Andrey E. Ryabinin - US grants

The mechanisms by which alcohol exerts deleterious effects on human memory are still poorly understood. Recent data indicate that alcohol blocks experience-dependent gene activation in the hippocampus, a brain structure which plays a critical role in the acquisition of memory. The goal of the present proposal is to test whether alcohol suppresses memory because of its selective inhibition of hippocampal activity and to investigate the neurochemical and pharmacological mechanisms of this inhibition. The first specific aim of the proposed research is to investigate the effects of acute alcohol intoxication on hippocampus-dependent and -independent learning and experience-induced neural activity. A series of behavioral studies using the contextual conditioning paradigm in rats will determine the selectivity of suppressive effects of ethanol on hippocampal functions and dissect the sensory specificity of these effects of alcohol. Experiments using expression of immediate early genes (IEGs) as an activity-dependent neuronal maker will address whether alcohol~s effects on hippocampal activity, determined by behavioral means, are accompanied by parallel changes in hippocampal gene expression. The second specific aim of this study is to investigate the neurochemical mechanisms leading to alcohol's suppression of hippocampus-dependent learning and neural activity. Experiments with pharmacological agents will determine whether GABAA receptor agonist and antagonists can modulate alcohol effects on hippocampus-dependent forms of fear conditioning and experience- induced hippocampal activity. Double-labeling studies will establish the neurotransmitter nature of hippocampal neurons in which alcohol changes experience-induced IEG expression. Western blotting and immunohistochemical analysis will be performed to assess what second messenger system mediates alcohol~s effects on hippocampal IEG expression and memory. Finally, the DNA binding activity of transcription factor complexes from hippocampus after learning and alcohol intoxication will be investigated in a series of band-ship experiments. Taken together, these studies will provide valuable insight at several levels of analysis of alcohol~s action on cognitive functions and eventually allow to start understanding of the mechanisms involved in alcohol-induced amnesia.

[unreadable] DESCRIPTION (provided by applicant): The mechanisms regulating alcohol consumption in large part are not well understood. A number of research groups have analyzed expression of inducible transcription factors (ITFs) to identify alcohol-sensitive brain regions. Early studies observed changes in expression of ITF c-Fos in a substantial number of brain regions after involuntary alcohol administration in rodents. In contrast, voluntary alcohol self-administration in rodents leads to c-Fos expression in a much smaller number of brain regions. Importantly, in these paradigms robust induction of c-Fos occurs only in the Edinger-Westphal nucleus (EW). EW is a compact brain structure involved in oculomotor adaptation, nociception, regulation of metabolism, feeding, body temperature and anxiety. This nucleus has gained attention as the primary source of neuropeptide urocortin (Ucn). Recent studies show that inbred strains of mice known for differences in alcohol consumption, and mice genetically selected to differ in measures of alcohol reward differ dramatically in the number of Ucn cells in EW. We hypothesize that the brain Ucn system contributes to regulation of alcohol consumption, and that this regulation occurs via EW projection to the lateral septum (LS). To test this hypothesis four Specific Aims are proposed. [unreadable] 1. To confirm relation between level of Ucn in EW and alcohol consumption in a genetically heterogeneous population of mice and in congenic mice carrying chromosomal loci regulating alcohol preference. [unreadable] 2. To investigate the effects of EW lesions on alcohol consumption in mice. [unreadable] 3. To investigate the role of Ucn projections to LS in alcohol consumption using retrograde neuroanatomical tracing and microinjection of Ucn and its antagonists into LS. [unreadable] 4. To investigate the effects of ethanol exposure on the level and activity of the CRH-R2 receptors in LS, and in the level of Ucn mRNA in EW (using receptor autoradiography and in situ hybridization). [unreadable] The long-term goal of these studies is to understand the mechanisms regulating alcohol consumption. Such knowledge is important for development of pharmacotherapy of alcoholism [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This proposal seeks to become a part of the INIA consortium "Neurobiologial Basis of Excessive Drinking", and focuses of the neuropeptide Urocortin 1 (Ucn1). Ucn1 is the most effective endogenous ligand of both corticotropin releasing factor (CRF) receptors CRF1 and CRF2. The main source of Ucn1 in the brain is the non-preganglionic Edinger-Westphal nucleus (npEW). One of the main projection areas of npEW is the lateral septum (LS). Recent evidence indicates that the Ucn1 system is extremely sensitive to alcohol, that differences in this system predispose animals to differences in alcohol consumption, and that manipulations of this system regulate alcohol intake. Based on this evidence we hypothesize that differences in Ucn1 activity are important determinants of excessive alcohol intake. In this project we propose to apply collaborative efforts to investigate three specific aims: (1) To identify genes showing consistently different expression in npEWand LS between selectively-bred high and low alcohol consuming animals using microarray technology. Following animal models will be explored: mice selectively bred for excessive drinking in the dark, mice selectively bred for excessive drinking in the scheduled fluid access procedure, mice selectively bred using the 2-bottle choice procedure, rats selectively bred using the 2-bottle choice procedure, and their respective control lines. Differences in gene expression will be confirmed using immunohistochemistry, in situ hybridization and quantitative RTPCR. (2) To test alcohol consumption in Ucn1 knockout mice using three behavioral models: DID - excessive drinking in the dark; SHAG - excessive drinking due to scheduled access; and the standard 2-bottle drinking procedure. We will also use microarray technology to investigate whether Ucn1 knockout mice developed compensations in genes identified in Specific Aim 1. (3) To test whether genes identified in animal models of excessive alcohol consumption in Specific Aim 1 and Specific Aim 2 are expressed in npEW and LS of human post-mortem brains, and whether they are differentially expressed between alcoholic subjects and controls. Human homologues of the identified genes will be tested by quantitative RT-PCR. Taken together, these studies will provide a thorough comprehensive analysis of the Ucn1 neurocircuit and its involvement in excessive alcohol consumption, and could provide groundwork for development of new approaches for Oregon Health treatments of alcoholism and alcohol abuse disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alcohol consumption and social behaviors have a complex relationship. Some view alcoholic beverages as 'social lubricants". On the other hand, alcohol abuse and alcoholism lead to severe social and marital problems, including neglect, abuse and aggression. Alcohol consumption is often regarded as a way to overcome sadness following loss of friend or spouse, and severe alcohol abuse often follows divorce or death of friend. Could the co-occurrence of alcohol and social problems be not a coincidence, but have an underlying biological mechanism? This application proposes to use a novel rodent model to address this question. More specifically, we hypothesize that neurochemical mechanisms regulating social behaviors and alcohol intake overlap and involve specific receptors to vasopressin and corticotropin-relasing factor (CRF). The link between social behavior and alcohol abuse has been difficult to address using animal models because most laboratory rodents don't display strong affiliative behaviors and don't show high alcohol drinking. However, the situation has changed with increased research on prairie and pine voles, microtine rodents that do display strong affiliative behaviors. Intriguingly, our pilot studies indicate that prairie voles show high alcohol consumption in a two-bottle preference test. Being the first identified genetically heterogeneous non-selectively bred social rodent model of subjects with high alcohol consumption, the prairie voles offer a unique opportunity to address the mechanisms of interactions between social and biological mechanisms leading to high alcohol intake. This exploratory grant proposes to begin addressing these mechanisms. In particular, we propose to address social, developmental, genetic and molecular mechanisms leading to high alcohol intake in four specific aims: 1) To investigate the extent of correlation between social affiliation and alcohol drinking in voles;2) To investigate whether pair-housed and isolate-housed peri-adolescent and adult prairie voles consume different amounts of alcohol;3) To investigate whether the correlation between high alcohol intake and social affiliation is genetically determined;4) To investigate whether vasopressin V1a and CRF2 receptors contribute to genetic predisposition to high alcohol drinking and measures of social affiliation. Understanding mechanisms leading to high alcohol consumption in this new rodent model could lay ground for innovative approaches to treatment of alcoholism and alcohol abuse-related disorders taking into account the social-, age- and genotype-specific particularities of an individual patient.

DESCRIPTION (provided by applicant): Methamphetamine (METH) is an addictive psychostimulant with extremely high relapse rates. Effective pharmacotherapies to treat METH addiction, and in particular to counter relapse have not yet been developed. Research in animal models implicates the corticotropin releasing factor (CRF) peptide system in the mechanisms of extinction and reinstatement of psychostimulant self- administration (SA). These studies suggest CRF receptors could be an important target for therapeutic development. The CRF peptide system is complex, consisting of two main types of receptors, CRF1 and CRF2, and four endogenous ligands, CRF, urocortin 1 (Ucn1), urocortin 2 (Ucn2) and urocortin 3 (Ucn3). Since many brain regions are innervated by several of these endogenous ligands and often contain both receptors, the contribution of individual components of the CRF system to extinction and relapse is difficult to resolve using pharmacological approaches. Recently developed knockout (KO) mice, deficient in components of the CRF system, provide a novel approach that can complement pharmacological studies and clarify the role of components of the CRF system in behaviors such as reinstatement of drug-seeking. Here we propose using CRF1KO, CRF2 KO and Ucn1 KO to study the role of components of the CRF system in extinction from, and reinstatement of SA of METH. We hypothesize that different components of the CRF system will be differentially involved in drug- versus stress-induced reinstatement of METH SA. The project will include 3 Specific Aims: (1) to compare rates of METH SA in CRF1, CRF2 and Ucn1 KO mice using operant intravenous procedures. We hypothesize that CRF1 KO will show slightly slower acquisition rates of METH SA than other genotypes, but that all three genotypes will ultimately reach a similar level of METH SA. (2) To compare rates of extinction from METH SA in CRF1, CRF2 and Ucn1 KO mice. We hypothesize that CRF2 KO mice will show faster rates of extinction from METH SA compared to other genotypes. (3) To compare reinstatement of METH SA in CRF1, CRF2 and Ucn1 KO mice following a priming dose of METH or exposure to stress (metabolic and physical). We hypothesize that drug-induced reinstatement will be attenuated in CRF1 KO, but not in CRF2 or Ucn1 KO mice, and that stress-induced reinstatement will be attenuated in CRF2 KO mice suggesting that it involves endogenous urocortins. Analysis of stress- induced reinstatement in Ucn1 KO mice will further delineate whether this behavior is mediated by Ucn1 versus Ucn2 or Ucn3 peptides. PUBLIC HEALTH RELEVANCE: Methamphetamine (METH) is an addictive psychostimulant with extremely high relapse rates. Effective pharmacotherapies to treat METH addiction, and in particular to counter relapse, have not yet been developed. This research will use an animal model of METH self-administration to examine the involvement of the corticotropin releasing factor (CRF) peptide system in the mechanisms of extinction and reinstatement of METH-seeking behavior. These studies will determine if CRF receptors could be an important target for therapeutic development.

Theperioculomotor area (pili) is the main source of neuropeptide Urocortin 1 (Ucn1 lin the brain. Ucn1 is an endogenous peptide related to the corticotropin-releasing factor (CRF) that has higher affinity to CRF receptors than CRF itself. In addition to the Ucn1-containing cells of pili (plllu), theplll area also contains a population of dopaminergic neurons (pillda). Experiments perfornied in the previous years of this R01 have strongly confirmed the involvement of pili neurons in the regulation of alcohol consumption. Definitive evidence for this involvement accumulated across four lines of research: 1) the piliu neurons are preferentially activated following alcohol self-administration in mice and rats;2) Ucn1 immunoreactivity in piliu is positively correlated with alcohol intake in selectively-bred mouse and rat lines;3) electrolytic lesions of pili selectively block alcohol preference in C57BLl6J mice;4) microinjection of Ucn1 into the lateral septum, one of the target areas of plllu, selectively attenuates alcohol intake. Taken together these studies robustly implicate the pili neurons in the regulation of alcohol consumption. This application proposes to extend these studies to understand the mechanisms of this regulation. We hypothesize that pllldapromotes alcohol consumption, while plllu decreases alcohol consumption through their respective central projections. The main goal of this study is to address our hypothesis by characterizing the projectioTIs from pilida and plllu, and by testing the role of pilida in alcohol intake. To achieve thisgoal we prOpose three specific aims: 1) To characterize the distribution of prOjections from the piliu and pilida area of inbred C57BLl6J (C57) mice using neuroanatdrnical tracing approaches;2) To test whether the effects of pili lesions on alcohol intake are independent of the functions of Ucn1 using Ucn1 knockout mice;3) To test the importance of pilida in alcohol consumption using intracranial injections of dopamine agonists into pili of C57 mice. These studies will provide novel important information indicating how these recently identified neurons robustly regulate alcohol intake.

DESCRIPTION (provided by applicant): Recent studies show that ghrelin antagonists acting on growth hormone secretagogue receptor (GHSR) robustly attenuate excessive alcohol self-administration and associated alcohol reward. Findings generated by US indicate that the ghrelin antagonist D-Lys3-GHRP-6 (DLys) strongly and preferentially decreases alcohol drinking in the mouse drinking-in-the-dark (DID) model of binge-like excessive alcohol consumption, and that this decrease is accompanied by selective suppression of c-Fos expression in the centrally-projecting Edinger-Westphal nucleus (EWcp). The EWcp is the main brain source of the neuropeptide urocortin 1 (Ucn1, a highly potent endogenous ligand of corticotropin releasing factor receptors) and has been shown to be highly sensitive to ethanol and be involved in regulation of alcohol intake. We hypothesize that ghrelin antagonists can be used to decrease alcohol intake across different animal models and that GHSR can serve as an important target for development of pharmacotherapy of excessive alcohol consumption. The goal of this proposal is to test this hypothesis and identify behavioral, anatomical and molecular mechanisms contributing to this decrease. This goal will be addressed in the three specific aims. In specific aim 1 we will test the ability of different doses of GHSR antagonists to decrease alcohol intake across different phases and different animal models of excessive alcohol consumption. In specific aim 2 we will investigate the anatomical substrates of ghrelin's effects on excessive alcohol drinking using knock-in GHSR null mutant mice and intracranial injections into specific brain regions. In specific aim 3 we will investigate the molecular mechanisms of ghrelin's involvement in regulation of excessive alcohol intake by analyzing levels of Ghsr mRNA after excessive alcohol intake in mice, rats and non-human primates and by testing whether signal transduction mechanisms induced by ethanol in EWcp are attenuated by administration of GHSR antagonists

Alcoholism is a health disorder characterized by a progression from experimentation, to excessive intake, and ultimately alcohol dependence accompanied by compulsive alcohol consumption. The relationship between alcohol consumption and dependence is complex. For example, it is assumed that increased alcohol intake associated with alcohol dependence leads to increased symptoms of physical withdrawal. However, studies performed in the PARC showed that natural genetic variants (e.g. inbred mouse strains) with increased susceptibility to high alcohol withdrawal convulsions tend to avoid alcohol consumption. More recent PARC studies found a strong positive genetic correlation between measures of behavioral disinhibition in the Go/No-go task with measures of alcohol withdrawal severity. This component uses a candidate gene approach to test the contribution of the corticotropin releasing factor (CRF) system to these relationships. The CRF system is known to contribute to mechanisms surrounding alcohol dependence. Our preliminary studies suggest that the CRF system is involved in regulation of alcohol withdrawal and behavioral inhibition. The CRF system contains four peptides: CRF, urocortin (Ucn)1, Ucn2 and Ucn3, two types of CRF receptors, and the CRF-binding protein. The role of genes encoding these peptides and proteins in ethanol withdrawal or in impulsive behaviors has not been investigated. The four aims of this component propose to bridge this gap by: 1) investigating withdrawal-induced convulsions after acute and chronic ethanol in CRF, Ucnl, CRFI receptor, CRF2 receptor KO mice and their wildtype (WT) llttermates, 2) investigating behavioral inhibition in the Go/No-go task in CRF, Ucnl, CRFI receptor, CRF2 receptor KO mice, and their WT llttermates, 3) identifying gene networks contributing to regulation of behavioral inhibition and alcohol withdrawal by expression microarray analysis in CRF, Ucnl, CRFI receptor, CRF2 receptor KO mice and their WT controls, 4) identifying polymorphisms in genes encoding CRF, Ucnl, Ucn2, Ucn3, CRFI, CRF2 and CRF-BP in inbred strains of mice known to have differences in signs of ethanol-induced withdrawal and measures of disinhibition in the Go/No-go task, and to test associations of these polymorphisms with these and other ethanol-related phenotypes.

DESCRIPTION (provided by applicant): Social affiliations play an important role in the onset and relapse of alcoholism and heavy drinking, yet this aspect of alcoholism and alcohol abuse has not been successfully modeled in rodents. Recently we developed the use of prairie voles (Microtus ochrogaster) to study negative (facilitating) and positive (inhibitory) social influences on alcohol consumption. Specifically, we demonstrated alcohol preference and heavy ethanol intake in this species, and showed that pair-housed prairie voles influence each other's drinking. When voles are introduced to alcohol in pairs they exhibit higher alcohol preference than when they are introduced to alcohol being single-housed. In these pairs, one vole influences (i.e., increases) alcohol consumption of its partner - modeling facilitating influences on alcohol drinking. In contrast, during pairing of high- and low-drinking voles that have previously experienced alcohol when they were single- housed, the vole with higher basal intake tends to decrease its drinking thereby matching its partner - modeling inhibitory social influences. Thus, the prairie vole could serve as the first rodent model of the effects of specific inter-personal affiliations on high alcohol drinking in both directions. We hypothesize that prairie voles influence their partner's rate of alcohol consumption via acoustic communication as do humans and that this influence is regulated by dominant/submissive relations, the opiate and the vasopressin (AVP) systems. We propose to test this hypothesis through following four Specific Aims. Specific Aim 1: To test whether the social influences on alcohol drinking occur by synchronizing alcohol drinking and are influenced by dominant-submissive interactions. Specific Aim 2: To test the whether USVs contribute to coordinated drinking in voles further increasing the validity of our model. Specific Aim 3: To test whether the efficacy of an established pharmacotherapy of alcoholism targeting the endogenous opiate system is modulated by social influences. Specific Aim 4: To test whether the AVP system known to regulate social affiliations in prairie voles contributes to regulation of coordinated drinking. Taken together this work will for the first time reveal information on the biological mechanisms regulating social influence on excessive alcohol drinking in a rodent model. PUBLIC HEALTH RELEVANCE: Social affiliations play a substantial role in the onset and relapse of alcoholism and heavy drinking, yet the mechanisms of this aspect of alcoholism are poorly understood. In this application we will use the prairie vole as a rodent model to study the mechanisms underlying effects of specific social affiliations on excessive alcohol use. The success of these studies may provide new insights into strategies for therapeutic treatments of alcoholism.

Project Summary / Abstract The overarching aim of this pre- and post-doctoral training program is to develop specialists who are able to conduct basic research at levels ranging from the molecular to the cognitive/clinical, on the biological mechanisms underlying the etiology, treatment and prevention of alcohol (ethanol) use disorders. Twenty-two members of the graduate faculty of the Oregon Health & Science University (OHSU) serve as preceptors for predoctoral students and postdoctoral research fellows in two graduate programs at OHSU?Behavioral Neuroscience, and the Neuroscience Graduate Program. Major research interests represent five areas of common interest: (1) genetic bases for ethanol responses and risk, (2) learned and unlearned determinants of ethanol reward, (3) neurobiological bases for the rewarding, aversive and neuropathological effects of ethanol, (4) neuroadaptive mechanisms associated with repeated and/or developmental ethanol exposure, ethanol dependence and sensitization, and (5) effects of ethanol on memory and cognition. In addition, comparison to the effects of other drugs of abuse is of significant interest. Technical strategies reflect four levels of analysis: I. Behavioral pharmacological/pharmacogenetic, II. Neurochemical/neurophysiological/ neuropharmacological, III. Cellular/molecular biological and IV. Cognitive neuroscience/social, including human/clinical level. Coordinated research efforts within the Portland Alcohol Research Center (PARC) and the Integrative Neuroscience Initiative on Alcoholism (INIA) have strengthened training by unifying investigators and creating multidimensional research projects. Training includes firm curricular grounding in the basic sciences, specific pharmacological training in ethanol and other abused drugs, and extensive and continuous participation in research. Six predoctoral trainees per year, beginning with 0-2 years of graduate experience, will be supported by the training grant for 2-3 years, and then by individual National Research Service Awards or their mentors' resources. Three postdoctoral trainees per year with 1-3 years of postdoctoral experience will be supported by the training grant for 2 years. We have a well-developed plan for improving the diversity of our trainees and all trainees are expected to complete an initial intensive course in the Responsible Conduct of Research, as well as continuing education in this area. Ample opportunities exist for our trainees to be involved in public education and outreach.

Project summary Reinforcing effects of alcohol are likely to be triggered by low doses of ethanol consumed once a subject is getting acquainted with alcoholic beverages. Moreover, it might be difficult to decipher mechanisms involving effects of high doses of alcohol without understanding mechanisms underlying effects of low doses. We have initiated mapping activity of immediate early genes in rodent brains almost 20 years ago. These initial studies found a number of brain regions in which a low dose of alcohol either induced c-Fos expression or suppressed basal or novelty- and stress-induced c-Fos expression. Subsequently, we also mapped brain regions in which c-Fos was induced after mice voluntarily consumed low doses of alcohol. However, these early studies were hampered by several problems with methodology at that time. These problems included: 1) The studies were performed during the bright phase of the circadian cycle, where activity and basal c-Fos expression is low, making it difficult to detect inhibition of neural activity, 2) Once staining was performed it was impossible to reuse the stained slices limiting double-labeling approaches and ability to identify neurochemical nature of subpopulations of neurons, 3) The peak time of c-Fos protein expression is relatively broad, therefore, c-Fos activation reflected not only effects of alcohol, but also presumably alcohol- independent behavior prior or after actions of alcohol. Recently developed technologies and approaches allow overcoming these technical difficulties. This exploratory grant proposes to use two comprehensive technologies to identify neuronal populations regulated by voluntary self-administration of low doses of alcohol in mice. This goal will be achieved in two Specific Aims. In Aim 1, we will create 3-dimentional maps of changes in activity of neurons following consumption of low doses of alcohol using the CLARITY technique. In Aim 2, we will create time-sensitive maps of changes in activity of neurons following consumption of low doses of alcohol using fluorescence-in situ-hybridization.

Project Summary Over a hundred million of Americans suffer from chronic pain and over ten million of Americans suffer from alcohol abuse or dependence. There is a bidirectional relationship between chronic pain and alcohol dependence. Thus, alcohol dependence is a major predictor of severity of chronic pain, and people with chronic pain conditions are more likely to use alcohol for pain relief. Unfortunately, a mechanistic understanding of alcohol-related pain sensitivity is lacking. Our studies have identified increased pain sensitivity in mice during withdrawal from voluntary alcohol self-administration. Consumption of alcohol in these mice reversed mechanical hypersensitivity produced by alcohol withdrawal. The increased pain sensitivity in mice undergoing withdrawal is consistent with increased pain in alcohol-dependent patients. In addition, we found increased pain sensitivity in control ?bystander? mice housed in the same room as mice undergoing alcohol withdrawal. The social transfer of hyperalgesia from mice undergoing withdrawal to the bystander mice involved olfactory cues. Such social transfer of hyperalgesia could affect co-dependent family members of alcoholic patients. Immunohistochemical analysis revealed differential activation of dorsomedial hypothalamus, anterior cingulate and anterior insular cortex in these animals. We hypothesize that the identified brain regions are differentially involved in alcohol withdrawal-induced hyperalgesia and socially-transferred hyperalgesia. The goal of this proposal is to address this hypothesis and further characterize the phenomena of alcohol withdrawal- and social transfer-induced hyperalgesia. This goal will be achieved in three Specific Aims: Aim 1 will further characterize the phenomenon of hyperalgesia in alcohol withdrawing and bystander mice by examining whether the observed thermal hyperalgesia is exaggerated in female bystander mice, involves negative affective states, anxiety or stress responses, or coexists with depression-like behaviors. Aim 2 will test whether alcohol-induced activation of neuronal populations within dorsomedial hypothalamus is necessary and sufficient for regulation of pain sensitivity and alcohol drinking behavior in mice. Aim 3 will test whether alcohol withdrawal and social transfer-induced activation of neurons of anterior cingulate and/or insula are necessary and sufficient for regulation of pain sensitivity.

Humans often consume alcoholic beverages to promote social interactions. On the other hand, alcohol abuse can have devastating effects on long-term social attachments. The mechanisms alcohol's effects on social relationships have been difficult to address. The situation changed with studies showing that many features of social bonding can be modeled using socially monogamous prairie voles (Microtus ochrogaster). Mechanisms regulating pair bonding in voles have later been shown to contribute to human social bonding, indicating strong construct validity of this animal model. We have initiated studies analyzing effects of social environment on alcohol consumption and, more recently, identified striking sex-dependent effects of voluntary alcohol consumption on the formation of male-female bonds in prairie voles. Specifically, voluntary alcohol consumption inhibited formation of partner preference in male animals, but facilitated partner preference in females. These findings indicated that alcohol acts through biological mechanisms to produce specific effects on adult social attachments. In parallel we found that alcohol drinking during cohabitation also produces sex-specific effects on c-Fos expression in the centrally-projecting Edinger-Westphal nucleus (EWcp). This application proposes to test whether EWcp contributes to alcohol's effects on pair bonding and further investigate alcohol's effects on pair bonding in three Specific Aims (SAs). In SA1 we will use CLARITY-based and standard immunohistochemistry to measure levels of several peptides and c-Fos across different brain regions during cohabitation in the presence or absence of alcohol. In SA2 we will test the causal role of EWcp activation in alcohol's effects on partner preference using the Designer Receptor(s) Exclusively Activated by Designer Drug(s) approach. Subsequent studies will test the roles of specific subpopulations of EWcp or brain regions identified in SA1. Finally, while our previous studies found effects of the first day of voluntary alcohol drinking on formation of pair-bonds, in SA3 we will test alcohol's effects on pair bonding following prolonged alcohol use and alcohol's effects on already established pair bonds.

Project Summary Post-traumatic stress disorder (PTSD) is twice as prevalent in females as in males, with a proportion of individuals also developing an alcohol use disorder (AUD). Using predator odor stress (PS) as an animal model of PTSD, we determined that two PS exposures significantly increased anxiety-related behavior and neuronal activation in the hippocampus (HC) of male and female C57BL/6J (B6) mice. Notably, intermittent PS significantly increased alcohol (ethanol) intake by 60% (males) and 71% (females), with heterogeneity in the response. Further, ?sensitivity? to PS-enhanced ethanol intake conferred significantly greater corticotropin releasing factor receptor-1 (CRFR1) protein levels in female versus male HC, consistent with evidence for sex differences in CRFR1 signaling following stress. The proposed studies build on the above evidence by testing the hypothesis that comorbidity of PTSD and AUD is due to increased CRFR1 expression in HC neurons projecting to mPFC and that sex differences in CRFR1 induction by PS contribute to this comorbidity. Aim 1 will determine whether sex differences exist in the association between PS-enhanced ethanol drinking and alteration in anxiety, heart rate (HR), and/or compulsive ethanol drinking in B6 mice. We predict that PS-enhanced drinking in ?sensitive? mice will be associated with an increase in anxiety, HR, and compulsive drinking and that there will be sex differences in the pattern of changes. Aim 2 will map changes in CRFR1 expression and neuronal activation by PS and by PS-enhanced drinking in crfr1-gfp mice. We predict that there will be sex differences in brain regional CRFR1-colabelled activity patterns in response to intermittent PS and in the relationship with ethanol intake. Aim 3 will manipulate the activity of CRFR1- expressing neurons using chemogenetic or pharmacologic approaches and determine the impact on PS- enhanced drinking. Two studies will use Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in crfr1-cre mice to test the necessity and sufficiency of CRFR1 in ventral CA1, with inhibitory (Gi) and excitatory (Gq) DREADDs, respectively. We predict that preventing PS-induced activation of ventral CA1 (Gi DREADD) will block PS-enhanced drinking only in ?sensitive? mice, whereas activating the ventral CA1 (Gq DREADD) will enhance ethanol intake in mice drinking ethanol without intermittent PS. A complementary study will determine whether systemic administration of a CRFR1 antagonist will reduce PS-enhanced drinking intake in B6 mice, with the prediction that the antagonist will be most effective in ?sensitive? mice. Aim 4 will determine whether manipulation of the projection from ventral CA1 to mPFC is important for PS-enhanced drinking in B6 mice, by injecting Gi DREADDs into ventral CA1 and clozapine-N-oxide into mPFC. We predict that preventing PS-induced activation of the ventral CA1 to mPFC projection will block PS-enhanced drinking. Collectively, the information will elucidate sex differences in mechanisms underlying sensitivity to PS- enhanced drinking that can be targeted for the treatment of PTSD-induced AUD.