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Background: Adolescent social isolation (ASI) has profound long-term effects on behavioral and neural development. Despite this, the specific long-term impact of ASI during different adolescent stages and across sexes remain underexplored., Methods: Our study addresses this gap by examining the effects of early- and late- adolescent social isolation on both male and female rats. Rats were either isolated (or group-housed) starting from PD 21 (early) or PD 42 (late) for three weeks and then rehoused into groups. In adulthood (PD 90), rats underwent a battery of tests: elevated plus-maze, open field, novel object recognition, social interaction and social recognition memory and hotplate tests. Finally, we analyzed oxytocin receptor binding in several regions in the brains of a second cohort of rats., Results: Both, male and female rats from the late adolescent social isolation (LASI) groups spent significantly less time interacting in the social interaction test. Additionally, we observed a general decrease in social recognition memory regardless of sex. Both male ASI groups demonstrated heightened thermal pain sensitivity, while the opposite was observed in early adolescent social isolation (EASI) female rats. In the brain, we observed changes in oxytocin receptor (OTR) binding in the paraventricular nucleus of the hypothalamus (PVN) and paraventricular nucleus of the thalamus (PVT) and central amygdala (CeA) with the largest changes in EASI and LASI female rats., Conclusion: Our model demonstrates long-lasting alterations on behavior and oxytocin receptor binding levels following ASI providing insights into the long-term effects of ASI in a time- and sex-specific manner., (© 2024. The Author(s).)

Structural and functional changes of the brain are assumed to contribute to excessive cocaine intake, craving, and relapse in cocaine use disorder (CUD). Epigenetic and transcriptional changes were hypothesized as a molecular basis for CUD-associated brain alterations. Here we performed a multi-omics study of CUD by integrating epigenome-wide methylomic (N = 42) and transcriptomic (N = 25) data from the same individuals using postmortem brain tissue of Brodmann Area 9 (BA9). Of the N = 1 057 differentially expressed genes (p < 0.05), one gene, ZFAND2A, was significantly upregulated in CUD at transcriptome-wide significance (q < 0.05). Differential alternative splicing (AS) analysis revealed N = 98 alternatively spliced transcripts enriched in axon and dendrite extension pathways. Strong convergent overlap in CUD-associated expression deregulation was found between our BA9 cohort and independent replication datasets. Epigenomic, transcriptomic, and AS changes in BA9 converged at two genes, ZBTB4 and INPP5E. In pathway analyses, synaptic signaling, neuron morphogenesis, and fatty acid metabolism emerged as the most prominently deregulated biological processes. Drug repositioning analysis revealed glucocorticoid receptor targeting drugs as most potent in reversing the CUD expression profile. Our study highlights the value of multi-omics approaches for an in-depth molecular characterization and provides insights into the relationship between CUD-associated epigenomic and transcriptomic signatures in the human prefrontal cortex., (© 2024. The Author(s).)

Background: Alcohol use disorder (AUD) has a profound public health impact. However, understanding of the molecular mechanisms that underlie the development and progression of AUD remains limited. Here, we investigated AUD-associated DNA methylation changes within and across 2 addiction-relevant brain regions, the nucleus accumbens and dorsolateral prefrontal cortex., Methods: Illumina HumanMethylation EPIC array data from 119 decedents (61 cases, 58 controls) were analyzed using robust linear regression with adjustment for technical and biological variables. Associations were characterized using integrative analyses of public annotation data and published genetic and epigenetic studies. We also tested for brain region-shared and brain region-specific associations using mixed-effects modeling and assessed implications of these results using public gene expression data from human brain., Results: At a false discovery rate of ≤.05, we identified 105 unique AUD-associated CpGs (annotated to 120 genes) within and across brain regions. AUD-associated CpGs were enriched in histone marks that tag active promoters, and our strongest signals were specific to a single brain region. Some concordance was found between our results and those of earlier published alcohol use or dependence methylation studies. Of the 120 genes, 23 overlapped with previous genetic associations for substance use behaviors, some of which also overlapped with previous addiction-related methylation studies., Conclusions: Our findings identify AUD-associated methylation signals and provide evidence of overlap with previous genetic and methylation studies. These signals may constitute predisposing genetic differences or robust methylation changes associated with AUD, although more work is needed to further disentangle the mechanisms that underlie these associations and their implications for AUD., (© 2024 The Authors.)

Excessive alcohol consumption remains a global public health crisis, with millions suffering from alcohol use disorder (AUD, or simply "alcoholism"), leading to significantly reduced life expectancy. This review examines the interplay between habitual and goal-directed behaviors and the associated neurobiological changes induced by chronic alcohol exposure. Contrary to a strict habit-goal dichotomy, our meta-analysis of the published animal experiments combined with a review of human studies reveals a nuanced transition between these behavioral control systems, emphasizing the need for refined terminology to capture the probabilistic nature of decision biases in individuals with a history of chronic alcohol exposure. Furthermore, we distinguish habitual responding from compulsivity, viewing them as separate entities with diverse roles throughout the stages of the addiction cycle. By addressing species-specific differences and translational challenges in habit research, we provide insights to enhance future investigations and inform strategies for combatting AUD., (© 2024. The Author(s).)

Substance use disorders (SUDs) are seen as a continuum ranging from goal-directed and hedonic drug use to loss of control over drug intake with aversive consequences for mental and physical health and social functioning. The main goals of our interdisciplinary German collaborative research centre on Losing and Regaining Control over Drug Intake (ReCoDe) are (i) to study triggers (drug cues, stressors, drug priming) and modifying factors (age, gender, physical activity, cognitive functions, childhood adversity, social factors, such as loneliness and social contact/interaction) that longitudinally modulate the trajectories of losing and regaining control over drug consumption under real-life conditions. (ii) To study underlying behavioural, cognitive and neurobiological mechanisms of disease trajectories and drug-related behaviours and (iii) to provide non-invasive mechanism-based interventions. These goals are achieved by: (A) using innovative mHealth (mobile health) tools to longitudinally monitor the effects of triggers and modifying factors on drug consumption patterns in real life in a cohort of 900 patients with alcohol use disorder. This approach will be complemented by animal models of addiction with 24/7 automated behavioural monitoring across an entire disease trajectory; i.e. from a naïve state to a drug-taking state to an addiction or resilience-like state. (B) The identification and, if applicable, computational modelling of key molecular, neurobiological and psychological mechanisms (e.g., reduced cognitive flexibility) mediating the effects of such triggers and modifying factors on disease trajectories. (C) Developing and testing non-invasive interventions (e.g., Just-In-Time-Adaptive-Interventions (JITAIs), various non-invasive brain stimulations (NIBS), individualized physical activity) that specifically target the underlying mechanisms for regaining control over drug intake. Here, we will report on the most important results of the first funding period and outline our future research strategy., (© 2024 The Author(s). Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Background: Alcohol use disorder (AUD) is associated with increased mortality and morbidity risk. A reason for this could be accelerated biological aging, which is strongly influenced by disease processes such as inflammation. As recent studies of AUD show changes in DNA methylation and gene expression in neuroinflammation-related pathways in the brain, biological aging represents a potentially important construct for understanding the adverse effects of substance use disorders. Epigenetic clocks have shown accelerated aging in blood samples from individuals with AUD. However, no systematic evaluation of biological age measures in AUD across different tissues and brain regions has been undertaken., Methods: As markers of biological aging (BioAge markers), we assessed Levine's and Horvath's epigenetic clocks, DNA methylation telomere length (DNAmTL), telomere length (TL), and mitochondrial DNA copy number (mtDNAcn) in postmortem brain samples from Brodmann Area 9 (BA9), caudate nucleus, and ventral striatum (N = 63-94), and in whole blood samples (N = 179) of individuals with and without AUD. To evaluate the association between AUD status and BioAge markers, we performed linear regression analyses while adjusting for covariates., Results: The majority of BioAge markers were significantly associated with chronological age in all samples. Levine's epigenetic clock and DNAmTL were indicative of accelerated biological aging in AUD in BA9 and whole blood samples, while Horvath's showed the opposite effect in BA9. No significant association of AUD with TL and mtDNAcn was detected. Measured TL and DNAmTL showed only small correlations in blood and none in brain., Conclusions: The present study is the first to simultaneously investigate epigenetic clocks, telomere length, and mtDNAcn in postmortem brain and whole blood samples in individuals with AUD. We found evidence for accelerated biological aging in AUD in blood and brain, as measured by Levine's epigenetic clock, and DNAmTL. Additional studies of different tissues from the same individuals are needed to draw valid conclusions about the congruence of biological aging in blood and brain., (© 2024 The Authors. Alcohol: Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

Background: Cocaine use disorder (CUD) is characterized by a loss of control over cocaine intake and is associated with structural, functional, and molecular alterations in the human brain. At the molecular level, epigenetic alterations are hypothesized to contribute to the higher-level functional and structural brain changes observed in CUD. Most evidence of cocaine-associated epigenetic changes comes from animal studies while only a few studies have been performed using human tissue., Methods: We investigated epigenome-wide DNA methylation (DNAm) signatures of CUD in human post-mortem brain tissue of Brodmann area 9 (BA9). A total of N = 42 BA9 brain samples were obtained from N = 21 individuals with CUD and N = 21 individuals without a CUD diagnosis. We performed an epigenome-wide association study (EWAS) and analyzed CUD-associated differentially methylated regions (DMRs). To assess the functional role of CUD-associated differential methylation, we performed Gene Ontology (GO) enrichment analyses and characterized co-methylation networks using a weighted correlation network analysis. We further investigated epigenetic age in CUD using epigenetic clocks for the assessment of biological age., Results: While no cytosine-phosphate-guanine (CpG) site was associated with CUD at epigenome-wide significance in BA9, we detected a total of 20 CUD-associated DMRs. After annotation of DMRs to genes, we identified Neuropeptide FF Receptor 2 (NPFFR2) and Kalirin RhoGEF Kinase (KALRN) for which a previous role in the behavioral response to cocaine in rodents is known. Three of the four identified CUD-associated co-methylation modules were functionally related to neurotransmission and neuroplasticity. Protein-protein interaction (PPI) networks derived from module hub genes revealed several addiction-related genes as highly connected nodes such as Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C) , Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) , and Jun Proto-Oncogene, AP-1 Transcription Factor Subunit (JUN) . In BA9, we observed a trend toward epigenetic age acceleration (EAA) in individuals with CUD remaining stable even after adjustment for covariates., Conclusion: Results from our study highlight that CUD is associated with epigenome-wide differences in DNAm levels in BA9 particularly related to synaptic signaling and neuroplasticity. This supports findings from previous studies that report on the strong impact of cocaine on neurocircuits in the human prefrontal cortex (PFC). Further studies are needed to follow up on the role of epigenetic alterations in CUD focusing on the integration of epigenetic signatures with transcriptomic and proteomic data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Poisel, Zillich, Streit, Frank, Friske, Foo, Mechawar, Turecki, Hansson, Nöthen, Rietschel, Spanagel and Witt.)

Adolescence is a critical time of social learning in which both the quantity and quality of social interactions shape adult behavior and social function. During adolescence, social instability such as disrupting or limiting social interactions can lead to negative life-long effects on mental health and well-being in humans. Animal models on social instability are critically important in understanding those underlying neurobiological mechanisms. However, studies in rats using these models have produced partly inconsistent results and can be difficult to generalize. Here we assessed in a sex and age consistent manner the long-term behavioural consequences of social instability stress (SIS - 1-hr daily isolation and change in cage mate between postnatal day (PD30-45)) in Wistar rats. Female and male rats underwent a battery of tests for anxiety-like, exploratory, and social behaviour over five days beginning either in adolescence (PD46) or in adulthood (PD70). Social instability led to reduced anxiety-like behaviour in the elevated plus maze in both sexes in adolescence and in adulthood. Social interactions were also reduced in rats that underwent SIS - an effect that was independent of sex and age when tested. SIS improved social recognition memory in both sexes whereas a sex-dependent effect was seen in the social novelty preference test where male rats that underwent SIS spent more time in social approach toward a novel peer than toward their cage mate. In comparison, control male and female groups did not differ in this test, in time spent with novel versus the cage mate. Thus, overall, social instability stress in Wistar rats altered the behavioural repertoire, with enduring alterations in social behaviour, enhanced exploratory behaviour, and reduced anxiety-like behaviour. In conclusion, the social instability stress paradigm may better be interpreted as a form of enrichment in Wistar rats than as a stressor., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Chronic alcohol consumption and alcohol use disorder have a tremendous impact on the patient's psychological and physiological health. There is evidence that chronic alcohol consumption influences SARS-CoV2 infection risk, but so far, the molecular mechanism underlying such an effect is unknown., Methods: We generated the expression data of SARS-CoV2 infection-relevant genes (Ace2, Tmprss2, and Mas) in different organs in rat models of chronic alcohol exposure and alcohol dependence. Ace2 and Tmprss2 represent the virus entry point, whereas Mas activates the anti-inflammatory response once the cells are infected., Results: Across three different chronic alcohol test conditions, we found a consistent upregulation of Ace2 gene expression in the lung, which has been shown to be the most affected organ in COVID-19 patients. Other organs such as liver, ileum, kidney, heart, and brain also showed upregulation of Ace2 and Mas gene expression but less consistently across the different animal models, while Tmprss2 expression was unaffected in all conditions., Conclusions: We conclude that alcohol-induced upregulation of Ace2 gene expression can lead to an elevated stochastic probability of virus entry into cells and may thus confer a molecular risk for SARS-CoV2 infection., (© 2023 The Authors. Alcohol: Clinical & Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

Alcohol Use Disorder (AUD) is a major contributor to global mortality and morbidity. Postmortem human brain tissue enables the investigation of molecular mechanisms of AUD in the neurocircuitry of addiction. We aimed to identify differentially expressed (DE) genes in the ventral and dorsal striatum between individuals with AUD and controls, and to integrate the results with findings from genome- and epigenome-wide association studies (GWAS/EWAS) to identify functionally relevant molecular mechanisms of AUD. DNA-methylation and gene expression (RNA-seq) data was generated from postmortem brain samples of 48 individuals with AUD and 51 controls from the ventral striatum (VS) and the dorsal striatal regions caudate nucleus (CN) and putamen (PUT). We identified DE genes using DESeq2, performed gene-set enrichment analysis (GSEA), and tested enrichment of DE genes in results of GWASs using MAGMA. Weighted correlation network analysis (WGCNA) was performed for DNA-methylation and gene expression data and gene overlap was tested. Differential gene expression was observed in the dorsal (FDR < 0.05), but not the ventral striatum of AUD cases. In the VS, DE genes at FDR < 0.25 were overrepresented in a recent GWAS of problematic alcohol use. The ARHGEF15 gene was upregulated in all three brain regions. GSEA in CN and VS pointed towards cell-structure associated GO-terms and in PUT towards immune pathways. The WGCNA modules most strongly associated with AUD showed strong enrichment for immune response and inflammation pathways. Our integrated analysis of multi-omics data sets provides further evidence for the importance of immune- and inflammation-related processes in AUD., (© 2022. The Author(s).)

(1) Background: Epigenome-wide association studies (EWAS) in peripheral blood have repeatedly found associations between tobacco smoking and aberrant DNA methylation (DNAm), but little is known about DNAm signatures of smoking in the human brain, which may contribute to the pathophysiology of addictive behavior observed in chronic smokers. (2) Methods: We investigated the similarity of DNAm signatures in matched blood and postmortem brain samples ( n = 10). In addition, we performed EWASs in five brain regions belonging to the neurocircuitry of addiction: anterior cingulate cortex (ACC), Brodmann Area 9, caudate nucleus, putamen, and ventral striatum ( n = 38-72). (3) Results: cg15925993 within the LOC339975 gene was epigenome-wide significant in the ACC. Of 16 identified differentially methylated regions, two ( PRSS50 and LINC00612/A2M-AS1 ) overlapped between multiple brain regions. Functional enrichment was detected for biological processes related to neuronal development, inflammatory signaling and immune cell migration. Additionally, our results indicate the association of the well-known AHRR CpG site cg05575921 with smoking in the brain. (4) Conclusion: The present study provides further evidence of the strong relationship between aberrant DNAm and smoking.

Alcohol use disorder (AUD) is closely linked to the brain regions forming the neurocircuitry of addiction. Postmortem human brain tissue enables the direct study of the molecular pathomechanisms of AUD. This study aims to identify these mechanisms by examining differential DNA-methylation between cases with severe AUD (n = 53) and controls (n = 58) using a brain-region-specific approach, in which sample sizes ranged between 46 and 94. Samples of the anterior cingulate cortex (ACC), Brodmann Area 9 (BA9), caudate nucleus (CN), ventral striatum (VS), and putamen (PUT) were investigated. DNA-methylation levels were determined using the Illumina HumanMethylationEPIC Beadchip. Epigenome-wide association analyses were carried out to identify differentially methylated CpG-sites and regions between cases and controls in each brain region. Weighted correlation network analysis (WGCNA), gene-set, and GWAS-enrichment analyses were performed. Two differentially methylated CpG-sites were associated with AUD in the CN, and 18 in VS (q < 0.05). No epigenome-wide significant CpG-sites were found in BA9, ACC, or PUT. Differentially methylated regions associated with AUD case-/control status (q < 0.05) were found in the CN (n = 6), VS (n = 18), and ACC (n = 1). In the VS, the WGCNA-module showing the strongest association with AUD was enriched for immune-related pathways. This study is the first to analyze methylation differences between AUD cases and controls in multiple brain regions and consists of the largest sample to date. Several novel CpG-sites and regions implicated in AUD were identified, providing a first basis to explore epigenetic correlates of AUD., (© 2021. The Author(s).)

There is increasing evidence for a daily rhythm of µ-opioid receptor (MOR) efficacy and the development of alcohol dependence. Previous studies show that β-arrestin 2 (bArr2) has an impact on alcohol intake, at least partially mediated via modulation of MOR signaling, which in turn mediates the alcohol rewarding effects. Considering the interplay of circadian rhythms on MOR and alcohol dependence, we aimed to investigate bArr2 in alcohol dependence at different time points of the day/light cycle on the level of bArr2 mRNA (in situ hybridization), MOR availability (receptor autoradiography), and MOR signaling (Damgo-stimulated G-protein coupling) in the nucleus accumbens of alcohol-dependent and non-dependent Wistar rats. Using a microarray data set we found that bArr2, but not bArr1, shows a diurnal transcription pattern in the accumbens of naïve rats with higher expression levels during the active cycle. In 3-week abstinent rats, bArr2 is up-regulated in the accumbens at the beginning of the active cycle (ZT15), whereas no differences were found at the beginning of the inactive cycle (ZT3) compared with controls. This effect was accompanied by a specific down-regulation of MOR binding in the active cycle. Additionally, we detect a higher receptor coupling during the inactive cycle compared with the active cycle in alcohol-dependent animals. Together, we report daily rhythmicity for bArr2 expression linked to an inverse pattern of MOR, suggesting an involvement for bArr2 on circadian regulation of G-protein coupled receptors in alcohol dependence. The presented data may have implications for the development of novel bArr2-related treatment targets for alcoholism., (© 2022 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Alcohol-dependent patients commonly show impairments in executive functions that facilitate craving and can lead to relapse. However, the molecular mechanisms leading to executive dysfunction in alcoholism are poorly understood, and new effective pharmacological treatments are desired. Here, using a bidirectional neuromodulation approach, we demonstrate a causal link between reduced prefrontal mGluR2 function and both impaired executive control and alcohol craving. A neuron-specific prefrontal mGluR2 knockdown in rats generated a phenotype of reduced cognitive flexibility and excessive alcohol seeking. Conversely, virally restoring prefrontal mGluR2 levels in alcohol-dependent rats rescued these pathological behaviors. In the search for a pharmacological intervention with high translational potential, psilocybin was capable of restoring mGluR2 expression and reducing relapse behavior. Last, we propose a FDG-PET biomarker strategy to identify mGluR2 treatment-responsive individuals. In conclusion, we identified a common molecular pathological mechanism for both executive dysfunction and alcohol craving and provided a personalized mGluR2 mechanism-based intervention strategy for medication development for alcoholism.

Background: Accumulating clinical evidence suggests that women with prior exposure to adverse childhood experiences are more susceptible to heavy drinking and other health-related behaviors. Yet, preclinical studies investigating sex-dependent effects of adolescent adverse social experiences (ASEs) on later alcohol-seeking behavior are lacking. This is mainly due to the unavailability of valid animal models and a shortage of studies that compare effects in males and females. Therefore, we sought to investigate the sex-dependent effects of ASE on adult alcohol-seeking behavior, locomotion, and reward sensitivity in male and female rats., Methods: We recently developed a rat model for childhood/adolescent peer rejection that allows us to study the long-term consequences of ASEs. Adolescent Wistar rats were reared from postnatal day (pd) 21 to pd 50 either within a group of Fischer 344 rats (ASE) or within a group of Wistar rats (control). Wistar rats housed with Fischer 344 rats do not reciprocate social play in adolescence. This reduced play across adolescence mimics peer rejection and results in chronic dysregulation of social and pain-related behaviors. We tested adult male and female rats in the reinstatement paradigm for cue-induced alcohol-seeking behavior, circadian locomotor activity, and sucrose consumption long after the termination of the peer rejection condition., Results: Peer rejection induced persistent sex-dependent changes in alcohol cue-induced reinstatement. Females showed an increased reinstatement effect while peer-rejected males demonstrated a decrease. Sex differences were observed in locomotor activity or reward sensitivity to sucrose., Conclusions: Peer rejection has long-lasting sex-dependent consequences on alcohol-seeking behavior without affecting locomotion or sweet reward sensitivity. Our results suggest that peer-rejected female rats represent a vulnerable population in which to study relapse-like behaviors that are similar to clinical findings, while males seem to buffer the peer rejection effect and demonstrate resilience to later life alcohol-seeking behaviors, as measured by the reinstatement effect. Finally, we provide a novel approach to investigate the molecular and neurobiological underpinnings of ASEs on alcohol and other drug-seeking behaviors., (© 2021 The Authors. Alcoholism: Clinical & Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcoholism.)

Background: In 4 decades, numerous nicotine replacement therapy products have been developed. Population pharmacokinetic models can support exposure-response modeling and inform nicotine replacement therapy product development, but only limited model-based cross-study population pharmacokinetic analyses for nicotine replacement therapy products have been published., Objectives: The aim of this retrospective analysis was to assess the population pharmacokinetics of nicotine across intravenous, oral, transdermal and oromucosal (mouth spray, chewing gum, lozenge and inhaler) routes and formulations in healthy smoking subjects., Methods: Data on 930 unique subjects (46,016 observations) from 29 single- and repeated-dose studies with multiple formulations across intravenous, oral, transdermal and oromucosal routes of administration were included. Data from intravenous and extravascular routes of administration were modelled separately for run efficiency reasons. For developing extravascular models, clearance and disposition parameters and their inter-individual variabilities were fixed to the estimates for intravenously delivered nicotine. Detectable pre-dose nicotine concentrations were modelled as a hypothetical nicotine bolus into the central compartment at the start of wash-out. Modelling repeated-dose oral and buccal administrations required a time-dependent increase in clearance or decrease in bioavailability to describe the data adequately., Results: Disposition of intravenous nicotine was best described by a three-compartment model with initial and terminal half-lives of 7 min and 4.5 h, respectively, and the absorption of single oral doses was best described with a first-order absorption rate constant of 1.55 h -1 . The data of buccal formulations were modelled with parallel oromucosal absorption and gastrointestinal absorption of a part of the dose that is swallowed. For transdermal nicotine, parallel zero- and first-order release from the patch and a transit-compartment absorption model best described the data. Key pharmacokinetic parameters were reliably estimated, with typical values for clearance (67 L/h for a 70-kg subject), volume of distribution (4.3 L/kg), oral bioavailability (40%) and transdermal bioavailability (76%) within expected ranges. The estimated fraction of the dose swallowed for buccal formulations ranged from 55% (gum) to 69% (lozenge)., Conclusions: Robust population pharmacokinetic models were developed for five nicotine replacement therapy product types and for intravenous and oral nicotine. These population pharmacokinetic models are used in exposure-response analyses and simulation-based nicotine replacement therapy product design.

A pronounced decrease of oxytocin and increase of oxytocin receptor binding sites were recently reported in male alcohol dependent rats and male alcohol dependent patients. Here we comment on this and emphasize that in female alcohol dependent rats and humans no changes occur in the oxytocin system. We therefore suggest specific intervention with oxytocin only in male subjects., (© 2020 The Authors. Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Background and Purpose: Alcohol and nicotine use disorders are commonly comorbid. Both alcohol and nicotine can activate opioid systems in reward-related brain regions, leading to adaptive changes in opioid signalling upon chronic exposure. The potential role of these adaptations for comorbidity is presently unknown. Here, we examined the contribution of μ and κ-opioid receptors to nicotine-induced escalation of alcohol self-administration in rats., Experimental Approach: Chronic nicotine was tested on alcohol self-administration and motivation to obtain alcohol. We then tested the effect of the κ antagonist CERC-501 and the preferential μ receptor antagonist naltrexone on basal and nicotine-escalated alcohol self-administration. To probe μ or κ receptor adaptations, receptor binding and G-protein coupling assays were performed in reward-related brain regions. Finally, dopaminergic activity in response to alcohol was examined, using phosphorylation of DARPP-32 in nucleus accumbens as a biomarker., Key Results: Nicotine robustly induced escalation of alcohol self-administration and motivation to obtain alcohol. This was blocked by naltrexone but not by CERC-501. Escalation of alcohol self-administration was associated with decreased DAMGO-stimulated μ receptor signalling in the ventral tegmental area (VTA) and decreased pDARPP-32 in the nucleus accumbens shell in response to alcohol., Conclusions and Implications: Collectively, these results suggest that nicotine contributes to escalate alcohol self-administration through a dysregulation of μ receptor activity in the VTA. These data imply that targeting μ rather than κ receptors may be the preferred pharmacotherapeutic approach for the treatment of alcohol use disorder when nicotine use contributes to alcohol consumption., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

One of the major risk factors for global death and disability is alcohol, tobacco, and illicit drug use. While there is increasing knowledge with respect to individual factors promoting the initiation and maintenance of substance use disorders (SUDs), disease trajectories involved in losing and regaining control over drug intake (ReCoDe) are still not well described. Our newly formed German Collaborative Research Centre (CRC) on ReCoDe has an interdisciplinary approach funded by the German Research Foundation (DFG) with a 12-year perspective. The main goals of our research consortium are (i) to identify triggers and modifying factors that longitudinally modulate the trajectories of losing and regaining control over drug consumption in real life, (ii) to study underlying behavioral, cognitive, and neurobiological mechanisms, and (iii) to implicate mechanism-based interventions. These goals will be achieved by: (i) using mobile health (m-health) tools to longitudinally monitor the effects of triggers (drug cues, stressors, and priming doses) and modify factors (eg, age, gender, physical activity, and cognitive control) on drug consumption patterns in real-life conditions and in animal models of addiction; (ii) the identification and computational modeling of key mechanisms mediating the effects of such triggers and modifying factors on goal-directed, habitual, and compulsive aspects of behavior from human studies and animal models; and (iii) developing and testing interventions that specifically target the underlying mechanisms for regaining control over drug intake., (© 2019 The Authors. Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

A major hypothesis in the addiction field suggests deficits in dopamine signaling during abstinence as a driving mechanism for the relapsing course of the disorder. Paradoxically, blockade of mu-opioid receptors (MORs) intended to suppress dopamine release and alcohol reward is a widely used treatment for preventing relapse in alcohol use disorder (AUD). To elucidate this apparent discrepancy, we systematically survey the literature on experimental studies in AUD subjects and animal models, which assessed striatal dopamine levels and D1, D2-like receptor, dopamine transporter and MOR via positron emission tomography (PET) and ex vivo receptor binding assays. The reported evidence indicates a changing dopaminergic signaling over time, which is associated with concomitant alterations in MOR, thus suggesting a highly dynamic regulation of the reward system during abstinence. Such a view can reconcile the various evidences from in vivo and postmortem studies, but makes developing an effective pharmacological intervention that specifically targets either dopamine receptors or the transporter system a daunting task., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Molecular changes induced by excessive alcohol consumption may underlie formation of dysphoric state during acute and protracted alcohol withdrawal which leads to craving and relapse. A main molecular addiction hypothesis is that the upregulation of the dynorphin (DYN)/κ-opioid receptor (KOR) system in the nucleus accumbens (NAc) of alcohol-dependent individuals causes the imbalance in activity of D1- and D2 dopamine receptor (DR) expressing neural circuits that results in dysphoria. We here analyzed post-mortem NAc samples of human alcoholics to assess changes in prodynorphin (PDYN) and KOR (OPRK1) gene expression and co-expression (transcriptionally coordinated) patterns. To address alterations in D1- and D2-receptor circuits, we studied the regulatory interactions between these pathways and the DYN/KOR system. No significant differences in PDYN and OPRK1 gene expression levels between alcoholics and controls were evident. However, PDYN and OPRK1 showed transcriptionally coordinated pattern that was significantly different between alcoholics and controls. A downregulation of DRD1 but not DRD2 expression was seen in alcoholics. Expression of DRD1 and DRD2 strongly correlated with that of PDYN and OPRK1 suggesting high levels of transcriptional coordination between these gene clusters. The differences in expression and co-expression patterns were not due to the decline in neuronal proportion in alcoholic brain and thereby represent transcriptional phenomena. Dysregulation of DYN/KOR system and dopamine signaling through both alterations in co-expression patterns of opioid genes and decreased DRD1 gene expression may contribute to imbalance in the activity of D1- and D2-containing pathways which may lead to the negative affective state in human alcoholics.

Rationale: The corticotropin-releasing hormone (CRH) system is a key mediator of stress-induced responses in alcohol-seeking behavior. Recent research has identified the central nucleus of the amygdala (CeA), a brain region involved in the regulation of fear and stress-induced responses that is especially rich in CRH-positive neurons, as a key player in mediating excessive alcohol seeking. However, detailed characterization of the specific influences that local neuronal populations exert in mediating alcohol responses is hampered by current limitations in pharmacological and immunohistochemical tools for targeting CRH receptor subtype 1 (CRHR1)., Objective: In this study, we investigated the effect of cell- and region-specific overexpression of CRHR1 in the CeA using a novel transgenic tool., Methods: Co-expression of CRHR1 in calcium-calmodulin-dependent kinase II (αCaMKII) neurons of the amygdala was demonstrated by double immunohistochemistry using a Crhr1-GFP reporter mouse line. A Cre-inducible Crhr1-expressing adeno-associated virus (AAV) was site-specifically injected into the CeA of αCaMKII-Cre ERT2 transgenic rats to analyze the role of CRHR1 in αCaMKII neurons on alcohol self-administration and reinstatement behavior., Results: Forty-eight percent of CRHR1-containing cells showed co-expression of αCaMKII in the CeA. AAV-mediated gene transfer in αCaMKII neurons induced a 24-fold increase of Crhr1 mRNA in the CeA which had no effect on locomotor activity, alcohol self-administration, or cue-induced reinstatement. However, rats overexpressing Crhr1 in the CeA increased responding in the stress-induced reinstatement task with yohimbine serving as a pharmacological stressor., Conclusion: We demonstrate that CRHR1 overexpression in CeA-αCaMKII neurons is sufficient to mediate increased vulnerability to stress-triggered relapse into alcohol seeking.

Approved pharmacological treatments for alcohol use disorder are limited in their effectiveness, and new drugs that can easily be translated into the clinic are warranted. One of those candidates is oxytocin because of its interaction with several alcohol-induced effects. Alcohol-dependent rats as well as post-mortem brains of human alcoholics and controls were analyzed for the expression of the oxytocin system by qRT-PCR, in situ hybridization, receptor autoradiography ([ 125 I]OVTA binding), and immunohistochemistry. Alcohol self-administration and cue-induced reinstatement behavior was measured after intracerebroventricular injection of 10 nM oxytocin in dependent rats. Here we show a pronounced upregulation of oxytocin receptors in brain tissues of alcohol-dependent rats and deceased alcoholics, primarily in frontal and striatal areas. This upregulation stems most likely from reduced oxytocin expression in hypothalamic nuclei. Pharmacological validation showed that oxytocin reduced cue-induced reinstatement response in dependent rats-an effect that was not observed in non-dependent rats. Finally, a clinical pilot study (German clinical trial number DRKS00009253) using functional magnetic resonance imaging in heavy social male drinkers showed that intranasal oxytocin (24 IU) decreased neural cue-reactivity in brain networks similar to those detected in dependent rats and humans with increased oxytocin receptor expression. These studies suggest that oxytocin might be used as an anticraving medication and thus may positively affect treatment outcomes in alcoholics.

Cue-reward associations form distinct memories that can drive appetitive behaviors and are involved in craving for both drugs and natural rewards. Distinct sets of neurons, so-called neuronal ensembles, in the infralimbic area (IL) of the medial prefrontal cortex (mPFC) play a key role in alcohol seeking. Whether this ensemble is specific for alcohol or controls reward seeking in general remains unclear. Here, we compared IL ensembles formed upon recall of drug (alcohol) or natural reward (saccharin) memories in male Wistar rats. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal, we found that cue-induced seeking of either alcohol or saccharin activated ensembles of similar size and organization, whereby these ensembles consist of largely overlapping neuronal populations. Thus, the IL seems to act as a general integration hub for reward seeking behavior, but also contains subsets of neurons that encode for the different rewards. SIGNIFICANCE STATEMENT Cue-reward associations form distinct memories that can act as drivers of appetitive behaviors and are involved in craving for natural rewards as well as for drugs. Distinct sets of neurons, so-called neuronal ensembles, in the infralimbic area of the mPFC play a key role in cue-triggered reward seeking. However, it is unclear whether these ensembles act as broadly tuned controllers of approach behavior or represent the learned associations between specific cues and rewards. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal we find largely overlapping neuronal populations. Repeated activation by two distinct events could reflect the linking of the two memory traces within the same neuron., Competing Interests: The authors declare no competing financial interests., (Copyright © 2018 the authors 0270-6474/18/383507-13$15.00/0.)

The ability of many drugs of abuse, including cocaine, to mediate reinforcement and drug-seeking behaviors is in part mediated by the corticotropin-releasing hormone (CRH) system, in which CRH exerts its effects partly via the CRH receptor subtype 1 (CRHR1) in extra-hypothalamic areas. In fact, CRHR1 expressed in regions of the mesolimbic dopamine (DA) system have been demonstrated to modify cocaine-induced DA release and alter cocaine-mediated behaviors. Here we examined the role of neuronal selectivity of CRHR1 within the mesolimbic system on cocaine-induced behaviors. First we used a transgenic mouse line expressing GFP under the control of the Crhr1 promoter for double fluorescence immunohistochemistry to demonstrate the cellular location of CRHR1 in both dopaminergic and D1 dopaminoceptive neurons. We then studied cocaine sensitization, self-administration, and reinstatement in inducible CRHR1 knockouts using the CreERT2/loxP in either dopamine transporter (DAT)-containing neurons (DAT-Crhr1) or dopamine receptor 1 (D1)-containing neurons (D1-Crhr1). For sensitization testing, mice received five daily injections of cocaine (15 mg/kg IP). For self-administration, mice received eight daily 2 h cocaine (0.5 mg/kg per infusion) self-administration sessions followed by extinction and reinstatement testing. There were no differences in the acute or sensitized locomotor response to cocaine in DAT-Crhr1 or D1-Crhr1 mice and their respective controls. Furthermore, both DAT-Crhr1 and D1-Crhr1 mice reliably self-administered cocaine at the level of controls. However, DAT-Crhr1 mice demonstrated a significant increase in cue-induced reinstatement relative to controls, whereas D1-Crhr1 mice demonstrated a significant decrease in cue-induced reinstatement relative to controls. These data demonstrate the involvement of CRHR1 in cue-induced reinstatement following cocaine self-administration, and implicate a bi-directional role of CRHR1 for cocaine craving.

The research domain criteria (RDoC) matrix has been developed to reorient psychiatric research towards measurable behavioral dimensions and underlying mechanisms. Here, we used a new genetic rat model with a loss-of-function point mutation in the dopamine transporter (DAT) gene ( Slc6a3 &#95;N157K) to systematically study the RDoC matrix. First, we examined the impact of the Slc6a3 &#95;N157K mutation on monoaminergic signaling. We then performed behavioral tests representing each of the five RDoC domains: negative and positive valence systems, cognitive, social and arousal/regulatory systems. The use of RDoC may be particularly helpful for drug development. We studied the effects of a novel pharmacological approach metabotropic glutamate receptor mGluR2/3 antagonism, in DAT mutants in a comparative way with standard medications. Loss of DAT functionality in mutant rats not only elevated subcortical extracellular dopamine concentration but also altered the balance of monoaminergic transmission. DAT mutant rats showed deficits in all five RDoC domains. Thus, mutant rats failed to show conditioned fear responses, were anhedonic, were unable to learn stimulus-reward associations, showed impaired cognition and social behavior, and were hyperactive. Hyperactivity in mutant rats was reduced by amphetamine and atomoxetine, which are well-established medications to reduce hyperactivity in humans. The mGluR2/3 antagonist LY341495 also normalized hyperactivity in DAT mutant rats without affecting extracellular dopamine levels. We systematically characterized an altered dopamine system within the context of the RDoC matrix and studied mGluR2/3 antagonism as a new pharmacological strategy to treat mental disorders with underlying subcortical dopaminergic hyperactivity., (© 2017. Published by The Company of Biologists Ltd.)

It has previously been shown that the inhibition of L-type calcium channels (LTCCs) decreases alcohol consumption, although the contribution of the central LTCC subtypes Cav1.2 and Cav1.3 remains unknown. Here, we determined changes in Cav1.2 (Cacna1c) and Cav1.3 (Cacna1d) mRNA and protein expression in alcohol-dependent rats during protracted abstinence and naive controls using in situ hybridization and western blot analysis. Functional validation was obtained by electrophysiological recordings of calcium currents in dissociated hippocampal pyramidal neurons. We then measured alcohol self-administration and cue-induced reinstatement of alcohol seeking in dependent and nondependent rats after intracerebroventricular (i.c.v.) injection of the LTCC antagonist verapamil, as well as in mice with an inducible knockout (KO) of Cav1.2 in Ca 2+ /calmodulin-dependent protein kinase IIα (CaMKIIα)-expressing neurons. Our results show that Cacna1c mRNA concentration was increased in the amygdala and hippocampus of alcohol-dependent rats after 21 days of abstinence, with no changes in Cacna1d mRNA. This was associated with increased Cav1.2 protein concentration and L-type calcium current amplitudes. Further analysis of Cacna1c mRNA in the CA1, basolateral amygdala (BLA), and central amygdala (CeA) revealed a dynamic regulation over time during the development of alcohol dependence. The inhibition of central LTCCs via i.c.v. administration of verapamil prevented cue-induced reinstatement of alcohol seeking in alcohol-dependent rats. Further studies in conditional Cav1.2-KO mice showed a lack of dependence-induced increase of alcohol-seeking behavior. Together, our data indicate that central Cav1.2 channels, rather than Cav1.3, mediate alcohol-seeking behavior. This finding may be of interest for the development of new antirelapse medications.

Blockade of the μ-opioid receptor (MOR) by naltrexone reduces relapse risk in a subpopulation of alcohol-dependent patients. Previous positron-emission-tomography (PET) studies using the MOR ligand [ 11 C]carfentanil have found increased MOR availability in abstinent alcoholics, which may reflect either increased MOR expression or lower endogenous ligand concentration. To differentiate between both effects, we investigated two cohorts of alcoholic subjects using either post-mortem or clinical PET analysis. Post-mortem brain tissue of alcohol-dependent subjects and controls (N=43/group) was quantitatively analyzed for MOR ([ 3 H]DAMGO)-binding sites and OPRM1 mRNA in striatal regions. [ 11 C]carfentanil PET was performed in detoxified, medication free alcohol-dependent patients (N=38), followed by a randomized controlled study of naltrexone versus placebo and follow-up for 1 year (clinical trial number: NCT00317031). Because the functional OPRM1 variant rs1799971:A>G affects the ligand binding, allele carrier status was considered in the analyses. MOR-binding sites were reduced by 23-51% in post-mortem striatal tissue of alcoholics. In the PET study, a significant interaction of OPRM1 genotype, binding potential (BP ND ) for [ 11 C]carfentanil in the ventral striatum, and relapse risk was found. Particularly in G-allele carriers, lower striatal BP ND was associated with a higher relapse risk. Interestingly, this effect was more pronounced in the naltrexone treatment group. Reduced MOR is interpreted as a neuroadaptation to an alcohol-induced release of endogenous ligands in patients with severe alcoholism. Low MOR availability may explain the ineffectiveness of naltrexone treatment in this subpopulation. Finally, low MOR-binding sites are proposed as a molecular marker for a negative disease course.

PPARγ is one of the three isoforms of the Peroxisome Proliferator-Activated Receptors (PPARs). PPARγ is activated by thiazolidinediones such as pioglitazone and is targeted to treat insulin resistance. PPARγ is densely expressed in brain areas involved in regulation of motivational and emotional processes. Here, we investigated the role of PPARγ in the brain and explored its role in anxiety and stress responses in mice. The results show that stimulation of PPARγ by pioglitazone did not affect basal anxiety, but fully prevented the anxiogenic effect of acute stress. Using mice with genetic ablation of neuronal PPARγ (PPARγ NestinCre ), we demonstrated that a lack of receptors, specifically in neurons, exacerbated basal anxiety and enhanced stress sensitivity. The administration of GW9662, a selective PPARγ antagonist, elicited a marked anxiogenic response in PPARγ wild-type (WT), but not in PPARγ NestinCre knock-out (KO) mice. Using c-Fos immunohistochemistry, we observed that acute stress exposure resulted in a different pattern of neuronal activation in the amygdala (AMY) and the hippocampus (HIPP) of PPARγ NestinCre KO mice compared with WT mice. No differences were found between WT and KO mice in hypothalamic regions responsible for hormonal response to stress or in blood corticosterone levels. Microinjection of pioglitazone into the AMY, but not into the HIPP, abolished the anxiogenic response elicited by acute stress. Results also showed that, in both regions, PPARγ colocalizes with GABAergic cells. These findings demonstrate that neuronal PPARγ is involved the regulation of the stress response and that the AMY is a key substrate for the anxiolytic effect of PPARγ., Significance Statement: Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) is a classical target for antidiabetic therapies with thiazolidinedione compounds. PPARγ agonists such as rosiglitazone and pioglitazone are in clinical use for the treatment of insulin resistance. PPARγ has recently attracted attention for its involvement in the regulation of CNS immune response and functions. Here, we demonstrate that neuronal PPARγ activation prevented the negative emotional effects of stress and exerted anxiolytic actions without influencing hypothalamic-pituitary-adrenal axis function. Conversely, pharmacological blockade or genetic deletion of PPARγ enhanced anxiogenic responses and increased vulnerability to stress. These effects appear to be controlled by PPARγ neuronal-mediated mechanisms in the amygdala., (Copyright © 2016 the authors 0270-6474/16/3612612-13$15.00/0.)

Schizophrenia is a severe neuropsychiatric disorder with impairments in social cognition. Several brain regions have been implicated in social cognition, including the nucleus caudatus, prefrontal and temporal cortex, and cerebellum. Oxytocin is a critical modulator of social cognition and the formation and maintenance of social relationships and was shown to improve symptoms and social cognition in schizophrenia patients. However, it is unknown whether the oxytocin receptor is altered in the brain. Therefore, we used qRT-PCR and Ornithine Vasotocin Analog ([ 125 I]OVTA)-based receptor autoradiography to investigate oxytocin receptor expression at both the mRNA and protein level in the left prefrontal and middle temporal cortex, left nucleus caudatus, and right posterior superior vermis in 10 schizophrenia patients and 6 healthy controls. Furthermore, to investigate confounding effects of long-term antipsychotic medication we treated rats with clozapine or haloperidol for 12weeks and assessed expression of the oxytocin receptor in cortical and subcortical brain regions. In schizophrenia patients, we found a downregulation of oxytocin receptor mRNA in the temporal cortex and a decrease in receptor binding in the vermis. In the other regions, the results showed trends in the same direction, without reaching statistical significance. We found no differences between antipsychotic-treated rats and controls. Downregulated expression and binding of the oxytocin receptor in brain regions involved in social cognition may lead to a dysfunction of oxytocin signaling. Our results support a dysfunction of the oxytocin receptor in schizophrenia, which may contribute to deficits of social cognition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Epidemiological surveys have indicated that anxiety disorders are more frequent in diabetic patients than in the general population. Similar results have been shown in animal studies using the streptozotocin (STZ)-induced diabetes model. The mechanisms underlying this relationship are not clearly understood, but it has been suggested that alterations in the dopaminergic neurotransmission, which plays an important role in the amygdaloid modulation of fear and anxiety, may be involved. The aim of this study was to ascertain whether or not the amygdaloid DA D1 receptors are involved in the increase of anxiety-like behavior observed in "diabetic" animals. Adult Wistar male rats were injected with STZ (50mg/kg, i.p.) in two consecutive days and subjected to the Shock-Probe Burying Test 10days after the beginning of treatment. STZ-treated rats showed a significant increase in immobility/freezing behavior whereas no effects were elicited in latency to bury, burying behavior itself and the number of shocks received during testing as compared with non-diabetic controls. These results suggest the triggering of a passive coping response in the STZ-treated rats. Interestingly, immobility/freezing behavior was reversed following the intra-amygdaloid dopamine D1 receptor blockade by the local microinfusion of SCH23390 (100ng/side). Autoradiographic experiments showed a selective increase of [(3)H]-SCH23390 binding in the ventral intercalated paracapsular islands of STZ-treated rats when compared to the non-treated control group. Our results suggest that a hyperdopaminergic state involving DA D1 receptors within the amygdala may have a role in the increase of anxiety observed in diabetic rats., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A major hypothesis in addiction research is that alcohol induces neuroadaptations in the mesolimbic dopamine (DA) system and that these neuroadaptations represent a key neurochemical event in compulsive drug use and relapse. Whether these neuroadaptations lead to a hypo- or hyperdopaminergic state during abstinence is a long-standing, unresolved debate among addiction researchers. The answer is of critical importance for understanding the neurobiological mechanism of addictive behavior. Here we set out to study systematically the neuroadaptive changes in the DA system during the addiction cycle in alcohol-dependent patients and rats. In postmortem brain samples from human alcoholics we found a strong down-regulation of the D1 receptor- and DA transporter (DAT)-binding sites, but D2-like receptor binding was unaffected. To gain insight into the time course of these neuroadaptations, we compared the human data with that from alcohol-dependent rats at several time points during abstinence. We found a dynamic regulation of D1 and DAT during 3 wk of abstinence. After the third week the rat data mirrored our human data. This time point was characterized by elevated extracellular DA levels, lack of synaptic response to D1 stimulation, and augmented motor activity. Further functional evidence is given by a genetic rat model for hyperdopaminergia that resembles a phenocopy of alcohol-dependent rats during protracted abstinence. In summary, we provide a new dynamic model of abstinence-related changes in the striatal DA system; in this model a hyperdopaminergic state during protracted abstinence is associated with vulnerability for relapse.

Background: Studies in humans and rodents have demonstrated under certain conditions some reinforcing properties of modafinil, a drug being examined clinically for its potential to treat psychostimulant abuse. However, the majority of rodent studies examining the abuse potential of modafinil have used high doses that may not be clinically relevant. In fact, recent work has indicated that doses similar to those administered to humans are not reinforcing in mice., Methods: The current study examined sex differences in the ability of low-dose modafinil (0.75mg/kg, IP) to induce a conditioned place preference in mice, and assessed sex-dependent alterations in dopamine D1, D2 and DAT binding sites in reward-related regions in naïve and modafinil-treated mice., Results: Low-dose modafinil failed to induce a conditioned place preference in male mice, while female mice demonstrated a significant modafinil place preference. Several dopamine binding differences were also detected in naïve and modafinil-treated mice, including sex differences in D1 and D2 availability in reward-related regions, and are discussed in relation to sex-dependent differences in the reinforcing effects of modafinil and psychostimulants in general., Conclusions: These findings implicate sex differences in the reinforcing properties of modafinil in mice, and indicate that clinical evaluation of the sex dependence of the reinforcing properties of modafinil in humans is warranted., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Loss of control over drinking is a key deficit in alcoholism causally associated with malfunction of the medial prefrontal cortex (mPFC), but underlying molecular and cellular mechanisms remain unclear. Cue-induced reinstatement of alcohol seeking activates a subset of mPFC neurons in rats, identified by their common expression of the activity marker cFos and comprised of both principal and interneurons. Here, we used cFos-lacZ and pCAG-lacZ transgenic rats for activity-dependent or nonselective inactivation of neurons, respectively, which by their lacZ encoded β-galactosidase activity convert the inactive prodrug Daun02 into the neurotoxin daunorubicin. We report that activity-dependent ablation of a neuronal ensemble in the infralimbic but not the prelimbic subregion induced excessive alcohol seeking. The targeted neuronal ensemble was specific for the cue-induced response because stress-induced reinstatement was not affected in these animals. Importantly, nonselective inactivation of infralimbic neurons, using pCAG-lacZ rats, was without functional consequence on the cue-induced reinstatement task. Thus, inhibitory control over alcohol seeking is exerted by distinct functional ensembles within the infralimbic cortex rather than by a general inhibitory tone of this region on the behavioral output. This indicates a high level of functional compartmentation within the rat mPFC whereat many functional ensembles could coexist and interact within the same subregion., Significance Statement: Hebb's (1949) idea of memories as being represented in local neuronal networks is supported by identification of transiently stable activity patterns within subgroups of neurons. However, it is difficult to link individual networks to specific memory tasks, for example a learned behavior. By a novel approach of activity-dependent ablation, here we identify a specific neuronal ensemble located in the infralimbic subregion of the medial prefrontal cortex that controls a seeking response for alcohol in rats. Our data demonstrate that functional output depends on specific neuronal ensembles within a given brain region rather than on the global activity of that region, which raises important questions about the interpretation of numerous earlier experiments using site-directed silencing or stimulation for elucidating brain function., (Copyright © 2015 the authors 0270-6474/15/3510750-12$15.00/0.)

Background: One of the most commonly used approaches to induce ethanol (EtOH) dependence in rodents is EtOH vapor inhalation. This procedure requires the co-administration of pyrazole-an inhibitor of the alcohol dehydrogenase-to obtain stable blood EtOH concentrations (BECs) during the entire induction course. However, pyrazole can produce unwanted side effects. Our goal was to obtain EtOH-dependent mice without pyrazole and to study their behavioral and molecular postdependent phenotype. In particular, we were interested in alterations in the corticotrophin-releasing hormone (CRH) and receptor (CRHR1 and CRHR2) system as a prominent role of CRH in driving the postdependent state via actions in the central extended amygdala (CeA) has been demonstrated in rats but not in postdependent mice., Methods: We established an alternative model of chronic intermittent EtOH (CIE) inhalation without the use of pyrazole in C57BL/6N mice. Our CIE exposure protocol involved 8 cycles. One cycle consisted of 8 hours with EtOH inhalation and 8 hours without EtOH. We then examined withdrawal symptoms. After 2 weeks of abstinence, we studied relapse, reinstatement of EtOH-seeking, and stress-induced EtOH self-administration. We also did transcriptional analysis of components of the CRH system during CIE, protracted abstinence, and after stress-induced EtOH self-administration., Results: CIE exposure without pyrazole resulted in reproducible BECs during the induction procedure. Mice showed strong withdrawal scores during 4 to 12 hours after the last CIE cycle and enhanced stress-induced EtOH self-administration. This postdependent phenotype during abstinence was accompanied by enhanced Crh and Crhr1 transcripts but no change in Crhr2 transcripts in the CeA. Cue-induced EtOH-seeking behavior and relapse (alcohol deprivation effect) were not affected by the inhalation procedure., Conclusions: We have established a CIE inhalation protocol without pyrazole in mice and showed excessive EtOH self-administration under mild stress and enhanced CRH/CRHR1 signaling in the CeA., (Copyright © 2015 by the Research Society on Alcoholism.)

The striatum plays a key role in motor learning. Striatal function depends strongly on dopaminergic neurotransmission, but little is known about neuroadaptions of the dopamine system during striatal learning. Using an established task that allows differentiation between acquisition and consolidation of motor learning, we here investigate D1 and D2-like receptor binding and transcriptional levels after initial and long-term training of mice. We found profound reduction in D1 binding within the dorsomedial striatum (DMS) after the first training session on the accelerated rotarod and a progressive reduction in D2-like binding within the dorsolateral striatum (DLS) after extended training. Given that similar phase- and region-specific striatal neuroadaptations have been found also during learning of complex procedural tasks including habit formation and automatic responding, the here observed neurochemical alterations are important for our understanding of neuropsychiatric disorders that show a dysbalance in the function of striatal circuits, such as in addictive behaviors.

Polymorphisms in the TPH2 gene coding for the serotonin synthesizing enzyme in the brain are considered as risk factors associated with depression and anxiety in humans. However, whether a certain variation in the TPH2 gene leads to decreased brain serotonin production and development of psychological abnormalities remains unresolved. We generated a new mouse model, carrying one Tph2-null allele and one Tph21473G-allele, coding for a hypoactive form of the enzyme. We tested these mice along with C57BL/6 mice (Tph2C/C), congenic C57BL/6 mice homozygous for the Tph21473G-allele (Tph2G/G), and heterozygous Tph2-deficient mice (Tph2C/-) for anxiety- and depression-like behavior, and evaluated brain serotonin metabolism and 5-HT1AR signaling by high-performance liquid chromatography and quantitative autoradiography, respectively. Progressive reduction in TPH2 activity had no effect on emotional behavior, and only slightly affected brain serotonin levels. However, serotonin degradation rate was drastically decreased in mice with reduced TPH2 activity, thereby compensating for the lowered rate of serotonin production in these mice. In addition, the hypothermic response to the 5-HT1AR agonist, 8-OH-DPAT, was attenuated in mice with reduced serotonin production. In contrast, 5-HT1A autoreceptor density and G-protein coupling were not changed in mice with gradual decrease in central serotonin. Taken together, these data suggest that in conditions of reduced serotonin production lowered serotonin degradation rate contributes to the maintenance of brain serotonin at levels sufficient for adequate behavior responses. These findings reveal that decreased TPH2 activity cannot be considered a reliable predisposition factor for impaired emotional behavior., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: L-type calcium channel (LTCC) activity in the brain is mediated by 2 subtypes, Ca(v)1.2 and Ca(v)1.3. The individual contributions of these LTCC subtypes to the long-term pharmacological and behavioral effects of nicotine are unknown., Methods: Using quantitative in situ hybridization, we examined expression levels of Ca(v)1.2 and Ca(v)1.3 in forebrain regions of mice treated with nicotine (0.175 mg/kg) or saline for 1 or 14 days and sacrificed 24 hr or 7 days following the last injection. Additionally, we treated mice with nicotine for 14 days and then administered the nonspecific LTCC antagonist nifedipine twice daily during a 7-day abstinence period prior to testing for nicotine sensitization to determine the effect of LTCC blockade on sensitization., Results: Ca(v)1.2 mRNA was unaffected 24 hr following a single nicotine exposure, whereas Ca(v)1.3 mRNA was upregulated in several brain regions. Following 14 days of nicotine treatment and 24 hr of abstinence, Ca(v)1.2 mRNA was downregulated throughout the areas examined, whereas Ca(v)1.3 mRNA had mostly returned to control values. Following 7 days of abstinence, a strong upregulation of Ca(v)1.2 transcripts was observed, whereas Ca(v)1.3 mRNA was largely unaffected. In our sensitization study, nifedipine administered during nicotine abstinence impaired subsequent nicotine sensitization., Conclusions: Our data suggest a differential involvement of Ca(v)1.2 and Ca(v)1.3 in nicotine-related processes. Ca(v)1.3 seems to be involved primarily during early exposure to nicotine. Ca(v)1.2 appears to play a role in the long-term molecular and behavioral changes that occur following chronic nicotine and abstinence. Nifedipine may counteract those nicotine-induced alterations in LTCC activity to impair nicotine sensitization.

The incubation of cocaine craving describes the time-dependent augmentation of cue-induced cocaine seeking during withdrawal from prolonged cocaine self-administration and requires time-dependent changes in neuroplasticity at the level of glutamatergic synapses in the nucleus accumbens (NAc). In contrast to most studies that use multiple cocaine-cue conditioning sessions, the present study tested mice with limited cocaine experience (i.e., a single conditioning session) in the incubation of cue-mediated cocaine seeking and its associated changes in the glutamate system. Mice that self-administered cocaine during a single session exhibited a time-dependent increase in their response for the drug-associated cue as compared to mice that self-administered saline. This behavior was associated with changes in AMPA and NMDA receptor binding characteristics. Furthermore, Group I metabotropic glutamate receptor (mGluR1) mRNA levels were altered in several brain regions, including the NAc. Because of the pivotal role of mGluR1 in the control of cocaine-induced plasticity, we investigated the role of mGluR1 in the formation of drug cue-mediated cocaine seeking. After prolonged withdrawal, mice in which an mGluR1 antagonist was administered following cocaine self-administration displayed increased cocaine seeking compared to vehicle-treated mice. These results suggest that limited cocaine experience is sufficient to induce neurobiological changes that enable an initially neutral cue to acquire motivational value that increases over time, an effect that likely involves glutamate signaling through mGluR1.

Corticotropin releasing factor (CRF) is the primary mediator of stress responses, and nociceptin/orphanin FQ (N/OFQ) plays an important role in the modulation of these stress responses. Thus, in this multidisciplinary study, we explored the relationship between the N/OFQ and the CRF systems in response to stress. Using in situ hybridization (ISH), we assessed the effect of body restraint stress on the gene expression of CRF and N/OFQ-related genes in various subdivisions of the amygdala, a critical brain structure involved in the modulation of stress response and anxiety-like behaviors. We found a selective upregulation of the NOP and downregulation of the CRF1 receptor transcripts in the CeA and in the BLA after body restraint. Thus, we performed intracellular electrophysiological recordings of GABAA-mediated IPSPs in the central nucleus of the amygdala (CeA) to explore functional interactions between CRF and N/OFQ systems in this brain region. Acute application of CRF significantly increased IPSPs in the CeA, and this enhancement was blocked by N/OFQ. Importantly, in stress-restraint rats, baseline CeA GABAergic responses were elevated and N/OFQ exerted a larger inhibition of IPSPs compared with unrestraint rats. The NOP antagonist [Nphe1]-nociceptin(1-13)NH2 increased the IPSP amplitudes in restraint rats but not in unrestraint rats, suggesting a functional recruitment of the N/OFQ system after acute stress. Finally, we evaluated the anxiety-like response in rats subjected to restraint stress and nonrestraint rats after N/OFQ microinjection into the CeA. Intra-CeA injections of N/OFQ significantly and selectively reduced anxiety-like behavior in restraint rats in the elevated plus maze. These combined results demonstrate that acute stress increases N/OFQ systems in the CeA and that N/OFQ has antistress properties.

Rationale: The rewarding effects of alcohol have been attributed to interactions between opioid and dopaminergic system within the mesolimbic reward pathway. We have previously shown that ablation of β-arrestin 2 (Arrb2), a crucial regulator of μ-opioid receptor function, attenuates alcohol-induced hyperlocomotion and c-fos activation in the nucleus accumbens., Objectives: Here, we further investigated the role of Arrb2 in modulating alcohol-induced dopamine (DA) release and conditioned place preference (CPP). We also assessed the functional importance of Arrb2 for μ-opioid receptor surface expression and signaling following an acute alcohol challenge., Methods: Alcohol-evoked (0.375, 0.75, and 1.5 g/kg intraperitoneally) DA release was measured by in vivo microdialysis in the shell of nucleus accumbens. Reward was assessed by the CPP paradigm. Receptor function was assessed by μ-receptor binding and [(35)S]GTP-γ-S autoradiography., Results: In Arrb2 knockout mice accumbal DA levels reach maximum response at a lower dose compared to wild-type (wt) animals. In line with these results, Arrb2 knockout mice display increased CPP for alcohol as compared to wt mice. Finally, Arrb2 mutant mice display increased μ-opioid receptor signaling in the ventral and dorsal striatum and amygdala in response to a low dose of alcohol, indicating impaired desensitization mechanisms in these mice., Conclusions: Our results show that Arrb2 modulates the response to low doses of alcohol on various levels including μ-opioid receptor signaling, DA release, and reward. They also reveal a clear dissociation between the effects of Arrb2 on psychomotor and reward behaviors.

Background: The comparability of gene expression between blood and brain tissues is a central issue in neuropsychiatric research where the analysis of molecular mechanisms in the brain is of high importance for the understanding of the diseases and the discovery of biomarkers. However, the accessibility of brain tissue is limited. Therefore, knowledge about how easily accessible peripheral tissue, e. g. blood, is comparable to and reflects gene expression of brain regions will help to advance neuropsychiatric research., Description: Gene expression in the blood, hippocampus (HC) and prefrontal cortex (PFC) of genetically identical rats was compared using a genome-wide Affymetrix gene expression microarray covering 29,215 expressed genes. A total of 56.8% of 15,717 expressed genes were co-expressed in blood and at least one brain tissue, while 55.3% of all genes were co-expressed in all three tissues simultaneously. The overlapping genes included a set of genes of relevance to neuropsychiatric diseases, in particular bipolar disorder, schizophrenia and alcohol addiction. These genes included CLOCK, COMT, FAAH, NPY, NR3C1, NRGN, PBRM1, TCF4, and SYNE., Conclusions: This study provides baseline data on absolute gene expression and differences between gene expression in the blood, HC and PFC brain tissue of genetically identical rats. The present data represents a valuable resource for future studies as it might be used for first information on gene expression levels of genes of interest in blood and brain under baseline conditions. Limitations of our study comprise possible contamination of brain tissue with blood and the non-detection of genes with very low expression levels. Genes that are more highly expressed in the brain than in the blood are of particular interest since changes in their expression, e.g. due to disease status, or treatment, are likely to be detected in an experiment. In contrast, genes with higher expression in the blood than in the brain are less informative since their higher baseline levels could superimpose variation in brain.

Medication development for cocaine-addicted patients is difficult, and many promising preclinical candidates have failed in clinical trials. One reason for the difficulty in translating preclinical findings to the human condition is that drug testing is typically conducted in behavioral procedures in which animals do not show addiction-like traits. Recently, a DSM-IV-based animal model has been developed that allows studying the transition to an addiction-like behavior. Changes in synaptic plasticity are involved in the transition to cocaine addiction. In particular, it has been shown that metabotropic glutamate receptor 2/3 (mGluR2/3)-mediated long-term depression is suppressed in the prelimbic cortex in addict-like rats. We therefore hypothesized that cocaine-seeking in addict-like rats could be treated with an mGluR2/3 agonist. Indeed, addict-like rats that were treated systemically with the mGluR2/3 agonist LY379268 (0, 0.3, and 3 mg/kg) showed a pronounced reduction in cue-induced reinstatement of cocaine-seeking. In an attempt to dissect the role played by mGluR2 and mGluR3 in cue-induced reinstatement, we analyzed the mRNA expression patterns in several relevant brain areas but did not find any significant differences between cocaine addict-like and non-addict-like rats, suggesting that the behavioral differences observed are due to translational rather than transcriptional regulation. Another possibility to study the contributions of mGluR2 and mGluR3 in mediating addictive-like behavior is the use of knockout models. Because mGluR2 knockouts cannot be used in operant procedures due to motoric impairment, we only tested mGluR3 knockouts. These mice did not differ from controls in reinstatement, suggesting that mGluR2 receptors are critical in mediating addictive-like behavior.

A key deficit in alcohol dependence is disrupted prefrontal function leading to excessive alcohol seeking, but the molecular events underlying the emergence of addictive responses remain unknown. Here we show by convergent transcriptome analysis that the pyramidal neurons of the infralimbic cortex are particularly vulnerable for the long-term effects of chronic intermittent ethanol intoxication. These neurons exhibit a pronounced deficit in metabotropic glutamate receptor subtype 2 (mGluR(2)). Also, alcohol-dependent rats do not respond to mGluR(2/3) agonist treatment with reducing extracellular glutamate levels in the nucleus accumbens. Together these data imply a loss of autoreceptor feedback control. Alcohol-dependent rats show escalation of ethanol seeking, which was abolished by restoring mGluR(2) expression in the infralimbic cortex via viral-mediated gene transfer. Human anterior cingulate cortex from alcoholic patients shows a significant reduction in mGluR(2) transcripts compared to control subjects, suggesting that mGluR(2) loss in the rodent and human corticoaccumbal neurocircuitry may be a major consequence of alcohol dependence and a key pathophysiological mechanism mediating increased propensity to relapse. Normalization of mGluR(2) function within this brain circuit may be of therapeutic value.

The identification and functional understanding of the neurocircuitry that mediates alcohol and drug effects that are relevant for the development of addictive behavior is a fundamental challenge in addiction research. Here we introduce an assumption-free construction of a neurocircuitry that mediates acute and chronic drug effects on neurotransmitter dynamics that is solely based on rodent neuroanatomy. Two types of data were considered for constructing the neurocircuitry: (1) information on the cytoarchitecture and neurochemical connectivity of each brain region of interest obtained from different neuroanatomical techniques; (2) information on the functional relevance of each region of interest with respect to alcohol and drug effects. We used mathematical data mining and hierarchical clustering methods to achieve the highest standards in the preprocessing of these data. Using this approach, a dynamical network of high molecular and spatial resolution containing 19 brain regions and seven neurotransmitter systems was obtained. Further graph theoretical analysis suggests that the neurocircuitry is connected and cannot be separated into further components. Our analysis also reveals the existence of a principal core subcircuit comprised of nine brain regions: the prefrontal cortex, insular cortex, nucleus accumbens, hypothalamus, amygdala, thalamus, substantia nigra, ventral tegmental area and raphe nuclei. Finally, by means of algebraic criteria for synchronizability of the neurocircuitry, the suitability for in silico modeling of acute and chronic drug effects is indicated. Indeed, we introduced as an example a dynamical system for modeling the effects of acute ethanol administration in rats and obtained an increase in dopamine release in the nucleus accumbens-a hallmark of drug reinforcement-to an extent similar to that seen in numerous microdialysis studies. We conclude that the present neurocircuitry provides a structural and dynamical framework for large-scale mathematical models and will help to predict chronic drug effects on brain function., (© 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction.)

Background: In alcoholism, excessive glutamatergic neurotransmission has long been implicated in the acute withdrawal syndrome and as a key signal for dependence-related neuroplasticity. Our understanding of this pathophysiological mechanism originates largely from animal studies, but human data are needed for translation into successful medication development., Methods: We measured brain glutamate levels during detoxification in alcohol-dependent patients (n = 47) and in healthy control subjects (n = 57) as well as in a rat model of alcoholism by state-of-the-art ¹H-magnetic magnetic resonance spectroscopy at 3 and 9.4 T, respectively., Results: We found significantly increased glutamate levels during acute alcohol withdrawal in corresponding prefrontocortical regions of treatment-seeking alcoholic patients and alcohol-dependent rats versus respective control subjects. The augmented spectroscopic glutamate signal is likely related to increased glutamatergic neurotransmission because, enabled by the high field strength of the animal scanner, we detected a profoundly elevated glutamate/glutamine ratio in alcohol-dependent rats during acute withdrawal. All dependence-induced metabolic alterations normalize within a few weeks of abstinence in both humans and rats., Conclusions: Our data provide first-time direct support from humans for the glutamate hypothesis of alcoholism, demonstrate the comparability of human and animal magnetic resonance spectroscopy responses, and identify the glutamate/glutamine ratio as potential biomarker for monitoring disease progression., (Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Amygdaloid dopamine D(2) receptors play an important role in the modulation of fear/anxiety. Their topographical distribution within the amygdala is however unclear, and their role in unconditioned fear/anxiety remains largely unknown. The aim of this paper was to study the intra-amygdaloid distribution of D(2) receptors and to ascertain their role in unconditioned anxiety. Chemical anatomical studies in the rat, using D(2) and D(3)in situ hybridization, quantitative receptor autoradiography with either [(3)H]raclopride or [(125)I]sulpiride, and D(2)-like immunocytochemistry showed that the highest density of dopamine D(2) receptors is present in the central amygdaloid nucleus, particularly within its latero-capsular division, in which a D(2) but not a D(3) mRNA signal was observed. However, although at considerably reduced densities dopamine D(2) receptors were also found in other locations within the amygdala, including the basolateral nucleus. Behaviorally, the infusion of raclopride (0.75-4 μg/side) in the area of the central amygdaloid nucleus resulted at low doses in the appearance of anxiogenic-like effects in the Shock-Probe Burying test, whereas no effects of raclopride treatment were found at any dose in the Elevated Plus-Maze and the Open-Field test. Our results indicate that amygdaloid dopamine D(2)-like receptors have a topographically differentiated distribution within the rat amygdala, the major location being in the central amygdaloid nucleus. D(2)-like receptors play a role in the modulation of anxiety responses involving a potential differential function of D(2)-like receptors in the central amygdaloid nucleus versus the basolateral amygdaloid nucleus., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)