male Wistar rats consumed 115 g of beer per day (1/4 of body weight), a visible ceiling effect (approximately 14 g/kg per day). In female rats (surgeon self-administration), pre-treatment with 5 mg/kg PNU combined with 2 g/kg ABAE treatment significantly improved adult beer self-administration acquisition, decreased extinction response rate, and improved beer-seeking behavior. Overall, there was no effect of low doses (2 g/kg) of AMAE on alcohol consumption in adults.

Conclusions:The data indicate that the α7 receptor is a critical target for the development of pharmacotherapeutics to treat/prevent the adverse effects of ABAE. The fact that the data indicate that it is a two-way system (NAM blocking, PAMS promotion) provides compelling evidence for the hypothesis that the α7 receptor is critical for the lasting changes produced by ABAE.

Keywords:Puberty, prevention, alcohol use by adolescents

Revelation:Nothing to disclose.

University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

Bottom:The use/use of multiple substances, in particular stimulants and opioids, is not a new phenomenon, and there is a growing awareness among healthcare providers, policy makers and fundamental scientists that polysubstance abuse is the norm rather than the exception. Indeed, while simultaneous injection of cocaine and heroin (i.e. "speedballs") has been common for decades, recent estimates show that stimulant/opioid mixes are increasing in popularity, with over 50% of opioid users seeking treatment. . Moreover, while concurrent use of stimulants and opioids has been largely one-sided (e.g., opioid users added cocaine to heroin), recent evidence suggests that this too may be changing, with increasing numbers and widespread reports of opioids, especially fentanyl, mixed with cocaine and other stimulants. While valuable in itself, the fact that the vast majority of preclinical research on substance use continues to focus on the effects of single drugs, studied separately, highlights the need for a better understanding of drug interactions in order to effectively treat individuals with substance use disorders.

Methods:Adult male Sprague-Dawley rats (N= 33) were surgically prepared with indwelling catheters in the left and right femoral veins. The catheters were inserted under the skin and connected to a two-channel vascular access knob that was located on the outside in the center of the scapula. A group of mice (N= 17) was used to assess the simultaneous access to two stimulant drugs (i.e. MDPV and cocaine), while the other group of rats (N= 16) was used to assess simultaneous access to a stimulant (methamphetamine) and opioid (fentanyl). Initially, rats had access to MDPV 0.032 mg/kg/inf and cocaine 0.71 mg/kg/inf or methamphetamine 0.1 mg/kg/inf or fentanyl 0.0032 mg/kg/inf in a 5-second parallel schedule for FR5 : After the response to these doses stabilized, two different sets of dose manipulations were performed: 1) the dose of the non-preferred drug was increased by ½ log unit and preference was redefined; and 2) the dose of the preferred drug was reduced by ½ log unit and the preference was redefined. Eventually, both drugs were replaced with saline, and infusion-paired stimuli were omitted in emergency situations to quench the response on both levers. When the response fell below 15% of the baseline on both levers, a series of trials of suggestion and suggestion + drug reinstatement, incl. In these tests, rats were pre-bolused with MDPV (0.32 mg/kg, IV), cocaine (3.2 mg/kg, IV), methamphetamine (1 mg/kg, IV), heroin (0.1 mg/kg, IV), , IV) or fentanyl (0.032 mg/kg, IV)? The trials were separated by at least 2 extinction sessions.

Results:When rats had access to both MDPV and cocaine, they showed an exclusive choice of either MDPV or cocaine. However, these preferences can be reversed by increasing the dose of the least preferred drugs or decreasing the dose of the most preferred drugs. When rats were given access to methamphetamine and fentanyl, some rats responded to both levers, while others showed an exclusive choice of one drug (usually methamphetamine). However, as observed when MDPV and cocaine were available, these preferences (or lack of them) were. When MDPV and cocaine test rats were tested under reinstatement conditions, stimulants (e.g. cocaine versus MDPV lever, while heroin did not restore responses to either lever Although stimulants also tended to restore more responses to methamphetamine than to fentanyl levels, just as fentanyl tended to restore more responses to fentanyl than to methamphetamine levels In rats with a history of response to both stimulants and opioids, stimulants also restored fentanyl responses, and opioids also restored methamphetamine responses, albeit at lower levels.

Conclusions:People with substance use disorders often use multiple drugs, often from different pharmacological classes. Current research indicates that when two drugs from the same class are available (e.g., MDPV and cocaine), mice treat them as substitutes, choosing a functionally higher dose. However, when drugs from different pharmacological classes are available (i.e., methamphetamine and fentanyl), some rats treat them as complementary when trying both drugs during a session, while others show a preference for one or the other sensitizing drug. doses as for MDPV and cocaine. Interestingly, recall tests suggest that response histories to drugs from different pharmacological classes reduce the pharmacological specificity of the original drugs, which may have implications for understanding drug-seeking behavior in individuals with multiple use disorder. Future research will explore how these relationships are affected by opioid addiction and withdrawal syndrome.

Keywords:Abuse of polysubstances, stimulants, opioids

Revelation:Nothing to disclose.

VA Puget Sound Health Care System, Seattle, Waszyngton, USA

Bottom:Alcohol use disorder (AUD) is a significant problem in the active-duty military. Excessive noradrenergic signaling from the brain contributes to the anxiety, irritability, and insomnia that characterize alcohol withdrawal. These symptoms of aversive hyperarousal are components of the "craving for relief" that often interferes with attempts to sober up. Prazosin is a brain penetrating alpha-1 adrenoceptor antagonist that has been shown to reduce alcohol consumption in preclinical studies1 and randomized controlled trials (RCTs) in citizens seeking treatment2. In several recent RCTs, the efficacy of prazosin was limited to participants with symptoms of alcohol withdrawal and/or cardiovascular evidence of increased noradrenergic signaling consistent with alcohol withdrawal3,4,5.

Methods:Participating AUD soldiers were recruited from a 12-week intensive military alcohol treatment program at Madigan Army Medical Center/Joint Base Lewis McChord where they agreed to abstinence. The 102 soldiers who were randomly assigned and received at least one dose of the study drug were entered into modified mixed intention-to-treat (ITT) models for repeated measures analysis. Participants were randomized to prazosin or placebo equivalent, titrated over 3 weeks to a maximum dose of 4 mg in the morning, 6 mg in the afternoon, and 10 mg at bedtime. After titration, participants stayed on the target dose for an additional 10 weeks. Randomization occurred approximately 2 weeks after entering the Army AUD treatment program. Primary outcome measures included drinking diary scores (standard serving units)/day, % drinking days, % heavy drinking days, and Pennsylvania Alcohol Craving Scale (PACS) scores. Pre-specified endpoints are 9 and 13 weeks post-randomization.

Results:Prazosin was significantly better than placebo in reducing daily drinking (PI number= 0.03). Prazosin vs. Placebo changes in % days drinking, % days heavy drinking and PACS scores were in the same direction but did not reach significance (allPI numbervalues ​​> than 0.105). A planned subgroup analysis with cardiovascular evidence of increased noradrenergic signaling indicative of alcohol withdrawal (baseline heart rate > 90 bpm) showed strong and significant reductions in prazosin compared to placebo per day (PI number= 0.010), % of days consumed (PI number= 0.036) and % days of heavy drinking (PI number= 0.001). Prazosin appeared to prevent relapses over the last four weeks of the protocol in this higher heart rate subgroup analysis.

Conclusions:These findings suggest that prazosin is effective for AUD in individuals showing increased noradrenergic signaling consistent with alcohol withdrawal, and support the recent demonstration by Sinha et al5 among citizens seeking treatment that prazosin is an effective drug for reducing alcohol consumption. with alcohol withdrawal symptoms. Our findings also support the evaluation of prazosin for relapse prevention in individuals with AUD who show signs and symptoms of alcohol withdrawal.

Keywords:Prazosin, Alcohol, Brain

Revelation:Nothing to disclose.

National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, Stany Zjednoczone

Bottom:Alcohol abuse is prevalent in many societies around the world and is associated with high morbidity and mortality, often leading to alcohol use disorder (AUD) and alcohol-related end-organ damage. The mechanisms underlying the development of AUD are largely unknown. However, a growing body of evidence suggests that alcohol consumption is strongly associated with changes in DNA methylation. Identification of alcohol-related methylomic variation may provide new insights into the pathophysiology and new targets for the treatment of AUD.

Methods:Here, we conducted the largest single-cohort epigenomic association study (EWAS) of alcohol consumption to date (N= 8161) and results confirmed in AUD populations with the appropriate endophenotypes, as well as alcohol-related animal models.

Results:The results showed that 2504 CpG was significantly associated with alcohol consumption (BonferroniPI number-value < 6.8 x 10-8) with five probes placed in SLC7A11 (PI number= 7,75 x 10-108), JDP2 (PI number= 1,44 x 10-56), GAZ5 (PI number= 2,71 x 10-47), TRA2B (PI number= 3,54 x 10-42) e SLC43A1 (PI number= 1.18 x 10-40). The genes annotated at the appropriate sites of the CpG are involved in liver and brain function, cellular response to alcohol, and alcohol-related diseases, including hypertension and Alzheimer's disease. Two-sample Mendelian randomization confirmed a causal relationship between drinking and AUD risk (IVWPI number= 5.37 x 10-09). The methylation-based predictor of alcohol consumption was able to distinguish AUD cases into two independent groups (PI number= 6.32 x 10-38 ePI number= 5.41 x 10-14). The upper probe EWAS cg06690548, located in the cystine/glutamate transporter SLC7A11, was replicated in an independent group of AUD and control participants (N= 615) and showed strong hypomethylation in AUD (PI number<10-17). Decreased CpG methylation in this probe was consistently associated with clinical measures, including increased days of heavy drinking (PI number<10-4), elevated liver enzymes (GGT (PI number= 1.03 x 10-21), ALT (PI number= 1,29 x 10-6) e AST (PI number= 1.97 x 10-8)) in patients with AUD. Post-mortem brain analyzes have documented increased expression of SLC7A11 in the frontal cortex of AUD individuals, and animal models have shown significantly increased expression in the liver, suggesting a mechanism by which alcohol leads to hypomethylation-induced overexpression of SLC7A11.

Conclusions:Taken together, the EWAS discovery sample and subsequent validation of the top probe in AUD suggest a strong role for aberrant glutamate signaling caused by methylome variation in SLC7A11. Our data are of interest given the significant role of glutamate signaling in the brain and liver and may represent an important target for therapeutic intervention.

Keywords:Alcohol, Alcohol consumption, Epigenetics, Addiction phenotypes, DNA methylation

Revelation:Nothing to disclose.

University of Washington, Seattle, Washington, United States

Bottom:Opioid overdose deaths continue to rise despite significant efforts to reduce opioid use disorders. Drug withdrawal and abstinence can contribute to aversive states that increase the likelihood of relapse to opioid use. Significant deficits in working memory and executive function occur within the first two weeks of opioid withdrawal and may persist for months in vulnerable individuals. Impaired cognition may also contribute to the changes in mood, stress reactivity, and reward processing seen in early opioid withdrawal. Aversive states, including drug withdrawal, may promote the release of dynorphins, endogenous ligands of kappa opioid receptors (KOR). Although the psychopathology of substance use disorders is complex, dynorphins and KORs are expressed in the mammalian cortex, and analysis of their role in mice is likely to provide useful information for the development of treatments for cognitive dysfunction.

Methods:Immunohistochemical analyzes using the phospho-KOR antibody determined the conditions leading to KOR activation in the prefrontal cortex (PFC) of male C57BL/6 mice. Using an inert, genetically encoded KOR-based sensor (kLight1.2a), we measured in vivo the dynamics of endogenous dynorphin release in the PFC. To investigate the role of the PFC KOR in working memory, we trained mice in an operant delay switching task to respond to a retracted lever, wait a fixed delay for a lever reposition, and then respond to an alternate lever. Mice were trained to constant performance with a 10-second re-entry delay and then injected into the PFC with artificial cerebrospinal fluid (ACSF) or the long-acting (∼3 weeks) KOR antagonist, norBNI (1.25 μg in 0.5 μl vehicle) ). After recovery from surgery, mice were administered either saline, KOR agonist (U50.488 5 mg/kg i.p.) or withdrawal via morphine precipitation. The contribution of locally released dynorphin to the prefrontal cortex was investigated by optical stimulation of prodynorphinCre neurons with channel rhodopsin-2 prior to immunohistochemistry and preservation.

Results:In male C57BL/6 mice, we found that acute morphine withdrawal induces dynorphin release in the medial prefrontal cortex (PFC) and impairs cognition by activating local kappa opioid receptors (KOR). Immunohistochemistry showed that morphine withdrawal increased dynorphin release in the prefrontal cortex. Using the KOR ligand sensor kLight1.2a, we determined the time course of endogenous dynorphin release in the prefrontal cortex in vivo after withdrawal of naloxone-precipitated morphine. Local activation of the KOR in the PFC resulted in a polyphasic memory processing impairment in the delayed operant switching behavioral task, manifested by a decrease in response count and accuracy during the early and late phases of the operant session. Local pretreatment in the PFC with the selective KOR antagonist norbinaltorphimine (norBNI) prevented the inhibitory effect of systemic KOR activation during the early and late sessions. Early but not late phase disruption was blocked by viral ablation of the PFC KORs, suggesting an anatomically distinct contribution of presynaptic and postsynaptic KORs. Morphine withdrawal also disrupted the effects of delayed metabolism, and this effect was blocked by pre-treatment of norBNI in PFC. Stimulation of prodynorfinCre neurons in the prefrontal cortex increased KOR PFC activation as measured by immunohistochemistry and disrupted delayed-switching performance, suggesting a local dynorphin/KOR circuit involved in cognitive dysfunction.

Conclusions:These results suggest that new therapies capable of blocking dynorphin signaling in the cerebral cortex may reduce the cognitive impairment seen in some psychiatric disorders, including adverse stress reactions during drug withdrawal.

Funding: Supported by NIH grants: P50-MH106428 (CC), P30-DA048736 (CC), R01-DA030074 (CC), R21-MH108839 (BL), T32-DA07278 (CC and AA).

Keywords:Opioid addiction, opioid peptides, Kappa opioid receptor

Revelation:Nothing to disclose.

Yale University School of Medicine, New Haven, Connecticut, Stany Zjednoczone

Bottom:People with cocaine use disorder (CU) experience prolonged craving during withdrawal. Chronic cocaine craving is closely related to the severity of recent cocaine use. One hypothesis is that hypodopaminergic states during withdrawal exhibit a negative reward prediction bias - reward deficiency - that accelerates and sustains craving and decreases response to physical reinforcement. Chronic cocaine craving is also associated with altered motivation and increased stress and anxiety. Our previous imaging studies have reported how UC responds to cocaine cues compared to drug cues in relation to chronic cocaine craving as assessed by the Cocaine Craving Questionnaire (CCQ) (Wang et al., 2020 Neuropsychopharm), both in cocaine cues for food (Zhang et al. al., 2020, Intl J Neuropsychopharm) to highlight cues (Zhornitsky et al., 2021, Biol Psychiatr: Global Open Sci) and receive a reward in the monetary incentive delay task (MIDT) (Zhornitsky et al. ., 2020, Intl J Neuropsychopharm). These studies cover the hippocampus and parahippocampus, hypothalamus, medial cortex, and ventral striatum, respectively, each of memory, motivation, limbic movement, and reward circuits. These data provide an opportunity to explore 1) how these local responses relate to exposure to drugs, food and stress stimuli; 2) how these peripheral responses relate to chronic cocaine craving and physiological arousal during task challenges;

Methods:A total of 56 recently screened UC patients and 51 age- and sex-matched HC patients participated in the study. the exact sample size varied according to the behavioral task. CU met the criteria for current cocaine addiction from the SCID for DSM IV. Recent cocaine use confirmed by urine drug test. They had been drug-free for about 10 days while in the hospital ward prior to the current fMRI scan. All participants were assessed for drug and alcohol use, including history of use and current use. CU also interviewed the 18-item CSSA to assess the degree of cocaine addiction. Chronic craving for cocaine was assessed with the CCQ-Brief every 2 to 3 days during hospitalization. Behavioral tasks including cocaine, eating, and stress reactivity in the block project and MIDT in the event project are described in the articles cited above. In all experiments, skin conductance was recorded simultaneously by fMRI. Image data preprocessing and modeling were performed according to published procedures, and the results were adjusted for the threshold value.

Results:(A) MIDT reward perception did not differentially involve the ventral striatum (SV) between the CU and HC, nor did the SV show reward activity in correlation with the CCQ score in the CU. (B) Exposure to cocaine and stress cues (compared to neutral) evoked greater activity in the hippocampus, and exposure to cocaine cues elicited less activation in the parahippocampal gyrus (PHG). Only parahippocampal activity correlated with the CCQ score in UC – the lower the PHG inactivation, the greater the chronic cocaine craving. (C) The hypothalamus showed reactivity to drugs and food stimuli in correlation with the CCQ score. (D) In ​​different tasks, hippocampal and PHG activity was correlated between food and drug stimuli and between food and stress reactivity, but not between drugs and stress reactivity. (E) Hypothalamic activation was correlated between drug and food stimulus reactivity, but not between drug and stress or between food stimulus reactivity and stress stimulus. Is (eat) stress signal reactivity involves the medial cingulate cortex (MCC) with respect to the CCQ score. However, among those studied, MCC activity was not correlated during exposure to drugs, food, and stress cues.

Conclusions:These findings indicate that (1) chronic craving for cocaine is reflected in PHG reactivity to drug cues, hypothalamic reactivity to drug and food cues, and MCC reactivity to stress cues, but not VS reward response; (2) cocaine exposure activates memory, motivation, and stress circuits; (3) the memory circuit is proportionally involved between food and drug reactivity and stress, but not between drug and stress reactivity; (4) the response of the incentive circuit is proportional between exposure to drug and food stimuli (but not to stress); and (5) the limbic motor circuit is unique in its stress reactivity in terms of its role in mirroring chronic cocaine craving. Collectively, the results highlight the distinct psychological and neural processes that underlie chronic cocaine craving.

Keywords:Cocaine addiction, reward, motivation, emotional stress, suggestion

Revelation:Nothing to disclose.

University of Kentucky, Lexington, Kentucky, United States

Bottom:Nicotine-seeking behavior elicits neuroimmune responses in the nucleus accumbens (NACore), including activation of microglia. In addition, adenosine 2A receptors (A2aR) regulate microglial proliferation, activation and survival. Systemic A2aR agonists effectively reduce ethanol self-administration, cocaine recall, and changes in synaptic plasticity by controlling presynaptic glutamate release in the striatum. These neuroimmune mechanisms, however, have not been studied for their role in nicotine-induced behaviors.

Methods:In the current study, male and female rats were subjected to nicotine self-administration (0.06 mg/kg/injection) followed by stimulus-induced extinction and recall training. A2aRs were chronically challenged (CGS 21680, 0.4 mg/kg ip) or antagonized (SCH 58261, 0.4 mg/kg ip) during extinction and immediately prior to reinstatement, then lever pressing for conditioned cues was compared with treated animals nicotine with animals. In addition, NAcore microglial morphology was quantified after self-administration of nicotine in both sexes.

Results:Here, we show sex-specific patterns of microglial activation where women showed greater NAcore microglial activation compared to men (PI number< 0.05). In addition, we found an increase in A2a receptor expression in the NAcore after chronic nicotine self-administration (PI number< 0.05). Finally, the A2a challenge significantly reduced the nicotine requirement, while the challenge did not change the nicotine requirement compared to the vehicle (PI number< 0,05).

Conclusions:These results indicate that A2a activation inhibits nicotine-seeking behavior, which is likely mediated by microglia. In addition, we have shown that women are more sensitive to microglial activation after voluntary nicotine ingestion. Ongoing research is determining whether A2a accumbens receptors control microglial activation and rapid, transient glutamate plasticity during nicotine-seeking behavior. In addition, we are currently using CX3CR1-cre transgenic mice in ongoing studies to determine whether chemogenetic activation or microglial inhibition controls nicotine seeking and lying glutamate plasticity.

Keywords:Neuroimmune activation, nicotine addiction, nucleus accumbens, sex differences, chemogenetics

Revelation:Nothing to disclose.

VA, University of Maryland, Baltimore, Maryland, United States

Bottom:The receptor-like protein tyrosine phosphatase PTPRD is highly expressed in neurons and concentrated at their presynaptic terminals. Activation of PTPRD by extracellular binding partners is thought to alter its intracellular phosphatase activity by altering the tyrosine phosphorylation of several synaptic proteins, including the GSK3 beta and GSK3 alpha tau kinases. Human PTPRD genomic variants are associated with several addiction-related phenotypes and neurofibrillary tangle densities in post-mortem Alzheimer's disease (AD) brains. Mice with reduced PTPRD expression exhibit addiction and AD-associated phenotypes. These and other results suggest that reducing PTPRD function would reduce addictive drug reward, and increasing PTPRD function would reduce the accumulation of neurofibrillary pathology.

Methods:Phosphatase assays using recombinant human PTPRD phosphatase and spectrophotometric detection of pNPP dephosphorylation products and orthophosphate release from phosphopeptides corresponding to potential substrates for PTPRD phosphatase. Candidate substrates named from brain phosphoproteomic studies of wild-type and PTPRD knockout mice. Place a preference for cocaine in mice. Self-administration of remifentanil (FR1) in rats.

Results:We now report that selected flavanols, especially quercetin, act as substrate-selective positive allosteric modulators of the PTPRD phosphatase's ability to dephosphorylate the phosphotyrosine (pY) GSK3 alpha/beta and thus reduce the activity of this tau hyperphosphorylator. In silico docking studies provide a new potential mechanism for this action. We report that NHB1109, a novel analogue of the PTPRD lead phosphatase inhibitor compound 7-BIA, exhibits good in vitro potency and selectivity, reasonable oral bioavailability and therapeutic index, and the ability to reduce cocaine-dependent place preference in mice and self-administration of remifentanil rats. Each of these results confirms that PTPRD is a new target for new therapeutic agents that inhibit or enhance its activity in a way that should benefit these three common human conditions. Comparisons of the effect of quercetin on the ability of PTPRD to dephosphorylate the pYGSK3 phosphopeptide with its effect on the dephosphorylation of other PTPRD substrates support a new concept of substrate-specific positive allosteric modulation.

Conclusions:PTPRD phosphatase inhibitors are good candidates to move into human trials to reduce the reward of stimulants and opiates. Positive allosteric modification of the phosphatase PTPRD and other data supports the study of quercetin to delay the progression of mild cognitive impairment in Alzheimer's disease. Our data with candidate substrates supports a new concept of positive allosteric modulation of substrate selectivity.

Keywords:Phosphatase, Psychostimulants, Opioids, Splątki neurofibrylarne, Alzheimer's disease

Revelation:Patent: royalties (automatic)

Yale University School of Medicine, New Haven, Connecticut, Stany Zjednoczone

Bottom:Although numerous studies have looked at changes in HPA axis activity and regulation in PTSD or major depressive disorder, little has investigated HPA axis regulation in relation to reward dysfunction, which can be present in both. One such study showed associations of a reduced response to reward with a single CRHR1 nucleotide variant, which is associated with an impaired response of the HPA axis to CRH challenge. Many previous studies have hypothesized that the main source of glucocorticoid signaling in the brain is peripherally produced adrenal cortisol, which then enters the brain. However, peripheral cortisol enters the brain at a slow rate and may only account for about 5% of brain cortisol, arguing that the local source of brain cortisol production is an important regulator. Using in vivo imaging of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a cortisol-producing enzyme in the brain, we sought to investigate reward function in relation to this putative marker of brain cortisol regulation. In addition, we used an objective measure of reward function to assess potential subclinical pathology in individuals with trauma exposure but who do not meet the criteria for PTSD, who often have low self-reported symptoms of loss (hedonia, numbness) related to reward function. prize. We used a well-validated reward probability task designed to objectively assess a person's propensity to respond to previously reinforced rewards. We performed a secondary analysis to assess whether, in trauma-exposed individuals, caudal 11beta-HSD1 availability is associated with reward response as an objective measure of reward function and as an endophenotype of bereavement symptoms.

Methods:In the original study, seventeen trauma-exposed individuals without PTSD (TE: 8 females, 33 +/- 8 years old) and sixteen PTSD patients (8 females, 37 +/- 10 years old) underwent positron emission tomography (PET) scans with [18F] AS2471907, radioligand for 11beta-HSD1. Participants received 93 +/- 14 MBq of [18F]AS2471907 as a bolus and were imaged for 150-240 minutes on a high-resolution research scanner. Of these subjects, twenty-six took part in the 25-minute probabilistic reward task (TE:N= 14, TEPT:N= 12) same day as PET scan [18F]AS2471907 (N= 21) or within 3 months after PET scan (N= 5). Participants in the TE group did not meet the criteria for PTSD on the physician-provided PTSD scale. Availability of 11beta-HSD1 was calculated as volume of distribution of [18F]AS2471907 (VT) using MA1 analysis and metabolite corrected arterial flow function. Analyzes focused on the caudate brain region based on previous work that observed altered caudate functional activity in relation to decreased reward response in MDD. The primary outcome variable of the probabilistic reward task was response bias, a well-validated and previously published indicator of systematic preference for the stimulus most commonly associated with higher reward. The median separation of the corresponding reward groups is constrained by the response error. The between-group difference in caudate 11beta-HSD1 availability was assessed by one-dimensional ANOVA for "Good Reward Response" vs. "Responding to reduced reward." A general linear modeling approach was used to compare the biased tail response of 11beta-HSD1 availability, mean 90-minute plasma cortisol, and 24-hour urinary cortisol.

Results:Preliminary results showed that the availability of 11beta-HSD1 in the tail was significantly higher (approximately 25%) in the "Good Reward Response" group compared to the "Reduced Reward Response" group (PI number= 0.037). In the TE group, a lower level of the 11beta-HSD1 caudal accessibility trend was associated with a poorer reward response or lower response error (R2 = 0.22,PI number= 0.092). There was no significant association of 11beta-HSD1 tail availability with 90-minute mean plasma cortisol (N= 13,PI number= 0.23). Preliminary results in a much smaller subgroup of subjects who completed the 24-hour urine collection (N= 7) showed a significant negative correlation between 11beta-HSD1 availability in the tail and urinary cortisol (R2 = 0.60,PI number= 0,041).

Conclusions:Overall, the availability of 11beta-HSD1 in the caudate nucleus, a region associated with reward function, was greater in the "Good response to reward" group, with a trend towards a positive correlation between the availability of 11beta-HSD1 in the caudate nucleus and greater deviation response. better reward mode. Previous work showed a significant association of higher 11beta-HSD1 availability in the prefrontal-frontal system with lower symptoms of PTSD loss (sum of emotional numbness and anhedonia), but there was a limited range of self-reported loss symptoms in the TE group that prevented similar analysis using psychometric. This analysis allowed for an objective assessment of reward function in the TE group. Although no correlation with plasma cortisol levels has been observed, and preliminary results of urinary cortisol collection cannot be interpreted without further data at present, 11beta-HSD1 alone may be a useful tool in investigating aspects of the clinical pathophysiology of RDoC dimensions. This may help to further define the different roles of cerebral and peripheral cortisol regulation in both clinical reward function in PTSD, MDD and subclinical reward function in traumatized individuals.

Keywords:RDoC, reward function, traumatic stress, HPA axis, neuroendocrine responses

Revelation:Nothing to disclose.

Johns Hopkins University School of Medicine, Baltimore, Maryland, Stany Zjednoczone

Bottom:Classic psychedelics (e.g. serotonin receptor 2A or 5-HT2AR, agonists including psilocybin and LSD) induce a wide range of acute changes in perception and cognition (Nichols, 2016). The corticostriatalthalamocortical loop model of psychedelic drug action (CSTC; Geyer & Vollenweider, 2001; Vollenweider & Geyer, 2008; Vollenweider & Preller, 2020) proposed that psychedelics disrupt the thalamic blockade of 5-H2 sensory information by, as a result, in the increased flow of sensory information to the cortex, leading to perceptual changes and cognitive impairments seen during acute psychedelic effects. Correspondingly, independent groups have found increased thalamocortical functional connectivity in non-task-related conditions while exposed to LSD in humans (Tagliazucchi et al., 2016; Müller et al., 2017; Preller et al., 2018, 2019). In particular, these studies treated the thalamus as a single structure, despite the known differential 5-HT2AR expression, functional specificity, and anatomic connectivity of the thalamic nuclei. Only a subset of the thalamic nuclei (e.g., reticular, dorsolateral, ventral, and pulmonary nuclei) express 5-HT2AR or are directly involved in the effects of psychedelic drugs in animal models (Inserra et al., 2021).

We used Model Independent Component Analysis (tICA) of resting-state fMRI (rsfMRI) data to identify functional subdivisions of the thalamus, and then examined psilocybin-induced changes in functional organization of the thalamus and cortical connectivity. We hypothesized that psilocybin would selectively alter the reticular and mesial organization of the dorsal nucleus and functional cortical connectivity.

Methods:Baseline rsfMRI data were collected from 38 clinically and psychiatrically healthy participants (22M/16F, Mage 54.9). The MRI data was minimally pre-processed using SPM12 (https://www.fil.ion.ucl.ac.uk/spm/) and thalamic spatial components were calculated from rsfMRI baseline data using the Group ICA fMRI toolkit (Calhoun et al., 2001). The ICA was spatially confined using a full-chamber occlusion mask from the Harvard-Oxford atlas (Desikan et al., 2006). The mean and between-individual spatial map variances for each of the 7 independent chamber components represented the previous or "standard" for the ICA model. The anatomical locations of each thalamic component were determined by calculating the overlap of each thalamic component and a histologically validated anatomical stereotactic atlas of the thalamus (Morel, Magnin, & Jeanmonod, 1997).

Eighteen participants (11M/7K, Mag 51.9) from the original cohort underwent 110-minute rsfMRI scans (corresponding to the peak effects of psilocybin) after blind oral administration of placebo and 10 mg/70 kg of psilocybin. tICA was used to generate topic and session specific component maps (placebo/psilocybin)N= 14 per person) and variance estimates. In voxel termsT- Tests were performed on each subsequent estimation map to identify voxels exhibiting significant "binding" (greater than zero component bias, FDRq=0.05). Changes in binding interference (measured as change in the number of active voxels in each scan) were tested using non-parametric Wilcoxon pairsT-tests (FDRq=0.05). Pearson correlations were used to test the association between session-to-session changes in the involvement of each thalamic component and between-session changes in self-reported subjective affect. Cortic-thalamic connectivity was compared between sessions using Fisher-transformed Pearson correlations between time courses of thalamic components and 10 resting-state cortical network time courses derived from the cortical ICA (N= 10; Smith et al., 2009). Double regression was performed on rsfMRI data after noise correction (Behzadi et al., 2007; Muschelli et al., 2014) to generate timeline components for each component of the thalamus and cortical network.

This study was conducted as part of a registered clinical trial (NCT02145091). All procedures were approved by the Johns Hopkins Medicine Institutional Review Board.

Results:Binding was lower during psilocybin than placebo in thalamic components corresponding to pulvinar (PI number[FDR]=0.03), dorsomedial (PI number[FDR]=0.032) and side central (PI number[FDR]=0.024) nuclei. Less functional connectivity between the pulmonary and occipital poles (PI number[FDR]=0.048) and between the central flank nucleus and the default mode network (PI number[FDR]=0.013) was observed during psilocybin. Increased reports of people experiencing "withdrawal" were negatively associated with lung involvement (R= -0,47,PI number= 0.051) and is positively associated with the lateral central nucleus with DMN connectivity (R= 0,53,PI number= 0.02). Increased involvement of the central lateral nucleus was positively associated with increased mystical experience (R= 0,53,PI number= 0,024).

Conclusions:A novel analytical approach revealed several anatomically specific, psilocybin-induced changes in thalamocortical organization and connectivity that have been linked to the subjective effects of psilocybin. These findings provide evidence for psilocybin-induced changes in specific thalamocortical circuits involved in the perceptual effects of classic psychedelics (involving the pulmonary and occipital poles), as well as circuits (involving the lateral central nucleus and the DMN) that may mediate subjective effects (mystical experience) ) appears to predict psychedelic therapeutic efficacy (Barrett & Griffiths, 2017).

Keywords:Psilocybin, thalamus, analysis of independent components, resting state fMRI, neuropsychopharmacology

Revelation:WavePaths, Ltd: Advisory Board (Auto)

University of California San Francisco, San Francisco, California, United States

Bottom:Abnormal associative learning is at the root of many mental illnesses. For example, learned associations between environmental stimuli and drug use strongly contribute to relapse. The best-known theoretical model of associative learning is reinforcement learning. Reinforcement learning proposes a simple but effective model for learning that a cue predicts a future reward: update the reward prediction by the reward prediction error (RPE) - the difference between the received and predicted reward. Although most experimental work on the neural mechanisms of reinforcement learning has focused on the mesolimbic dopamine circuit and its RPE coding, the extensive computational literature argues that the RPE dopamine circuit cannot function in isolation and that the brain likely contains complementary learning systems. For example, reinforcement learning algorithms have parameters such as learning speed that they also need to learn. Since the amount of net learning from RPE is the product of RPE multiplied by the reward learning rate, optimal tuning of the learning rate (the amount by which the RPE updates the reward prediction) can be extremely beneficial in aligning learning with the environment. Recently, several neural signals have been found to correlate with learning speed, including the anterior cingulate cortex (ACC), dorsal serotonergic system, dorsal prefrontal cortex, and anterior insula.

Here, we develop an alternative approach to learning rate control and test the hypothesis that the orbitofrontal cortex (OFC) controls reward learning rate. We tested this hypothesis for several reasons. First, the OFC receives input from all the areas listed above. Second, we noted earlier that suppression of the vmOFC reward response was sufficient to reduce the rate of behavioral learning. Third, OFC activity correlates with stimulus action, a key variable thought to regulate the rate of learning. We thus tested whether vmOFC neurons function as a controller of learning rate by signaling the relative importance of rewards and whether this signaling is at least partially mediated by one of their primary inputs from the medial thalamus.

Methods:To study reward responses in a large number of single ventral/medial OFC (vmOFC) neurons during reward anticipation learning, we used two-photon calcium imaging during a discriminant Pavlovian trait conditioning task in head-fixed mice (Namboodiri et al., 2019). All experiments were approved by the institutional IACUC.

This study included 33 wild-type C57/BL6 mice (14 females). Behavioral experiments were performed under water scarcity conditions where animals were kept at ∼85–90% of their pre-deprivation weight. Animals underwent surgery to inject a virus that induces the expression of a calcium sensor (AAVDJ-CaMKIIα-GCaMP6s) into the vmOFC and, in some cases, to inject a control AAV5-CaMKIIα-eNpHR3.0-mCherry or -opsin into the medial thalamus to study the effect of its inhibition on vmOFC neural coding.

Complementary conditioning was performed exactly as before (Namboodiri et al., 2019), with an auditory tone (3 kHz pulsed tone or 12 kHz steady tone, 75–80 dB) of 2 s duration combined with a sucrose reward (10–12, 5 %), ~2.5 µL) 1 second after tone change. This paired reward tone (CS + ) was randomly interspersed with another tone (12 kHz steady tone or 3 kHz pulsating tone, 75–80 dB) that did not predict reward (CS - ). In another experiment, random drops of sucrose were administered with random drops of quinine (1.5–2.5 mM) in a 3:1 ratio.

We recorded a total of over 7,000 vmOFC neurons in various experiments with and without inhibition of the medial thalamic inputs to the vmOFC. These neurons were first grouped based on their responses to feature adaptation to reveal 9 groups/subpopulations of neurons (Namboodiri et al., 2019). The reward response of these neurons was analyzed in different sessions at the beginning of the learning, at the end of the learning, a session where the reward probability was reduced to 50%, and a session where random and unpredictable sucrose and quinine were administered to the animals. Data were analyzed under these conditions using standard statistical methods.

Results:Using a novel neural learning rate control test, we show that the vmOFC reward response is consistent with a trial-by-trial (one-tailed) trial-by-trial learning rate controller.PI number< 10-16 for the whole population). We then show that in line with learning rate signalling, some subpopulations of neurons in vmOFC decrease their reward responses when there is low uncertainty in the environment (bilateralPI number<10-9 in some subpopulations). We then show that this vmOFC outcome response is consistent with an outcome-relevant signal when conditioned rewards were randomly interspersed with a conditioned highly significant (bilateral) aversive stimulus.PI number<10-9 in some subpopulations). Finally, we show that medial thalamic inputs to vmOFC also signal relative significance and causally control relative significance signaling in vmOFC.

Conclusions:In conclusion, we show that responses to vmOFC rewards control the rate of learning by signaling the relative importance of the reward. These results suggest that the contribution of the medial thalamus and OFC dysfunction to mental illness is due, at least in part, to impaired rate control of learning.

Keywords:Orbitofrontal cortex, medial orbitofrontal cortex, associative learning, two-photon calcium imaging

Revelation:Nothing to disclose.

Texas A&M University, College Station, Texas, United States

Bottom:Phase fear and long-term anxiety may be pathophysiologically distinct dimensions that underlie and explain the clinically significant variability of internalizing disorders. Working with animals supports this distinction, with predictable threat cues triggering short-term anxiety behaviors and unpredictable threat cues triggering anxiety-like states. The NIH research domain criteria also emphasize the distinction between acute hazard and potential hazard. The No-Threat, Predictable, Unpredictable (NPU) task can be used to quantify individual differences in response to predictable and unpredictable threats. Previous work using this example internalization task has mainly focused on categorical diagnoses. Knowing how predictable and unpredictable threat responses are related to diagnostic fear and anxiety may help refine phenotypic definitions in internalizing disorders. In addition, studying predictable and unpredictable threats with a prospective response can help identify predictive trajectories for fear and anxiety and can facilitate early intervention or preventive action.

Methods:The participants were 52 people (31 women, M age 24.46 years, SD 9.33) with various internalizing psychopathology. At hour 1, participants performed the NPU Shock Threat Task while the electroencephalogram (EEG) and eye blinks were recorded. Participants reported diagnostic fear (PANAS-X fear subscale) and anxiety (combination of trait anxiety/STAI, worry/PSWQ, and social anxiety/SPIN) at time 1 and time 2 (M = 1.68 years later, SD = 0, 68). We assessed the associations between psychophysiological response to predictable and unpredictable threat at Time 1 a) simultaneous Fear and Anxiety (Time 1) and b) Future Anxiety and Anxiety (Time 2) (controlling for Time 1 Fear and Anxiety).

Results:Greater electrocortical response (negative component, negativity prime, SPN) to anticipated threat cues was associated with increased fear at time 1 (control of anxiety at time 1), β=-0.256,PI number= 0.04, while higher SPNs for unpredictable distress signals only predicted increased anxiety at Time 2 (controlling anxiety and fear at Time 1), β==−0.272,PI number= 0.01. Increased surprise amplification for predictable threat cues predicted increased fear at Time 2 (controlling fear and anxiety at Time 1), β=0.646,PI number= 0,04.

Conclusions:Increased electrocortical response to predictable threat cues was associated with higher diagnostic fear, and greater startle response to predictable threat cues predicted an increase in fear at Time 2. Higher electrocortical response to unpredictable threat cues predicted greater distress at Time 2. 2. The results indicate a neurobiological basis for dimensional fear and anxiety and may be relevant to the development of an evidence-based clinical system.

Keywords:Anxiety and Depression, Research Domain Criteria (RDoC), EEG/ERP Electrophysiology, Tremor, Shock Threat

Revelation:Aptinyx Inc.: Consultant (only)

University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States

Bottom:As a consequence of COVID-19, an increasing number of teenagers are at risk of social isolation. While social isolation at any stage of development can have devastating long-term consequences for physical and mental health, a growing body of evidence shows a clear and profound impact of adolescent social isolation on a wide range of behavioral and physiological parameters. Goal-directed behavior and value-based decision-making are essential for healthy daily functioning, and deficits are strongly associated with poor functional outcomes in people living with mental illness. Here, we investigate the effects of adolescent social isolation in male and female mice on later goal-directed behavior and value-based decision making in adulthood.

Methods:For the adolescent social isolation model, male and female C57bl6/J mice were housed and weaned on PND21 in single or group housing (3 mice of the same sex/cage).N= 8-15 by gender and housing status). After PND60, mice were deprived of food and trained in a self-initiated two-choice operant task. The task trials were divided into 3 distinct phases: (1) Start: entering a centrally lit door initiated the trial and two retractable levers were extended, (2) Choice: rats must respond to one of the two levers within 10 s (a skip response is not considered account), (3) score evaluation: the levers have been withdrawn and the score (a prize with a variable volume of chocolate or vanilla liquid depending on the task step, described below) has been delivered. After a 1-second delay between trials, the next trial started. To test initial learning, mice were first trained to respond to an active lever that was boosted with 15 L of pure reward. The reaction of the idle lever was 4 seconds. After task acquisition, mice were trained for 10 days in a series reversal task in which the active lever was reversed after 8/10 correct answers. We then tested how isolated and socially housed mice used information about reward value and time cost to guide their decisions. First, mice were given rewards of different volumes (15 µl vs. 0, 5 or 10 µl) in the serial reversal task, with the 8/10 leverage selection reinforced by the larger 15 µl reward, resulting in an emergency reversal. Each test session was separated by at least 2 sessions of 15 ul vs. 0 ul of training sessions. After 3 sessions at each reward volume contrast, different time delays (1.5, 3, or 4.5 s) were applied to the larger 15 ul reward to see how cost was factored into the decision.

Results:Adolescents' social isolation influenced the acquisition of surgeons in different ways (two-way ANOVA interactionPI number= 0.02), elastic performance in the serial inversion problem (interaction of a three-way linear mixed-effects modelPI number< 0.01) and relative value of rewards (three-way linear interaction of the mixed-effects model).PI number= 0.03) and cost (three-way interaction of the linear mixed-effects modelPI number= 0.02). In female mice that were socially isolated during adolescence, they learned the operant task faster, but were more exploratory in their choice of behavior during the serial recall task. In contrast, adolescent social isolation in males delayed operant learning, but upon task takeover, previously isolated mice showed greater sensitivity to reward value, choosing the higher reward option more consistently, even when the reward difference was small and the time cost was high.

Conclusions:Together, these data show that adolescent social isolation in mice has divergent but lasting effects on later decision making in male and female mice. These gender differences may be the result of contrasting adaptations to social isolation or gender differences in the stress of social isolation. Ongoing work is creating neuroeconomic models to further analyze the effects of social isolation on choice behavior as well as to explore the role of underlying cortical-cortical circuits.

Keywords:Decision Making, Adolescent Social Isolation, Social Isolation Stress, Values ​​Based Decision Making, Adolescent Stress

Revelation:Nia Therapeutics: Founder (husband)

Emory University School of Medicine, Atlanta, Georgia, United States

Bottom:Given the high percentage of trauma victims who develop adverse post-traumatic neuropsychiatric sequelae (APNS), identifying individuals at risk and endophenotypes associated with susceptibility to APNS is of paramount importance. Previous neuroimaging studies have identified the inferior ventral prefrontal cortex (vmPFC) and hippocampal activation during inhibition paradigms as contributing factors to the development of PTSD. However, sample sizes were small and many questions remain regarding gender effects, relevance to other APNS, and the possibility of identifying more scalable biomarkers for neuroimaging outcomes. We wanted to answer these questions in this larger prospective trial.

Methods:Acutely traumatized individuals were recruited from emergency departments (EDs) for deep phenotyping in the AURORA study (N= 261.91 million). The subjects performed the Go/NoGo fMRI task 2 weeks after the injury. Contrast estimates for Go vs NoGo trials were extracted for vmPFC and bilateral hippocampus. APNS ratings included PTSD at 6 months (6 months), impulsivity and sleep problems at 8 weeks (8 weeks), and depression, anxiety, and alcohol use at 6 months. Our first goal was to replicate our previous findings of vmPFCs related to reduced hippocampal inhibition and activation as risk factors for PTSD. Considering the gender-specific effects of stress on limbic regions, we analyzed the interaction of gender and vmPFC and hippocampal activation, in addition to the main effects, using linear regression models. Second, we investigated whether vmPFC or hippocampal activation is specifically associated with PTSD or reflects a diagnostic risk factor in the APNS. Finally, we examined scaled measures related to lower vmPFC and hippocampal activation, for which we used the My Brain Test for neurocognitive assessment of response inhibition, e.g. Gradual Onset Continuous Performance Task (CPT) d', a measure of consistent hits for false positives.

Results:Neuroimaging and clinical data forN= 261 subjects (91 M/170 F; 46 Hispanic/89 White/114 Black/11 Other; mean age 33.6 years, SD 12.6) were included in the analyses. Linear regression analyzes showed a significant model for vmPFC (eat(2,260) = 4,33,PI number= 0.014), with female gender (B = -0.132,PI number= 0.032) and lower vmPFC activation (B = -0.123,PI number= 0.046) predicting PTSD symptoms after 6 months. An important model for the hippocampus (eat(1,260) = 4,61,PI number= 0.033) showed that hippocampal activation and gender influence the prediction of PTSD symptoms after 6 months (B = -0.132,PI number= 0.033). In particular, activation of the lower hippocampus in men was associated with greater PTSD symptoms at 6 months (R= -0,234,PI number= 0.025). Subsequently, lower vmPFC activation was associated with higher impulsivity (R= -0,157,PI number= .011), a compromise to (R= -0,131,PI number= 0.049) at 8 weeks, but no symptoms related to alcohol use, depression or anxiety at 6 months. Indeed, lower vmPFC activation was associated with lower levels of alcohol consumption at 6 months (R= ,149,PI number= 0.016). Lower hippocampal activation was not associated with other post-traumatic sequelae. Finally, in men, a significant correlation was observed between CPT d' and bilateral hippocampal activation (N= 85,R= ,29,PI number= 0,007).

Conclusions:This large ED study replicates and partially extends our previous findings. Lower hippocampal activation was particularly associated with later PTSD symptoms in men, while vmPFC activation predicted APNS diagnoses, particularly those in the disinhibitory domain, i.e. PTSD, impulsiveness, and sleep problems, suggesting a potential diagnostic mechanism of interest for early interventions. For the hippocampus, a correlation with motor neurocognitive ratings of response inhibition was observed, again only in men. This measure is much easier to collect than an fMRI scan and may be further explored as a method of identifying people at risk or as a potential target for early intervention.

Keywords:Emergency department, functional magnetic resonance imaging (fMRI), trauma exposure, ventral prefrontal cortex, hippocampus

Revelation:Nothing to disclose.

University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States

Bottom:Anhedonia - a central deficit in motivation and pleasure - remains one of the most difficult psychiatric symptoms to treat. Given the centrality of anhedonia in many mental disorders, refined interventions and validated biomarkers of anhedonia treatment response are key public health needs. The current study addresses a gap in our understanding of the relationships between treatment-related changes in anhedonia (and related symptoms), brain function, and inflammation.

Methods:An outpatient diagnostic sample of 73 adults (18–49 years, 51 women), recruited for clinically significant anhedonia, performed 7T fMRI scans before and after treatment involving a face-matching task known to elicit strong responses in neural circuits. threat processing and emotion regulation (Hariri et al., 2002). Participants were randomly assigned to 15 weeks of anhedonia activation therapy (BATA) or mindfulness-based cognitive therapy (MBCT) and regularly reported symptoms of anhedonia (SHAPS), depression (BDI) and perceived stress (PSS) during treatment. In addition, whole blood is drawn before each examination to assess peripheral inflammation (interleukin-6, IL-6). A subgroup of participants (~25) had mid-treatment assessments, so some participants received up to four tests and blood draws. Linear model permutation analysis toolkit (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/PALM/) was used to generate task-based whole-brain functional activation maps for the main fMRI task contrast (i.e. Faces > Shapes). A 2x2 mixed-effects ANOVA examined the effect of time (post vs. before) and treatment interaction (BATA vs. MBCT) over time. Significant effects were detected using the Unlimited Cluster Improvement (TFCE) method by controlling for the known error rate (FWE)PI number< 0.05. BOLD sign change values ​​were calculated and extracted from significant groups for each participant and time point for use in hierarchical linear models (HLMs). HLM studied the effects of treatment and time on self-reported symptoms, task-based brain activation, and IL-6. Additional HLM models examined how individual changes in task-related and IL-6 brain activation were related to symptoms to understand how these variables change together over time.

Results:There was no treatment for the effect of time on any outcome of interest (ie, symptoms, task-fMRI, IL-6). Both treatment groups showed significant improvements in self-reported anhedonia, depression and anxiety over time (PI number’s < .0001). Moreover, IL-6 showed a downward trend over time in all participants (PI number= 0.08). Post-treatment, compared with pre-treatment, increased activation was observed in a large cluster (k = 723) spanning the midline of the posterior cingulate gyrus, dorsoanterior cingulate gyrus, and supplementary motor cortex for the main fMRI contrast task (Faces > Shapes.PI number< 0.05, corrected by FWE). HLM using all time points confirmed that the signal percentage change in this cluster significantly increased over time throughout the sample (ɣ= 0,01039,PI number< 0.01). HLMs assessing intra-personal correlations showed that greater than normal cingulate activation was associated with lower symptoms of anhedonia at a given time point (ɣ= -4,0945,PI number< 0.01), depression (ɣ= -5,5513,PI number< 0.05) i stres (ɣ= -5,6443,PI number<0.001). In addition, lower-than-normal levels of IL-6 were associated with less anhedonia at a given time point (ɣ= 17,8700,PI number= 0.05), although it was not associated with depression or anxiety (PI numbers > 0.05). IL-6 and cingulate activation were not significantly involved in the subject (PI number> 0,05).

Conclusions:BATA and MBCT were equally effective in reducing patient-reported anhedonia, depression, and perceived stress in the anhedonia diagnostic sample. In addition, both treatments tended to decrease IL-6 levels. Across the trial, participants showed increased tooth activation over time in a task commonly used to study the neural circuits involved in emotion and threat processing. The cingulate gyrus is involved in mediating complex behaviors and is connected to various internal brain networks (including cognitive control and attention networks). The results are consistent with previous research showing that cognitive behavioral interventions reduce symptoms and inflammation, and increase activity in areas of the brain related to emotion regulation and cognitive control. Finally, personal associations suggest possible ways in which psychotherapy could alter brain and immune function to reduce psychiatric symptoms in patients with anhedonia.

Keywords:7T fMRI, inflammation, anhedonia

Revelation:Nothing to disclose.

Texas A and M University, College Station, Texas, United States

Bottom:Efforts to promote equality, inclusion and diversity within the CPDD are critical to increase innovation and excellence in addiction science and relevance to societal needs and public health. In line with these goals, the survey was developed to support and accelerate these efforts with a more flexible approach to characterizing diversity and measuring inclusion. This project explores the correlations between our membership survey and attendance at annual meetings in 2020.

Methods:For the membership survey, CPDD directory individuals were asked to take part in a 10-point online survey (N= 657). The demographic questions used a wide range of options regarding origin/ethnicity/race, gender identity and sexual orientation. In addition, a self-identification option was included ("None of these describe me. I describe myself as ____.") and the option to choose whether or not to respond. Respondents reported membership status, time since completion of final degree, perception of welcome to the organization, and had the opportunity to provide feedback on efforts to increase diversity and inclusion in the CPDD. Attendance survey at the meeting (N= 1189) was more limited and included membership, country of origin, ethnicity (but not race), gender, and whether or not they were attending the meeting for the first time.

Results:Previous research approaches allowed only one of six ethnic/racial options and male and female or other. The majority of members in the survey identified themselves using conventional survey methods, using 6 ethnicities/race and either male or female. However, many chose various other options related to their Hispanic ethnicity (13.8%), non-white identity (24.4%), non-binary and/or transracial identity (1.2%). A significant proportion of minority members reported their own self-identification preferences. Responses to the inclusion rate ("How welcome are you in CPDD?") suggest a relationship with certain demographic factors, such as ethnicity and sexual orientation. People who identify as Hispanic and people who identify as female are less likely to be members than those who identify as non-Hispanic or male. Most meeting attendees were non-members, and rates were higher for those who identified as female and Hispanic.

Conclusions:Our findings suggest that the use of expanded response options may more comprehensively characterize the diversity of CPDD members and their need to attend meetings. They also suggest that efforts should focus on moving from participation to inclusion for those who identify as Hispanic and/or women. Our goal is to use these findings to develop a data-driven approach to guide efforts to promote a more open and equitable scientific community.

Keywords:Diversity, inclusion and exclusion, connection

Revelation:Nothing to disclose.

Feinberg School of Medicine, Northwestern University, Chicago, Chicago, Illinois, Stany Zjednoczone

Bottom:Apathy and inhibition are two common neuropsychiatric symptoms (NPS) in dementia, particularly in behavioral variant frontotemporal dementia (bvFTD), but also in Alzheimer's type dementia (DAT). They are very painful, predict institutionalization and are extremely difficult to treat. The present diagnostic study examined the associations between these symptoms in bvFTD and DAT and brain structure as measured by T1 MRI in three major functional networks, Salience (SN), cognitive control networks (CCN) and default mode networks (DMN). Given previous neuroimaging results, we hypothesized that apathy would be associated with SN abnormalities and disinhibition would be associated with SN and NCC abnormalities.

Methods:157 people with a clinical diagnosis of ATD participated (N= 94; 36 women) or bvFTD (N= 63; 22 women) on the initiative of Neuroimaging of Alzheimer's disease and the initiative of Neuroimaging of frontocephalic lobe degeneration, respectively. All participants had a 3T T1 MRI and data from the Neuropsychiatric Inventory Questionnaire (NPI-Q), a caregiver-rated measure of NPS in dementia. We used the Freesurfer 6.0 and Glasser atlas (Glasser et al., 2016) to divide each MRI into 360 plots and computed 7 statistics per bundle: gray matter volume, area, cortical thickness, intrinsic curvature, mean curvature, index of curvature, and finger fold index . Using a functional network atlas (Ji et al., 2019), we identified diagrams representing SN, CCN and DMN nodes. The statistics for each pair of plots in each network were correlated to create morphometric intra-subject similarity networks (MSN; Seidlitz et al., 2019) for each of the SN, CCN and DMN. We removed the automatic and negative correlations and performed a density threshold of 0.40 (King and Wood, 2020) for each weighted and untargeted MSN. After normalizing the MSNs, we computed the following network metrics using the Brain Connectivity Toolkit: Transitivity of network separation, which reflects the number of nodes clustered together (normalized average clustering factor), Overall network integration performance (inverse mean shortest path length). For each NPS, we calculated ANCOVA 2 (present vs. absent) x 2 (diagnosis) by controlling for age, sex, estimated total intracranial volume (eTIV), days between MRI and QIN (MRI-NPI_days), and the sum of the clinical scale of the Dementia Rating Framework (CDR) -SB). We also regressed the overall performance and transitivity of each subnetwork (SN, CCN, DMN) by age and eTIV. The residuals from these regressions were correlated with the severity of apathy and inhibition (0=none, 1=mild, 2=moderate, 3=severe) using the Spearman correlation, controlling for age, MRI-NPI_days and CDR-SB.

Results:ANCOVA analysis showed the presence of apathy associated with SN transitivity (eat= 3,95,PI number= .049) and global MV efficiency (eat= 5,17,PI number= 0.024), both of which were lower in people with apathy. SN transients were also lower in DAT than in bvFTD (eat= 5,17,PI number= 0.024). Those with inhibition had lower CCN global effectiveness than those without (eat= 6,31,PI number= 0.013). There was an interaction of inhibition and diagnosis on the overall performance of SN (eat= 4,88,PI number= 0.029), with higher SN efficiency in bvFTD without disinhibition. After the Bonferroni correction (PI number< 0.016), the effect of inhibition on overall NCC performance remained significant. Spearman's correlations of apathy severity with network measurements were n.s. However, Spearman's correlations with the severity of inhibition were significant for the global NS performance (rho = -0.163,PI number= 0.044), CCN transitivity (rho = -0.210,PI number= 0.009) and overall CCN efficiency (rho = -0.228,PI number= 0.005). Both CCN correlations survived the Bonferroni correction. Sensitivity analyzes showed that removing CDR-SB as a covariate did not affect the results.

Conclusions:This is the first study to investigate how MSNs relate to NPS in dementia. Our findings are consistent with neuroimaging evidence that both apathy and inhibition are associated with damage to SN regions, particularly the medial prefrontal and orbital cortex, and that inhibition can also occur after damage to the dorsal prefrontal cortex. It should be noted that while MSNs have been shown to capture known cortical cytoarchitecture and axonal connectivity, they are merely an indicator of anatomical connectivity. Importantly, our diagnostic study showed that the presence and severity of symptoms were related to the similarity of the structural network, regardless of the clinical diagnosis. This finding has therapeutic implications, particularly those focused on modulating brain networks, as these results suggest that there are common underlying structural changes associated with these symptoms, regardless of the clinical diagnosis of dementia.

Keywords:Neuropsychiatric symptoms (NPS), metadiagnostics, Alzheimer's dementia, behavioral variant frontotemporal dementia, network neuroscience

Revelation:Nothing to disclose.

University of Montreal, Montreal, Canada

Bottom:In uncertain environments, we balance exploration and exploration: we mainly explore possibilities that we hope will be satisfying, but sometimes we also explore uncertain alternatives that could be even better. Exploration is associated with dysregulation of predictive choice activity in the prefrontal cortex, which may be a powerful means of random selection to promote exploratory discovery and learning. Although the mechanisms behind this disorganization remain unknown, scanning is also associated with changes in pupil size at constant luminance – a peripheral marker of neuromodulatory mechanisms that could theoretically cause disorganization in predictive choice activity. Alternatively, pupil size may simply monitor variables such as uncertainty and instability that increase the likelihood of scanning without predicting the neural correlates of scanning as such.

Methods:Here, we examined how pupil size at constant luminance (1) varied with exploration and exploration and (2) prefrontal neuronal activity was predicted at and between target conditions. We analyzed student data from a large neural dataset (574 units, 21,793 trials, 28 sessions, 2 male monkeys). (Note that we are ethically mandated to minimize animal numbers, so we cannot account for sex in individual primate experiments.) Portions of this dataset have been analyzed previously (Ebitz, Albarran, & Moore, 2018), although all analyzes here are new and the data students have not been previously reported. The monkeys performed a classic exploration task known as the restless k-armed thug: they chose from 3 options whose reward value varied randomly, unpredictably, and independently over time. This encouraged the monkeys to explore rewarding options when they were found, but also to explore uncertain alternatives that might turn out to be the best at any given time. To determine when the monkeys explored (vs. explored), we modeled them as a hidden target state behavior in a hidden Markov model. We previously reported that these labels of model-inferred target states explain more differences in neural activity than rewards, subjective value, choice to switch or stay, and other relevant variables.

Results:Consistent with previous studies, we found that pupil size at constant luminance was larger during exploratory choices compared to exploratory choices (both monkeys: pairedT-test:PI number< 0,0001,T(27)=4.95, mean shift=0.23, 95% CI=0.13 to 0.32; monkey 1:PI number< 0,0005,T(9)=5.50, mean=0.4, 95% CI=0.24 to 0.57; monkey 2:PI number< 0,02,T(17) = 2.85, mean = 0.13, 95% CI = 0.03 to 0.23). In fact, inter-trial variation in pupil size predicted the likelihood that the trial was exploratory (GLM logistics, beta = 0.009,PI number< 0.0001). However, this effect was not limited to exploratory attempts. In 10 trials, before the monkeys started to explore, pupil size increased slowly (both monkeys: GLM, beta     0.016,PI number< 0,003,N= 28; monkey 1: beta = 0.020,PI number< 0,02,N= 10; monkey 2: beta = 0.013,PI number< 0,05,N= 18). This was not an artifact of some target-state label mismatch: there was no ramp for other behavioral and neural measures that differed between exploration and exploration, such as movement speed or exploration neural signatures.

We next asked whether pupil size predicts predictive choice activity in the prefrontal cortex. We trained decoders to anticipate firing rate selections for units recorded simultaneously in each session. Selection decoding accuracy decreased during exploit selections compared to exploit selections (parT-test,PI number< 0,0001,T(27) = 8.65), and increasing pupil size predicted poorer selection decoding accuracy (GLM: beta = −0.032,PI number< 0,0001,N= 28). This was not a trivial consequence of averaging in probing trials (where pupil size was larger and decoding accuracy lower) and probe trials (where pupil size was smaller and decoding accuracy was higher) because it was also true and similar in magnitude only in exploratory trials (beta=-0.037,PI number< 0,002,N= 28). We previously reported that scanning also increased "dispersion" between trials (i.e.PI number< 0,0001,N= 28) and again only in exploratory trials (beta = 0.03,PI number< 0,0005,N= 28). Taken together, pupil size predicted trial-by-sample variability in predictive choice activity in the prefrontal cortex, although it increased before exploration and these neural measures did not.

Conclusions:The fact that pupil size increased prior to exploration may suggest that the student is paying attention to variables that make exploration more likely, rather than predicting exploratory brain states per se. However, the student also predicted trial changes after exploration trials of neural signatures during exploration, in the absence of any apparent change in behavior. These results are difficult to reconcile if we imagine the brain as a linear system, but perhaps make more sense if the exploration represents a critical "turning point" for the prefrontal cortex - a transition to a new dynamic regime where all choices are made. it may happen. In this non-linear model, pupillary mechanisms may shift the brain into a mode where tipping points are more likely, but the ultimate trigger to initiate exploration and disorganization of prefrontal activity is the positive feedback loop. complex and non-linear, depending only partly on the mechanisms associated with the pupil.

Keywords:Explore-Explore Dilemma Value-Based Decision Making Pupilometer Neurophysiology Explore

Revelation:Nothing to disclose.

University of Texas at Austin, Austin, Texas, United States

Bottom:Οι θεραπείες πρώτης γραμμής για τις διαταραχές της διάθεσης, του άγχους και του τραυματικού στρες περιλαμβάνουν φαρμακολογικές και ψυχοθεραπευτικές παρεμβάσεις, αλλά πολλά άτομα δεν ανταποκρίνονται επαρκώς και διατηρούν σημαντικά υπολειπόμενα συμπτώματα και λειτουργική έκπτωση. Οι παρεμβάσεις που βασίζονται σε μη επεμβατικές νευροτροποποιητικές συσκευές, συμπεριλαμβανομένης της επαναλαμβανόμενης διακρανιακής μαγνητικής διέγερσης (rTMS) και της διακρανιακής ηλεκτρικής διέγερσης, είναι προσεγγίσεις δεύτερης γραμμής που έχουν δείξε