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.

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.

Dynamic c-Fos changes in mouse brain during acute and protracted withdrawal from chronic intermittent ethanol exposure and relapse drinking.

Alcohol dependence promotes neuroadaptations in numerous brain areas, leading to escalated drinking and enhanced relapse vulnerability. We previously developed a mouse model of ethanol dependence and relapse drinking in which repeated cycles of chronic intermittent ethanol (CIE) vapor exposure drive a significant escalation of voluntary ethanol drinking. In the current study, we used this model to evaluate changes in neuronal activity (as indexed by c-Fos expression) throughout acute and protracted withdrawal from CIE (combined with or without a history of ethanol drinking). We analyzed c-Fos protein expression in 29 brain regions in mice sacrificed 2, 10, 26, and 74 hours or 7 days after withdrawal from 5 cycles of CIE. Results revealed dynamic time- and brain region-dependent changes in c-Fos activity over the time course of withdrawal from CIE exposure, as compared with nondependent air-exposed control mice, beginning with markedly low expression levels upon removal from the ethanol vapor chambers (2 hours), reflecting intoxication. c-Fos expression was enhanced during acute CIE withdrawal (10 and 26 hours), followed by widespread reductions at the beginning of protracted withdrawal (74 hours) in several brain areas. Persistent reductions in c-Fos expression were observed during prolonged withdrawal (7 days) in prelimbic cortex, nucleus accumbens shell, dorsomedial striatum, paraventricular nucleus of thalamus, and ventral subiculum. A history of ethanol drinking altered acute CIE withdrawal effects and caused widespread reductions in c-Fos that persisted during extended abstinence even without CIE exposure. These data indicate that ethanol dependence and relapse drinking drive long-lasting neuroadaptations in several brain regions.

Contribution of Dynorphin and Orexin Neuropeptide Systems to the Motivational Effects of Alcohol.

Understanding the neural systems that drive alcohol motivation and are disrupted in alcohol use disorders is of critical importance in developing novel treatments. The dynorphin and orexin/hypocretin neuropeptide systems are particularly relevant with respect to alcohol use and misuse. Both systems are strongly associated with alcohol-seeking behaviors, particularly in cases of high levels of alcohol use as seen in dependence. Furthermore, both systems also play a role in stress and anxiety, indicating that disruption of these systems may underlie long-term homeostatic dysregulation seen in alcohol use disorders. These systems are also closely interrelated with one another - dynorphin/kappa opioid receptors and orexin/hypocretin receptors are found in similar regions and hypocretin/orexin neurons also express dynorphin - suggesting that these two systems may work together in the regulation of alcohol seeking and may be mutually disrupted in alcohol use disorders. This chapter reviews studies demonstrating a role for each of these systems in motivated behavior, with a focus on their roles in regulating alcohol-seeking and self-administration behaviors. Consideration is also given to evidence indicating that these neuropeptide systems may be viable targets for the development of potential treatments for alcohol use disorders.

Role of the Dynorphin/Kappa Opioid Receptor System in the Motivational Effects of Ethanol.

Evidence has demonstrated that dynorphin (DYN) and the kappa opioid receptor (KOR) system contribute to various psychiatric disorders, including anxiety, depression, and addiction. More recently, this endogenous opioid system has received increased attention as a potential therapeutic target for treating alcohol use disorders. In this review, we provide an overview and synthesis of preclinical studies examining the influence of alcohol (ethanol [EtOH]) exposure on DYN/KOR expression and function, as well as studies examining the effects of DYN/KOR manipulation on EtOH's rewarding and aversive properties. We then describe work that has characterized effects of KOR activation and blockade on EtOH self-administration and EtOH dependence/withdrawal-related behaviors. Finally, we address how the DYN/KOR system may contribute to stress-EtOH interactions. Despite an apparent role for the DYN/KOR system in motivational effects of EtOH, support comes from relatively few studies. Nevertheless, review of this literature reveals several common themes: (i) rodent strains genetically predisposed to consume more EtOH generally appear to have reduced DYN/KOR tone in brain reward circuitry; (ii) acute and chronic EtOH exposure typically up-regulate the DYN/KOR system; (iii) KOR antagonists reduce behavioral indices of negative affect associated with stress and chronic EtOH exposure/withdrawal; and (iv) KOR antagonists are effective in reducing EtOH consumption, but are often more efficacious under conditions that engender high levels of consumption, such as dependence or stress exposure. These results support the contention that the DYN/KOR system plays a significant role in contributing to dependence- and stress-induced elevation in EtOH consumption. Overall, more comprehensive analyses (on both behavioral and mechanistic levels) are needed to provide additional insight into how the DYN/KOR system is engaged and adapts to influence the motivation effects of EtOH. This information will be critical for the development of new pharmacological agents targeting KORs as promising novel therapeutics for alcohol use disorders and comorbid affective disorders.

Stress Facilitates the Development of Cognitive Dysfunction After Chronic Ethanol Exposure.

BACKGROUND: Chronic exposure to stress or alcohol can drive neuroadaptations that alter cognition. Alterations in cognition may contribute to alcohol use disorders by reducing cognitive control over drinking and maintenance of abstinence. Here we examined effects of combined ethanol (EtOH) and stress exposure on prefrontal cortex (PFC)-dependent cognition. METHODS: Adult male C57BL/6J mice were trained to drink EtOH (15%, v/v) on a 1 h/d 1-bottle schedule. Once stable, mice were exposed to cycles of chronic intermittent EtOH (CIE) or air-control vapor exposure (Air), followed by test cycles of 1 h/d EtOH drinking. During test drinking, mice received no stress (NS) or 10 minutes of forced swim stress (FSS) 4 hours before each test. This schedule produced 4 experimental groups: control, Air/NS; EtOH-dependent no stress, CIE/NS; nondependent stress, Air/FSS; or EtOH-dependent stress, CIE/FSS. After 2 cycles of CIE and FSS exposure, we assessed PFC-dependent cognition using object/context recognition and attentional set shifting. At the end of the study, mice were perfused and brains were collected for measurement of c-Fos activity in PFC and locus coeruleus (LC). RESULTS: CIE/FSS mice escalated EtOH intake faster than CIE/NS and consumed more EtOH than Air/NS across all test cycles. After 2 cycles of CIE/FSS, mice showed impairments in contextual learning and extradimensional set-shifting relative to other groups. In addition to cognitive dysfunction, CIE/FSS mice demonstrated widespread reductions in c-Fos activity within prelimbic and infralimbic PFC as well as LC. CONCLUSIONS: Together, these findings show that interactions between EtOH and stress exposure rapidly lead to disruptions in signaling across cognitive networks and impairments in PFC-dependent cognitive function.

Oxytocin Reduces Ethanol Self-Administration in Mice.

BACKGROUND: Excessive ethanol (EtOH) consumption remains an important health concern and effective treatments are lacking. The central oxytocin system has emerged as a potentially important therapeutic target for alcohol and drug addiction. These studies tested the hypothesis that oxytocin reduces EtOH consumption. METHODS: Male C57BL/6J mice were given access to EtOH (20% v/v) using a model of binge-like drinking ("drinking in the dark") that also included the use of lickometer circuits to evaluate the temporal pattern of intake as well as 2-bottle choice drinking in the home cage. In addition, EtOH (12% v/v) and sucrose (5% w/v) self-administration on fixed- and progressive-ratio schedules were also evaluated. A wide range of systemically administered oxytocin doses were tested (0 to 10 mg/kg) in these models. RESULTS: Oxytocin (0, 0.3, 1, 3, or 10 mg/kg) dose dependently reduced EtOH consumption (maximal 45% reduction) in the binge drinking model, with lower effective doses having minimal effects on general locomotor activity. Oxytocin's effect was blocked by pretreatment with an oxytocin receptor antagonist, and the pattern of contacts (licks) at the EtOH bottle suggested a reduction in motivation to drink EtOH. Oxytocin decreased 2-bottle choice drinking without altering general fluid intake. Oxytocin also reduced operant responding for EtOH and sucrose in a dose-related manner. However, oxytocin decreased responding and motivation (breakpoint values) for EtOH at doses that did not alter responding for sucrose. CONCLUSIONS: These results indicate that oxytocin reduces EtOH consumption in different models of self-administration. The effects are not likely due to a general sedative effect of the neuropeptide. Further, oxytocin reduces motivation for EtOH at doses that do not alter responding for a natural reward (sucrose). While some evidence supports a role for oxytocin receptors in mediating these effects, additional studies are needed to further elucidate underlying mechanisms. Nevertheless, these results support the therapeutic potential of oxytocin as a treatment for alcohol use disorder.

Supersensitive Kappa Opioid Receptors Promotes Ethanol Withdrawal-Related Behaviors and Reduce Dopamine Signaling in the Nucleus Accumbens.

BACKGROUND: Chronic ethanol exposure reduces dopamine transmission in the nucleus accumbens, which may contribute to the negative affective symptoms associated with ethanol withdrawal. Kappa opioid receptors have been implicated in withdrawal-induced excessive drinking and anxiety-like behaviors and are known to inhibit dopamine release in the nucleus accumbens. The effects of chronic ethanol exposure on kappa opioid receptor-mediated changes in dopamine transmission at the level of the dopamine terminal and withdrawal-related behaviors were examined. METHODS: Five weeks of chronic intermittent ethanol exposure in male C57BL/6 mice were used to examine the role of kappa opioid receptors in chronic ethanol-induced increases in ethanol intake and marble burying, a measure of anxiety/compulsive-like behavior. Drinking and marble burying were evaluated before and after chronic intermittent ethanol exposure, with and without kappa opioid receptor blockade by nor-binaltorphimine (10mg/kg i.p.). Functional alterations in kappa opioid receptors were assessed using fast scan cyclic voltammetry in brain slices containing the nucleus accumbens. RESULTS: Chronic intermittent ethanol-exposed mice showed increased ethanol drinking and marble burying compared with controls, which was attenuated with kappa opioid receptor blockade. Chronic intermittent ethanol-induced increases in behavior were replicated with kappa opioid receptor activation in naïve mice. Fast scan cyclic voltammetry revealed that chronic intermittent ethanol reduced accumbal dopamine release and increased uptake rates, promoting a hypodopaminergic state of this region. Kappa opioid receptor activation with U50,488H concentration-dependently decreased dopamine release in both groups; however, this effect was greater in chronic intermittent ethanol-treated mice, indicating kappa opioid receptor supersensitivity in this group. CONCLUSIONS: These data suggest that the chronic intermittent ethanol-induced increase in ethanol intake and anxiety/compulsive-like behaviors may be driven by greater kappa opioid receptor sensitivity and a hypodopaminergic state of the nucleus accumbens.

Ibudilast reduces alcohol drinking in multiple animal models of alcohol dependence.

Neuroinflammatory signaling pathways in the central nervous system are of current interest as potential pharmacotherapy targets for alcohol dependence. In this study, we examined the ability of ibudilast, a non-selective phosphodiesterase inhibitor, to reduce alcohol drinking and relapse in alcohol-preferring P rats, high-alcohol drinking HAD1 rats, and in mice made dependent on alcohol through cycles of alcohol vapor exposure. When administered twice daily, ibudilast reduced alcohol drinking in rats by approximately 50% and reduced drinking by alcohol-dependent mice at doses which had no effect in non-dependent mice. These findings support the viability of ibudilast as a possible treatment for alcohol dependence.

Chronic intermittent ethanol exposure and withdrawal alters (3α,5α)-3-hydroxy-pregnan-20-one immunostaining in cortical and limbic brain regions of C57BL/6J mice.

BACKGROUND: The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP; allopregnanolone) has been studied during withdrawal from ethanol (EtOH) in humans, rats, and mice. Serum 3α,5α-THP levels decreased, and brain levels were not altered following acute EtOH administration (2 g/kg) in male C57BL/6J mice; however, the effects of chronic intermittent ethanol (CIE) exposure on 3α,5α-THP levels have not been examined. Given that CIE exposure changes subsequent voluntary EtOH drinking in a time-dependent fashion following repeated cycles of EtOH exposure, we conducted a time-course analysis of CIE effects on 3α,5α-THP levels in specific brain regions known to influence drinking behavior. METHODS: Adult male C57BL/6J mice were exposed to 4 cycles of CIE to induce EtOH dependence. All mice were sacrificed and perfused at 1 of 2 time points, 8 or 72 hours following the final exposure cycle. Free-floating brain sections (40 µm; 3 to 5 sections/region/animal) were immunostained and analyzed to determine relative levels of cellular 3α,5α-THP. RESULTS: Withdrawal from CIE exposure produced time-dependent and region-specific effects on immunohistochemical detection of 3α,5α-THP levels across cortical and limbic brain regions. A transient reduction in 3α,5α-THP immunoreactivity was observed in the central nucleus of the amygdala 8 hours after withdrawal from CIE (-31.4 ± 9.3%). Decreases in 3α,5α-THP immunoreactivity were observed 72 hours following withdrawal in the medial prefrontal cortex (-25.0 ± 9.3%), nucleus accumbens core (-29.9 ± 6.6%), and dorsolateral striatum (-18.5 ± 6.0%), while an increase was observed in the CA3 pyramidal cell layer of the hippocampus (+42.8 ± 19.5%). Sustained reductions in 3α,5α-THP immunoreactivity were observed at both time points in the lateral amygdala (8 hours -28.3 ± 12.8%; 72 hours -27.5 ± 12.4%) and in the ventral tegmental area (8 hours -26.5 ± 9.9%; 72 hours -31.6 ± 13.8%). CONCLUSIONS: These data suggest that specific neuroadaptations in 3α,5α-THP levels may be present in regions of brain that mediate anxiety, stress, and reinforcement relevant to EtOH dependence. The changes that occur at different time points likely modulate neurocircuitry involved in EtOH withdrawal as well as the elevated drinking observed after CIE exposure.

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.

Calpain inhibition prevents ethanol-induced alterations in spinal motoneurons.

Long-term exposure of ethanol (EtOH) alters the structure and function in brain and spinal cord. The present study addresses the mechanisms of EtOH-induced damaging effects on spinal motoneurons in vitro. Altered morphology and biochemical changes of such damage were demonstrated by in situ Wright staining and DNA ladder assay. EtOH at low to moderate (25-50 mM) concentrations induced damaging effects in the motoneuronal scaffold which involved activation of proteases like µ-calpain and caspase-3. Caspase-8 was seen only at higher (100 mM) EtOH concentration. Further, pretreatment with calpeptin, a potent calpain inhibitor, confirmed the involvement of active proteases in EtOH-induced damage to motoneurons. The lysosomal enzyme cathepsin D was also elevated in the motoneurons by EtOH, and this effect was significantly attenuated by inhibitor treatment. Overall, EtOH exposure rendered spinal motoneurons vulnerable to damage, and calpeptin provided protection, suggesting a critical role of calpain activation in EtOH-induced alterations in spinal motoneurons.

Similar ethanol drinking in adolescent and adult C57BL/6J mice after chronic ethanol exposure and withdrawal.

BACKGROUND: Increasing evidence shows that excessive alcohol consumption during adolescence increases vulnerability to alcohol use disorders in adulthood. The aim of this study was to examine differences between adolescent and adult C57BL/6J mice in drinking behavior and blood ethanol (EtOH) concentrations (BECs) after chronic EtOH exposure and withdrawal. METHODS: Male adolescent (PND = 28 to 30) and adult (PND = 70) C57BL/6J mice were allowed to consume EtOH in a 2-bottle choice paradigm (15% EtOH vs. water) for 3 weeks (Baseline drinking, Test 1, and Test 2), which were interspersed with 2 cycles (Cycles I and II) of chronic EtOH vapor or air inhalation (16 hours) and withdrawal (8 hours). BECs were determined during both cycles. RESULTS: Chronic EtOH exposure led to increased EtOH intake during Test 1 and Test 2 in both adolescent and adult mice compared with air-exposed controls, and no differences between age groups were observed. During Cycle I adult mice showed higher BECs compared with adolescents. During Cycle II, BECs were lower in adult mice as compared to Cycle I, and BECs in adolescent mice did not change between the 2 cycles. CONCLUSIONS: Chronic EtOH exposure followed by withdrawal periods increases EtOH consumption similarly in both adolescent and adult mice, despite differences in BECs.

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.

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.

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.

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 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.

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.