Animal Models of Excessive Alcohol Consumption in Rodents.

The development of animal models that demonstrate excessive levels of alcohol consumption has played an important role in advancing our knowledge about neurobiological underpinnings and environmental circumstances that engender such maladaptive behavior. The use of these preclinical models has also provided valuable opportunities for discovering new and novel therapeutic targets that may be useful in the treatment of alcohol use disorder (AUD). While no single model can fully capture the complexities of AUD, the goal is to develop animal models that closely approximate characteristics of heavy alcohol drinking in humans to enhance their translational value and utility. A variety of experimental approaches have been employed to produce the desired phenotype of interest-robust and reliable excessive levels of alcohol drinking. Here we provide an updated review of five animal models that are commonly used. The models entail procedural manipulations of scheduled access to alcohol (time of day, duration, frequency), periods of time when access to alcohol is withheld, and history of alcohol exposure. Specially, the models involve (a) scheduled access to alcohol, (b) scheduled periods of alcohol deprivation, (c) scheduled intermittent access to alcohol, (d) scheduled-induced polydipsia, and (e) chronic alcohol (dependence) and withdrawal experience. Each of the animal models possesses unique experimental features that engender excessive levels of alcohol consumption. Both advantages and disadvantages of each model are described along with discussion of future work to be considered in developing more optimal models. Ultimately, the validity and utility of these models will lie in their ability to aid in the discovery of new and novel potential therapeutic targets as well as serve as a platform to evaluate treatment strategies that effectively reduce excessive levels of alcohol consumption associated with AUD.

Transcriptome changes in the nucleus of the solitary tract induced by repeated stress, alcohol dependence, or stress-induced drinking in dependent mice.

Stress increases alcohol consumption in dependent animals and contributes to the development of alcohol use disorder. The nucleus of the solitary tract (NTS) is a critical brainstem region for integrating and relaying central and peripheral signals to regulate stress responses, but it is not known if it plays a role in alcohol dependence- or in stress-induced escalations in alcohol drinking in dependent mice. Here, we used RNA-sequencing and bioinformatics analyses to study molecular adaptations in the NTS of C57BL/6J male mice that underwent an ethanol drinking procedure that uses exposure to chronic intermittent ethanol (CIE) vapor, forced swim stress (FSS), or both conditions (CIE + FSS). Transcriptome profiling was performed at three different times after the last vapor cycle (0-hr, 72-hr, and 168-hr) to identify changes in gene expression associated with different stages of ethanol intoxication and withdrawal. In the CIE and CIE + FSS groups at 0-hr, there was upregulation of genes enriched for cellular response to type I interferon (IFN) and type I IFN- and cytokine-mediated signaling pathways, while the FSS group showed upregulation of neuronal genes. IFN signaling was the top gene network positively correlated with ethanol consumption levels in the CIE and CIE + FSS groups. Results from different analyses (differential gene expression, weighted gene coexpression network analysis, and rank-rank hypergeometric overlap) indicated that activation of type I IFN signaling would be expected to increase ethanol consumption. The CIE and CIE + FSS groups also shared an immune signature in the NTS as has been demonstrated in other brain regions after chronic ethanol exposure. A temporal-based clustering analysis revealed a unique expression pattern in the CIE + FSS group that suggests the interaction of these two stressors produces adaptations in synaptic and glial functions that may drive stress-induced drinking.

Targeting muscarinic receptors for the treatment of alcohol use disorders: Opportunities and hurdles for clinical development.

Emerging evidence suggests muscarinic acetylcholine receptors represent novel targets to treat alcohol use disorder. In this review, we draw from literature across medicinal chemistry, molecular biology, addiction and learning/cognition fields to interrogate the proposition for muscarinic receptor ligands in treating various aspects of alcohol use disorder, including cognitive dysfunction, motivation to consume alcohol and relapse. In support of this proposition, we describe cholinergic dysfunction in the pathophysiology of alcohol use disorder at a network level, including alcohol-induced adaptations present in both human post-mortem brains and reverse-translated rodent models. Preclinical behavioural pharmacology implicates specific muscarinic receptors, in particular, M4 and M5 receptors, as potential therapeutic targets worthy of further interrogation. We detail how these receptors can be selectively targeted in vivo by the use of subtype-selective allosteric modulators, a strategy that overcomes the issue of targeting a highly conserved orthosteric site bound by acetylcholine. Finally, we highlight the intense pharma interest in allosteric modulators of muscarinic receptors for other indications that provide an opportunity for repurposing into the alcohol use disorder space and provide some currently unanswered questions as a roadmap for future investigation.

Alcohol Dependence Modifies Brain Networks Activated During Withdrawal and Reaccess: A c-Fos-Based Analysis in Mice.

BACKGROUND: High-level alcohol consumption causes neuroplastic changes in the brain that promote pathological drinking behavior. Some of these changes have been characterized in defined brain circuits and cell types, but unbiased approaches are needed to explore broader patterns of adaptations. METHODS: We used whole-brain c-Fos mapping and network analysis to assess patterns of neuronal activity during alcohol withdrawal and following reaccess in a well-characterized model of alcohol dependence. Mice underwent 4 cycles of chronic intermittent ethanol to increase voluntary alcohol consumption, and a subset underwent forced swim stress to further escalate consumption. Brains were collected either 24 hours (withdrawal) or immediately following a 1-hour period of alcohol reaccess. c-fos counts were obtained for 110 brain regions using iDISCO and ClearMap. Then, we classified mice as high or low drinkers and used graph theory to identify changes in network properties associated with high-drinking behavior. RESULTS: During withdrawal, chronic intermittent ethanol mice displayed widespread increased c-Fos expression relative to air-exposed mice, independent of forced swim stress. Reaccess drinking reversed this increase. Network modularity, a measure of segregation into communities, was increased in high-drinking mice after alcohol reaccess relative to withdrawal. The cortical amygdala showed increased cross-community coactivation during withdrawal in high-drinking mice, and cortical amygdala silencing in chronic intermittent ethanol mice reduced voluntary drinking. CONCLUSIONS: Alcohol withdrawal in dependent mice causes changes in brain network organization that are attenuated by reaccess drinking. Olfactory brain regions, including the cortical amygdala, drive some of these changes and may play an important but underappreciated role in alcohol dependence.

Oxytocin Reduces Sensitized Stress-Induced Alcohol Relapse in a Model of Posttraumatic Stress Disorder and Alcohol Use Disorder Comorbidity.

BACKGROUND: There is high comorbidity of posttraumatic stress disorder (PTSD) and alcohol use disorder with few effective treatment options. Animal models of PTSD have shown increases in alcohol drinking, but effects of stress history on subsequent vulnerability to alcohol relapse have not been examined. Here we present a mouse model of PTSD involving chronic multimodal stress exposure that resulted in long-lasting sensitization to stress-induced alcohol relapse, and this sensitized stress response was blocked by oxytocin (OT) administration. METHODS: Male and female mice trained to self-administer alcohol were exposed to predator odor (TMT) + yohimbine over 5 consecutive days or left undisturbed. After reestablishing stable alcohol responding/intake, mice were tested under extinction conditions, and then all mice were exposed to TMT or context cues previously associated with TMT before a reinstatement test session. Separate studies examined messenger RNA expression of Oxt and Oxtr in hypothalamus following chronic stress exposure. A final study examined the effects of systemic administration of OT on stress-induced alcohol relapse in mice with and without a history of chronic stress experience. RESULTS: Chronic stress exposure produced long-lasting sensitization to subsequent stress-induced alcohol relapse that also generalized to stress-related context cues and transcriptional changes in hypothalamic OT system. OT injected before the reinstatement test session completely blocked the sensitized stress-induced alcohol relapse effect. CONCLUSIONS: Collectively, these results provide support for the therapeutic potential of OT, along with highlighting the value of utilizing this model in evaluating other pharmacological interventions for treatment of PTSD/alcohol use disorder comorbidity.

Preclinical and clinical evidence for suppression of alcohol intake by apremilast.

Treatment options for alcohol use disorders (AUDs) have minimally advanced since 2004, while the annual deaths and economic toll have increased alarmingly. Phosphodiesterase type 4 (PDE4) is associated with alcohol and nicotine dependence. PDE4 inhibitors were identified as a potential AUD treatment using a bioinformatics approach. We prioritized a newer PDE4 inhibitor, apremilast, as ideal for repurposing (i.e., FDA approved for psoriasis, low incidence of adverse events, excellent safety profile) and tested it using multiple animal strains and models, as well as in a human phase IIa study. We found that apremilast reduced binge-like alcohol intake and behavioral measures of alcohol motivation in mouse models of genetic risk for drinking to intoxication. Apremilast also reduced excessive alcohol drinking in models of stress-facilitated drinking and alcohol dependence. Using site-directed drug infusions and electrophysiology, we uncovered that apremilast may act to lessen drinking in mice by increasing neural activity in the nucleus accumbens, a key brain region in the regulation of alcohol intake. Importantly, apremilast (90 mg/d) reduced excessive drinking in non-treatment-seeking individuals with AUD in a double-blind, placebo-controlled study. These results demonstrate that apremilast suppresses excessive alcohol drinking across the spectrum of AUD severity.

Agmatine reduces alcohol drinking and produces antinociceptive effects in rodent models of alcohol use disorder.

Alcohol use disorder (AUD) is a chronic, relapsing disorder characterized by an escalation of drinking and the emergence of negative affective states over time. Within this framework, alcohol may be used in excessive amounts to alleviate withdrawal-related symptoms, such as hyperalgesia. Future effective therapeutics for AUD may need to exhibit the ability to reduce drinking as well as to alleviate co-morbid conditions such as pain, and to take mechanistic sex differences into consideration. Agmatine is an endogenous neuromodulator that has been previously implicated in the regulation of reward and pain processing. In the current set of studies, we examined the ability of agmatine to reduce escalated ethanol drinking in complementary models of AUD where adult male and female mice and rats were made dependent via chronic, intermittent ethanol vapor exposure (CIE). We also examined the ability of agmatine to modify thermal and mechanical sensitivity in alcohol-dependent male and female rats. Agmatine reduced alcohol drinking in a dose-dependent fashion, with somewhat greater selectivity in alcohol-dependent female mice (versus non-dependent female mice), but equivalent efficacy across male mice and both groups of male and female rats. In mice and female rats, this efficacy did not extend to sucrose drinking, indicating some selectivity for ethanol reinforcement. Female rats made dependent on alcohol demonstrated significant hyperalgesia symptoms, and agmatine produced dose-dependent antinociceptive effects across both sexes. While additional mechanistic studies into agmatine are necessary, these findings support the broad-based efficacy of agmatine to treat co-morbid excessive drinking and pain symptoms in the context of AUD.

Alcohol dependence and the ventral hippocampal influence on alcohol drinking in male mice.

Examining neural circuits underlying persistent, heavy drinking provides insight into the neurobiological mechanisms driving alcohol use disorder. Facilitated by its connectivity with other parts of the brain such as the nucleus accumbens (NAc), the ventral hippocampus (vHC) supports many behaviors, including those related to reward seeking and addiction. These studies used a well-established mouse model of alcohol (ethanol) dependence. After surgery to infuse DREADD-expressing viruses (hM4Di, hM3Dq, or mCherry-only) into the vHC and position guide cannula above the NAc, male C57BL/6J mice were treated in the CIE drinking model that involved repeated cycles of chronic intermittent alcohol (CIE) vapor or air (CTL) exposure alternating with weekly test drinking cycles in which mice were offered alcohol (15% v/v) 2 h/day. Additionally, smaller groups of mice were evaluated for either cFos expression or glutamate release using microdialysis procedures. In CIE mice expressing inhibitory (hM4Di) DREADDs in the vHC, drinking increased as expected, but CNO (3 mg/kg intraperitoneally [i.p.]) given 30 min before testing did not alter alcohol intake. However, in CTL mice expressing hM4Di, CNO significantly increased alcohol drinking (∼30%; p < 0.05) to levels similar to the CIE mice. The vHC-NAc pathway was targeted by infusing CNO into the NAc (3 or 10 µM/side) 30 min before testing. CNO activation of the pathway in mice expressing excitatory (hM3Dq) DREADDs selectively reduced consumption in CIE mice back to CTL levels (∼35-45%; p < 0.05) without affecting CTL alcohol intake. Lastly, activating the vHC-NAc pathway increased cFos expression and evoked significant glutamate release from the vHC terminals in the NAc. These data indicate that reduced activity of the vHC increases alcohol consumption and that targeted, increased activity of the vHC-NAc pathway attenuates excessive drinking associated with alcohol dependence. Thus, these findings indicate that the vHC and its glutamatergic projections to the NAc are involved in excessive alcohol drinking.

TLR3 activation with poly I:C exacerbates escalated alcohol consumption in dependent male C57BL/6J mice.

Background: Activation of TLR3 receptors, which are sensitive to viral infection, has emerged as a possible mechanism that increases alcohol intake in rodents.Objectives: These studies examined whether a history of ethanol dependence exacerbated the increase in drinking driven by the TLR3 agonist poly I:C.Methods: Male C57BL/6J mice (>10 per group) were given access to ethanol (20% v/v) 2 hours a day following a history of home cage drinking or after having been rendered ethanol-dependent using a chronic intermittent ethanol (CIE) vapor model. After testing multiple doses, a 5 mg/kg repeated poly I:C challenge was used to probe the effects of repeated immune challenge, alone or in conjunction with repeated cycles of CIE, on voluntary drinking. An ethanol (12% v/v) operant self-administration model was used to test the effects of poly I:C on stress-induced reinstatement of ethanol seeking and consumption.Results: Poly I:C in naive animals resulted in transient, modest increases in ethanol intake in the home cage and in self-administration (p < 0.05). However, poly I:C challenge resulted in sensitized stress-induced ethanol consumption and evoked a strong and persistent escalation of drinking in mice with a history of dependence (p < 0.05 for both).Conclusion: Activation of viral immune defense may affect ethanol consumption in dependence and sensitivity to future stressors. As patients who suffer from alcohol use disorder are at a heightened risk for viral infection, this interaction could generate risk factors for exacerbating behaviors associated with Alcohol Use Disorders via an immune mechanism.

Assessing negative affect in mice during abstinence from alcohol drinking: Limitations and future challenges.

Alcohol use disorder (AUD) is frequently comorbid with mood disorders, and these co-occurring neuropsychiatric disorders contribute to the development and maintenance of alcohol dependence and relapse. In preclinical models, mice chronically exposed to alcohol display anxiety-like and depressive-like behaviors during acute withdrawal and protracted abstinence. However, in total, results from studies using voluntary alcohol-drinking paradigms show variable behavioral outcomes in assays measuring negative affective behaviors. Thus, the main objective of this review is to summarize the literature on the variability of negative affective behaviors in mice after chronic alcohol exposure. We compare the behavioral phenotypes that emerge during abstinence across different exposure models, including models of alcohol and stress interactions. The complicated outcomes from these studies highlight the difficulties of assessing negative affective behaviors in mouse models designed for the study of AUD. We discuss new behavioral assays, comprehensive platforms, and unbiased machine-learning algorithms as promising approaches to better understand the interaction between alcohol and negative affect in mice. New data-driven approaches in the understanding of mouse behavior hold promise for improving the identification of mechanisms, cell subtypes, and neurocircuits that mediate negative affect. In turn, improving our understanding of the neurobehavioral basis of alcohol-associated negative affect will provide a platform to test hypotheses in mouse models that aim to improve the development of more effective strategies for treating individuals with AUD and co-occurring mood disorders.

Dynorphin/Kappa Opioid Receptor Activity Within the Extended Amygdala Contributes to Stress-Enhanced Alcohol Drinking in Mice.

BACKGROUND: While there is high comorbidity of stress-related disorders and alcohol use disorder, few effective treatments are available and elucidating underlying neurobiological mechanisms has been hampered by a general lack of reliable animal models. Here, we use a novel mouse model demonstrating robust and reproducible stress-enhanced alcohol drinking to examine the role of dynorphin/kappa opioid receptor (DYN/KOR) activity within the extended amygdala in mediating this stress-alcohol interaction. METHODS: Mice received repeated weekly cycles of chronic intermittent ethanol exposure alternating with weekly drinking sessions ± forced swim stress exposure. Pdyn messenger RNA expression was measured in the central amygdala (CeA), and DYN-expressing CeA neurons were then targeted for chemogenetic inhibition. Finally, a KOR antagonist was microinjected into the CeA or bed nucleus of the stria terminalis to examine the role of KOR signaling in promoting stress-enhanced drinking. RESULTS: Stress (forced swim stress) selectively increased alcohol drinking in mice with a history of chronic intermittent ethanol exposure, and this was accompanied by elevated Pdyn messenger RNA levels in the CeA. Targeted chemogenetic silencing of DYN-expressing CeA neurons blocked stress-enhanced drinking, and KOR antagonism in the CeA or bed nucleus of the stria terminalis significantly reduced stress-induced elevated alcohol consumption without altering moderate intake in control mice. CONCLUSIONS: Using a novel and robust model of stress-enhanced alcohol drinking, a significant role for DYN/KOR activity within extended amygdala circuitry in mediating this effect was demonstrated, thereby providing further evidence that the DYN/KOR system may be a valuable target in the development of more effective treatments for individuals presenting with comorbidity of stress-related disorders and alcohol use disorder.

The histone methyltransferase G9a mediates stress-regulated alcohol drinking.

The epigenetic enzyme G9a is a histone methyltransferase that dimethylates lysine 9 on histone H3 (H3K9me2), and in the adult nucleus accumbens (NAc), G9a regulates multiple behaviors associated with substance use disorder. We show here that chronic intermittent ethanol (CIE) exposure in male mice reduced both G9a and H3K9me2 levels in the adult NAc, but not dorsal striatum. Viral-mediated reduction of G9a in the NAc had no effects on baseline volitional ethanol drinking or escalated alcohol drinking produced by CIE exposure; however, NAc G9a was required for stress-regulated changes in ethanol drinking, including potentiated alcohol drinking produced by activation of the kappa-opioid receptor. In addition, we observed that chronic systemic administration of a G9a inhibitor, UNC0642, also blocked stress-potentiated alcohol drinking. Together, our findings suggest that chronic alcohol use, similar to other abused substances, produces a NAc-selective reduction in G9a levels that serves to limit stress-regulated alcohol drinking. Moreover, our findings suggest that pharmacological inhibition of G9a might provide a novel therapeutic approach to treat stress-induced alcohol drinking, which is a major trigger of relapse in individuals suffering from AUD.

Activation of hypothalamic oxytocin neurons reduces binge-like alcohol drinking through signaling at central oxytocin receptors.

Preclinical and clinical evidence suggests that exogenous administration of oxytocin (OT) may hold promise as a therapeutic strategy for reducing heavy alcohol drinking. However, it remains unknown whether these effects are mediated by stimulation of endogenous sources of OT and signaling at oxytocin receptors (OTR) in brain or in the periphery. To address this question, we employed a targeted chemogenetic approach to examine whether selective activation of OT-containing neurons in the paraventricular nucleus of the hypothalamus (PVN) alters alcohol consumption in a binge-like drinking ("Drinking-in-the-Dark"; DID) model. Adult male Oxt-IRES-Cre mice received bilateral infusion of a Cre-dependent virus containing an excitatory DREADD (AAV8-hSyn-DIO-hM3Dq-mCherry) or control virus (AAV8-hSyn-DIO-mCherry) into the PVN. Chemogenetic activation of PVNOT+ neurons following clozapine-N-oxide injection reduced binge-like alcohol drinking in a similar manner as systemic administration of the neuropeptide. Pretreatment with a brain-penetrant OTR antagonist (L-368,899) reversed this effect while systemic administration of a peripherally restricted OTR antagonist (Atosiban) did not alter reduced alcohol drinking following chemogenetic activation of PVNOT+ neurons. Altogether, these data are the first to demonstrate that targeted activation of hypothalamic (endogenous) OT reduces alcohol consumption, providing further evidence that this neuropeptide plays a role in regulation of alcohol self-administration behavior. Further, results indicate that the ability OT to reduce alcohol drinking is mediated by signaling at OTR in the brain.

Effects of ceftriaxone on ethanol drinking and GLT-1 expression in ethanol dependence and relapse drinking.

Repeated cycles of chronic intermittent ethanol (CIE) exposure increase voluntary consumption of alcohol (ethanol) in mice. Previous reports from our laboratory show that CIE increases extracellular glutamate in the nucleus accumbens (NAc) and that manipulating accumbal glutamate concentrations will alter ethanol drinking, indicating that glutamate homeostasis plays a crucial role in ethanol drinking in this model. A number of studies have shown that ceftriaxone increases GLT-1 expression, the major glutamate transporter, and that treatment with this antibiotic reduces ethanol drinking. The present studies examined the effects of ceftriaxone on ethanol drinking and GLT-1 in a mouse model of ethanol dependence and relapse drinking. The results show that ceftriaxone did not influence drinking at any dose in either ethanol-dependent or non-dependent mice. Further, ceftriaxone did not increase GLT-1 expression in the accumbens core or shell, with the exception of the ethanol-dependent mice receiving the highest dose of ceftriaxone. Interestingly, ethanol-dependent mice treated with only vehicle displayed reduced expression of GLT-1 in the accumbens shell and of the presynaptic mGlu2 receptor in the accumbens core. The reduced expression of the major glutamate transporter (GLT-1), as well as a receptor that regulates glutamate release (mGlu2), may help explain, at least in part, increased glutamatergic transmission in this model of ethanol dependence and relapse drinking.

Interaction of chronic intermittent ethanol and repeated stress on structural and functional plasticity in the mouse medial prefrontal cortex.

Stress is a risk factor that plays a considerable role in the development and maintenance of alcohol (ethanol) abuse and relapse. Preclinical studies examining ethanol-stress interactions have demonstrated elevated ethanol drinking, cognitive deficits, and negative affective behaviors in mice. However, the neural adaptations in prefrontal cortical regions that drive these aberrant behaviors produced by ethanol-stress interactions are unknown. In this study, male C57BL/6J mice were exposed to chronic intermittent ethanol (CIE) and repeated forced swim stress (FSS). After two cycles of CIE x FSS, brain slices containing the prelimbic (PrL) and infralimbic (IfL) cortex were prepared for analysis of adaptations in dendritic spines and synaptic plasticity. In the PrL cortex, total spine density was increased in mice exposed to CIE. Immediately following induction of long-term potentiation (LTP), the fEPSP slope was increased in the PrL of CIE x FSS treated mice, indicative of a presynaptic adaptation on post-tetanic potentiation (PTP). In the IfL cortex, CIE exposure regardless of FSS experience resulted in an increase in spine density. FSS alone or when combined with CIE exposure increased PTP following LTP induction. Repeated FSS episodes increased IfL cortical paired-pulse facilitation, a second measure of presynaptic plasticity. In summary, CIE exposure resulted in structural adaptations while repeated stress exposure drove metaplastic changes in presynaptic function, demonstrating distinct morphological and functional changes in PrL and IfL cortical neurons. Thus, the structural and functional adaptations may be one mechanism underlying the development of excessive drinking and cognitive deficits associated with ethanol-stress interactions.

Bioinformatics identification and pharmacological validation of Kcnn3/KCa2 channels as a mediator of negative affective behaviors and excessive alcohol drinking in mice.

Mood disorders are often comorbid with alcohol use disorder (AUD) and play a considerable role in the development and maintenance of alcohol dependence and relapse. Because of this high comorbidity, it is necessary to determine shared and unique genetic factors driving heavy drinking and negative affective behaviors. In order to identify novel pharmacogenetic targets, a bioinformatics analysis was used to quantify the expression of amygdala K+ channel genes that covary with anxiety-related phenotypes in the well-phenotyped and fully sequenced family of BXD strains. We used a model of stress-induced escalation of drinking in alcohol-dependent mice to measure negative affective behaviors during abstinence. A pharmacological approach was used to validate the key bioinformatics findings in alcohol-dependent, stressed mice. Amygdalar expression of Kcnn3 correlated significantly with 40 anxiety-associated phenotypes. Further examination of Kcnn3 expression revealed a strong eigentrait for anxiety-like behaviors and negative correlations with binge-like and voluntary alcohol drinking. Mice treated with chronic intermittent alcohol exposure and repeated swim stress consumed more alcohol in their home cages and showed hypophagia on the novelty-suppressed feeding test during abstinence. Pharmacologically targeting Kcnn gene products with the KCa2 (SK) channel-positive modulator 1-EBIO decreased drinking and reduced feeding latency in alcohol-dependent, stressed mice. Collectively, these validation studies provide central nervous system links into the covariance of stress, negative affective behaviors, and AUD in the BXD strains. Further, the bioinformatics discovery tool is effective in identifying promising targets (i.e., KCa2 channels) for treating alcohol dependence exacerbated by comorbid mood disorders.

Evaluation of the effect of doxasozin and zonisamide on voluntary ethanol intake in mice that experienced chronic intermittent ethanol exposure and stress.

The comorbidity between alcohol use disorder and post-traumatic stress disorder represents a serious health care burden with few effective treatment options. The current study was designed to evaluate the effect of an alpha 1 receptor antagonist (doxazosin) and a novel anticonvulsant (zonisamide) in a model of alcohol (ethanol) dependence and stress exposure. The main dependent variable was voluntary ethanol intake in mice that experienced chronic intermittent ethanol (CIE) exposure and forced swim stress (FSS) alone, and in combination. Adult male and female C57BL/6J mice had access to a single bottle of 15% (v/v) ethanol for 1-hr in the home cage, 3-hr into the dark phase of the light/dark cycle. Once stable ethanol intake was established (~4 weeks), mice were separated into four groups (CTL, CIE, FSS, CIE + FSS). Mice in the FSS condition received 10-min FSS exposure 4-hr prior to drinking sessions (remaining mice were not disturbed). During baseline and the first two test cycles, all mice received vehicle (saline) injections (IP) 30-min before ethanol access. As previously observed, FSS increased ethanol drinking in dependent (CIE-exposed) mice but not in nondependent control (CTL) mice. In the following test cycles mice were evaluated for ethanol intake after administration of doxazosin, zonisamide or their combination. Results indicated that the three doses of doxazosin evaluated significantly reduced voluntary ethanol intake in all mice. Zonisamide had a more modest effect and may require a more prolonged treatment regime. The combined administration of both compounds was not more effective than each drug alone. This study suggests that doxazosin is reliable at reducing voluntary ethanol intake in mice independently of their history of ethanol dependence and stress exposure.

Transcriptome Analysis of Alcohol Drinking in Non-Dependent and Dependent Mice Following Repeated Cycles of Forced Swim Stress Exposure.

Chronic stress is a known contributing factor to the development of drug and alcohol addiction. Animal models have previously shown that repeated forced swim stress promotes escalated alcohol consumption in dependent animals. To investigate the underlying molecular adaptations associated with stress and chronic alcohol exposure, RNA-sequencing and bioinformatics analyses were conducted on the prefrontal cortex (CTX) of male C57BL/6J mice that were behaviorally tested for either non-dependent alcohol consumption (CTL), chronic intermittent ethanol (CIE) vapor dependent alcohol consumption, repeated bouts of forced swim stress alone (FSS), and chronic intermittent ethanol with forced swim stress (CIE + FSS). Brain tissue from each group was collected at 0-h, 72-h, and 168-h following the final test to determine long-lasting molecular changes associated with maladaptive behavior. Our results demonstrate unique temporal patterns and persistent changes in coordinately regulated gene expression systems with respect to the tested behavioral group. For example, increased expression of genes involved in "transmitter-gated ion channel activity" was only determined for CIE + FSS. Overall, our results provide a summary of transcriptomic adaptations across time within the CTX that are relevant to understanding the neurobiology of chronic alcohol exposure and stress.

Brain regional gene expression network analysis identifies unique interactions between chronic ethanol exposure and consumption.

Progressive increases in ethanol consumption is a hallmark of alcohol use disorder (AUD). Persistent changes in brain gene expression are hypothesized to underlie the altered neural signaling producing abusive consumption in AUD. To identify brain regional gene expression networks contributing to progressive ethanol consumption, we performed microarray and scale-free network analysis of expression responses in a C57BL/6J mouse model utilizing chronic intermittent ethanol by vapor chamber (CIE) in combination with limited access oral ethanol consumption. This model has previously been shown to produce long-lasting increased ethanol consumption, particularly when combining oral ethanol access with repeated cycles of intermittent vapor exposure. The interaction of CIE and oral consumption was studied by expression profiling and network analysis in medial prefrontal cortex, nucleus accumbens, hippocampus, bed nucleus of the stria terminalis, and central nucleus of the amygdala. Brain region expression networks were analyzed for ethanol-responsive gene expression, correlation with ethanol consumption and functional content using extensive bioinformatics studies. In all brain-regions studied the largest number of changes in gene expression were seen when comparing ethanol naïve mice to those exposed to CIE and drinking. In the prefrontal cortex, however, unique patterns of gene expression were seen compared to other brain-regions. Network analysis identified modules of co-expressed genes in all brain regions. The prefrontal cortex and nucleus accumbens showed the greatest number of modules with significant correlation to drinking behavior. Across brain-regions, however, many modules with strong correlations to drinking, both baseline intake and amount consumed after CIE, showed functional enrichment for synaptic transmission and synaptic plasticity.

The role of oxytocin in alcohol and drug abuse.

The neuropeptide oxytocin (OXT) plays a key role in adaptive processes associated with reward, tolerance, memory and stress responses. Through interactions with brain reward and stress systems, OXT is known to play a role in several neuropsychiatric disorders, particularly those that involve altered social integration, such as alcohol and drug addiction (Heilig et al., 2016). As such, there is growing interest in the oxytocin system as a potential therapeutic target for the treatment of alcohol and substance use disorders. Accumulating preclinical evidence suggests that administration of OXT influences the development of tolerance, sensitization and withdrawal symptoms, and modulates numerous alcohol/drug-seeking and alcohol/drug-taking behaviors. Further, there is some evidence to suggest that OXT may help to reverse neuroadaptations that occur as a result of chronic alcohol or drug exposure. To date, there have been only a handful of clinical studies conducted in alcohol and drug dependent populations. This review summarizes the preclinical and clinical literature on the effects of OXT administration on alcohol- and drug-related behaviors. In addition, we discuss OXT interactions with the hypothalamic-pituitaryadrenal axis and multiple neurotransmitter systems within addiction circuitry.