Mu opioid receptors (MORs) are widely distributed throughout brain reward circuits and their role in drug and social reward is well established. Substantial evidence has implicated MOR and the endogenous opioid system in alcohol reward, but circuit mechanisms of MOR‐mediated alcohol reward and intake behavior remain elusive, and have not been investigated by genetic approaches. We recently created conditional knockout (KO) mice targeting the Oprm1 gene in GABAergic forebrain neurons. These mice (Dlx‐MOR KO) show a major MOR deletion in the striatum, whereas receptors in midbrain (including the Ventral Tegmental Area or VTA) and hindbrain are intact. Here, we compared alcohol‐drinking behavior and rewarding effects in total (MOR KO) and conditional KO mice. Concordant with our previous work, MOR KO mice drank less alcohol in continuous and intermittent two‐bottle choice protocols. Remarkably, Dlx‐MOR KO mice showed reduced drinking similar to MOR KO mice, demonstrating that MOR in the forebrain is responsible for the observed phenotype. Further, alcohol‐induced conditioned place preference was detected in control but not MOR KO mice, indicating that MOR is essential for alcohol reward and again, Dlx‐MOR KO recapitulated the MOR KO phenotype. Taste preference and blood alcohol levels were otherwise unchanged in mutant lines. Together, our data demonstrate that MOR expressed in forebrain GABAergic neurons is essential for alcohol reward‐driven behaviors, including drinking and place conditioning. Challenging the prevailing VTA‐centric hypothesis, this study reveals another mechanism of MOR‐mediated alcohol reward and consumption, which does not necessarily require local VTA MORs but rather engages striatal MOR‐dependent mechanisms.

Alcohol use disorder (AUD) is a widespread disease with limited treatment options. Targeting the neuroimmune system is a new avenue for developing or repurposing effective pharmacotherapies. Alcohol modulates innate immune signaling in different cell types in the brain by altering gene expression and the molecular pathways that regulate neuroinflammation. Chronic alcohol abuse may cause an imbalance in neuroimmune function, resulting in prolonged perturbations in brain function. Likewise, manipulating the neuroimmune system may change alcohol-related behaviors. Psychiatric disorders that are comorbid with AUD, such as post-traumatic stress disorder, major depressive disorder, and other substance use disorders, may also have underlying neuroimmune mechanisms; current evidence suggests that convergent immune pathways may be involved in AUD and in these comorbid disorders. In this review, we provide an overview of major neuroimmune cell-types and pathways involved in mediating alcohol behaviors, discuss potential mechanisms of alcohol-induced neuroimmune activation, and present recent clinical evidence for candidate immune-related drugs to treat AUD.

Alcohol use disorder (AUD) is a widespread disease with limited treatment options. Targeting the neuroimmune system is a new avenue for developing or repurposing effective pharmacotherapies. Alcohol modulates innate immune signaling in different cell types in the brain by altering gene expression and the molecular pathways that regulate neuroinflammation. Chronic alcohol abuse may cause an imbalance in neuroimmune function, resulting in prolonged perturbations in brain function. Likewise, manipulating the neuroimmune system may change alcohol-related behaviors. Psychiatric disorders that are comorbid with AUD, such as post-traumatic stress disorder, major depressive disorder, and other substance use disorders, may also have underlying neuroimmune mechanisms; current evidence suggests that convergent immune pathways may be involved in AUD and in these comorbid disorders. In this review, we provide an overview of major neuroimmune cell-types and pathways involved in mediating alcohol behaviors, discuss potential mechanisms of alcohol-induced neuroimmune activation, and present recent clinical evidence for candidate immune-related drugs to treat AUD.

Variations in pattern and extent of cognitive and motor impairment occur in alcoholism (ALC). Causes of such heterogeneity are elusive and inconsistently accounted for by demographic or alcohol consumption differences. We examined neurological and nutritional factors as possible contributors to heterogeneity in impairment. Participants with ALC (n = 96) and a normal comparison group (n = 41) were examined on six cognitive and motor domains. Signs of historically determined subclinical Wernicke's encephalopathy were detected using the Caine et al. criteria, which were based on postmortem examination and chart review of antemortem data of alcoholic cases with postmortem evidence for Wernicke's encephalopathy. Herein, four Caine criteria provided quantification of dietary deficiency, cerebellar dysfunction, low general cognitive functioning and oculomotor abnormalities in 86 of the 96 ALC participants. Subgroups based on Caine criteria yielded a graded effect, where those meeting more criteria exhibited greater impairment than those meeting no to fewer criteria. These results could not be accounted for by history of drug dependence. Multiple regression indicated that compromised performance on ataxia, indicative of cerebellar dysfunction, predicted non‐mnemonic and upper motor deficits, whereas low whole blood thiamine level, consistent with limbic circuit dysfunction, predicted mnemonic deficits. This double dissociation indicates biological markers that contribute to heterogeneity in expression of functional impairment in ALC. That non‐mnemonic and mnemonic deficits are subserved by the dissociable neural systems of frontocerebellar and limbic circuitry, both commonly disrupted in ALC, suggests neural mechanisms that can differentially affect selective functions, thereby contributing to heterogeneity in pattern and extent of dysfunction in ALC.

Variations in pattern and extent of cognitive and motor impairment occur in alcoholism (ALC). Causes of such heterogeneity are elusive and inconsistently accounted for by demographic or alcohol consumption differences. We examined neurological and nutritional factors as possible contributors to heterogeneity in impairment. Participants with ALC (n = 96) and a normal comparison group (n = 41) were examined on six cognitive and motor domains. Signs of historically determined subclinical Wernicke's encephalopathy were detected using the Caine et al. criteria, which were based on postmortem examination and chart review of antemortem data of alcoholic cases with postmortem evidence for Wernicke's encephalopathy. Herein, four Caine criteria provided quantification of dietary deficiency, cerebellar dysfunction, low general cognitive functioning and oculomotor abnormalities in 86 of the 96 ALC participants. Subgroups based on Caine criteria yielded a graded effect, where those meeting more criteria exhibited greater impairment than those meeting no to fewer criteria. These results could not be accounted for by history of drug dependence. Multiple regression indicated that compromised performance on ataxia, indicative of cerebellar dysfunction, predicted non-mnemonic and upper motor deficits, whereas low whole blood thiamine level, consistent with limbic circuit dysfunction, predicted mnemonic deficits. This double dissociation indicates biological markers that contribute to heterogeneity in expression of functional impairment in ALC. That non-mnemonic and mnemonic deficits are subserved by the dissociable neural systems of frontocerebellar and limbic circuitry, both commonly disrupted in ALC, suggests neural mechanisms that can differentially affect selective functions, thereby contributing to heterogeneity in pattern and extent of dysfunction in ALC.

Chronic, excessive alcohol use alters brain gene expression patterns, which could be important for initiating, maintaining, or progressing the addicted state. It has been proposed that pharmaceuticals with opposing effects on gene expression could treat alcohol use disorder (AUD). Computational strategies comparing gene expression signatures of disease to those of pharmaceuticals show promise for nominating novel treatments. We reasoned that it may be sufficient for a treatment to target the biological pathway rather than lists of individual genes perturbed by AUD. We analyzed published and unpublished transcriptomic data using gene set enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to identify biological pathways disrupted in AUD brain and by compounds in the Library of Network-based Cellular Signatures (LINCS L1000) and Connectivity Map (CMap) databases. Several pathways were consistently disrupted in AUD brain, including an up-regulation of genes within the Complement and Coagulation Cascade, Focal Adhesion, Systemic Lupus Erythematosus, and MAPK signaling, and a down-regulation of genes within the Oxidative Phosphorylation pathway, strengthening evidence for their importance in AUD. Over 200 compounds targeted genes within those pathways in an opposing manner, more than twenty of which have already been shown to affect alcohol consumption, providing confidence in our approach. We created a user-friendly web-interface that researchers can use to identify drugs that target pathways of interest or nominate mechanism of action for drugs. This study demonstrates a unique systems pharmacology approach that can nominate pharmaceuticals that target pathways disrupted in disease states such as AUD and identify compounds that could be repurposed for AUD if sufficient evidence is attained in preclinical studies

Adolescent alcohol drinking has been linked to increased risk for drug abuse during adulthood. Nicotine microinjected directly into the posterior ventral tegmental area (pVTA) stimulates dopamine (DA) release in the nucleus accumbens (NAc) shell. The α7 nicotinic acetylcholine receptor (nAChR) is a potent regulator of dopaminergic activity in the pVTA. The current experiments examined the effects of peri-adolescent ethanol (EtOH) drinking on the ability of intra-pVTA nicotine to stimulate DA release during adulthood and alterations in α7 nAChR expression within the pVTA. Alcohol-preferring (P) female rats consumed EtOH and/or water during adolescence (post-natal day [PND] 30-60) or adulthood (PND 90-120). Thirty days following removal of EtOH, subjects received microinjections of 1 μM, 10 μM, or 50 μM nicotine into the pVTA concurrently with microdialysis for extracellular DA in the NAc shell. Brains were harvested from an additional cohort after PND 90 for quantification of α7 nAChR within the pVTA. The results indicated that only adolescent EtOH consumption produced a leftward and upward shift in the dose response curve for nicotine to stimulate DA release in the NAc shell. Investigation of α7 nAChR expression within the pVTA revealed a significant increase in animals that consumed EtOH during adolescence compared to naïve animals. The data suggests that peri-adolescent EtOH consumption produced cross-sensitization to the effects of nicotine during adulthood. The interaction between adolescent EtOH consumption and inflated adult risk for drug dependency could be predicated, at least in part, upon alterations in α7 nAChR expression within the mesolimbic reward pathway.

Adolescent alcohol drinking has been linked to increased risk for drug abuse during adulthood. Nicotine microinjected directly into the posterior ventral tegmental area (pVTA) stimulates dopamine (DA) release in the nucleus accumbens (NAc) shell. The α7 nicotinic acetylcholine receptor (nAChR) is a potent regulator of dopaminergic activity in the pVTA. The current experiments examined the effects of peri-adolescent ethanol (EtOH) drinking on the ability of intra-pVTA nicotine to stimulate DA release during adulthood and alterations in α7 nAChR expression within the pVTA. Alcohol-preferring (P) female rats consumed EtOH and/or water during adolescence (post-natal day [PND] 30–60) or adulthood (PND 90–120). Thirty days following removal of EtOH, subjects received microinjections of 1 μM, 10 μM, or 50 μM nicotine into the pVTA concurrently with microdialysis for extracellular DA in the NAc shell. Brains were harvested from an additional cohort after PND 90 for quantification of α7 nAChR within the pVTA. The results indicated that only adolescent EtOH consumption produced a leftward and upward shift in the dose response curve for nicotine to stimulate DA release in the NAc shell. Investigation of α7 nAChR expression within the pVTA revealed a significant increase in animals that consumed EtOH during adolescence compared to naïve animals. The data suggests that peri-adolescent EtOH consumption produced cross-sensitization to the effects of nicotine during adulthood. The interaction between adolescent EtOH consumption and inflated adult risk for drug dependency could be predicated, at least in part, upon alterations in α7 nAChR expression within the mesolimbic reward pathway.

Myeloid differentiation primary response protein (MyD88) is a critical neuroimmune adaptor protein in TLR (Toll-like receptor) and IL-1R (Interleukin-1 receptor) signaling complexes. These two pro-inflammatory families play an important role in the neurobiology of alcohol use disorder, specifically MyD88 regulates ethanol drinking, ethanol-induced sedation, and ethanol-induced deficits in motor coordination. In this study, we examined the role of MyD88 in mediating the effects of IL-1β and ethanol on GABAergic transmission in the central amygdala (CeA) of male mice using whole-cell patch-clamp recordings in combination with pharmacological (AS-1, a mimetic that prevents MyD88 recruitment by IL-1R) and genetic (Myd88 knockout mice) approaches. We demonstrate through both approaches that IL-1β and ethanol’s modulatory effects at CeA GABA synapses are not dependent on MyD88. Myd88 knockout potentiated IL-1β’s actions in reducing postsynaptic GABA_A receptor function. Pharmacological inhibition of MyD88 modulates IL-1β’s action at CeA GABA synapses similar to Myd88 knockout mice. Additionally, ethanol-induced CeA GABA release was greater in Myd88 knockout mice compared to wildtype controls. Thus, MyD88 is not essential to IL-1β or ethanol regulation of CeA GABA synapses but plays a role in modulating the magnitude of their effects, which may be a potential mechanism by which it regulates ethanol-related behaviors

The voltage-gated sodium channel subunit β4 (SCN4B) regulates neuronal activity by modulating channel gating and has been implicated in ethanol consumption in rodent models and human alcoholics. However, the functional role for Scn4b in ethanol-mediated behaviors is unknown. We determined if genetic global knockout or targeted knockdown of Scn4b in the central nucleus of the amygdala (CeA) altered ethanol drinking or related behaviors. We used four different ethanol consumption procedures (continuous and intermittent two-bottle choice, drinking-in-the dark, and chronic intermittent ethanol vapor) and found that male and female Scn4b knockout mice did not differ from their wild-type littermates in ethanol consumption in any of the tests. Knockdown of Scn4b mRNA in the CeA also did not alter two-bottle choice ethanol drinking. However, Scn4b knockout mice demonstrated longer duration of the loss of righting reflex induced by ethanol, gaboxadol, pentobarbital, and ketamine. Knockout mice showed slower recovery to basal levels of handling-induced convulsions after ethanol injection, which is consistent with the increased sedative effects observed in these mice. However, Scn4b knockout mice did not differ in the severity of acute ethanol withdrawal. Acoustic startle responses, ethanol-induced hypothermia, and clearance of blood ethanol also did not differ between the genotypes. There were also no functional differences in the membrane properties or excitability of CeA neurons from Scn4b knockout and wild-type mice. Although we found no evidence that Scn4b regulates ethanol consumption in mice, it was involved in the acute hypnotic effects of ethanol and other sedatives.

The voltage‐gated sodium channel subunit β4 (SCN4B) regulates neuronal activity by modulating channel gating and has been implicated in ethanol consumption in rodent models and human alcoholics. However, the functional role for Scn4b in ethanol‐mediated behaviors is unknown. We determined if genetic global knockout (KO) or targeted knockdown of Scn4b in the central nucleus of the amygdala (CeA) altered ethanol drinking or related behaviors. We used four different ethanol consumption procedures (continuous and intermittent two‐bottle choice (2BC), drinking‐in‐the dark and chronic intermittent ethanol vapor) and found that male and female Scn4b KO mice did not differ from their wild‐type (WT) littermates in ethanol consumption in any of the tests. Knockdown of Scn4b mRNA in the CeA also did not alter 2BC ethanol drinking. However, Scn4b KO mice showed longer duration of the loss of righting reflex induced by ethanol, gaboxadol, pentobarbital and ketamine. KO mice showed slower recovery to basal levels of handling‐induced convulsions after ethanol injection, which is consistent with the increased sedative effects observed in these mice. However, Scn4b KO mice did not differ in the severity of acute ethanol withdrawal. Acoustic startle responses, ethanol‐induced hypothermia and clearance of blood ethanol also did not differ between the genotypes. There were also no functional differences in the membrane properties or excitability of CeA neurons from Scn4b KO and WT mice. Although we found no evidence that Scn4b regulates ethanol consumption in mice, it was involved in the acute hypnotic effects of ethanol and other sedatives.

Excessive alcohol (ethanol) consumption negatively impacts social, emotional, as well as cognitive function and well-being. Thus, identifying behavioral and/or biological predictors of excessive ethanol consumption is important for developing prevention and treatment strategies against alcohol use disorders (AUDs). Sex differences in alcohol consumption patterns are observed in humans, primates, and rodents. Selectively bred high alcohol-drinking rat lines, such as the “HAD-1” lines are recognized animal models of alcoholism. The present work examined sex differences in alcohol consumption, object recognition, and exploratory behavior in male and female HAD-1 rats. Naïve male and female HAD-1 rats were tested in an object recognition test (ORT) prior to a chronic 24 h intermittent ethanol access procedure for five weeks. Object recognition parameters measured included exploratory behavior, object investigation, and time spent near objects. During the initial training trial, rearing, active object investigation and amount of time spent in the object-containing section was significantly greater in female HAD-1 rats compared to their male counterparts. During the subsequent testing trial, time spent in the object-containing section was greater in female, compared to male, rats; but active object investigation and rearing did not statistically differ between females and males. In addition, female HAD-1 rats consumed significantly more ethanol than their male counterparts, replicating previous findings. Moreover, across all animals there was a significant positive correlation between exploratory behavior in ORT and ethanol consumption level. These results indicate there are significant sex differences in cognitive performance and alcohol consumption in HAD-1 rats, which suggests neurobiological differences as well.

Ultrasonic vocalizations (USVs) have been established as an animal model of emotional status and are often utilized in drug abuse studies as motivational and emotional indices. Further USV functionality has been demonstrated in our recent work showing accurate identification of selectively-bred high versus low alcohol-consuming male rats ascertained exclusively from 22 to 28 kHz and 50–55 kHz FM USV acoustic parameters. With the hypothesis that alcohol-sensitive sex differences could be revealed through USV acoustic parameters, the present study examined USVs and alcohol consumption in male and female selectively bred high-alcohol drinking (HAD-1) rats. For the current study, we examined USV data collected during a 12-week experiment in male and female HAD-1 rats. Experimental phases included Baseline (2 weeks), 4-h EtOH Access (4 weeks), 24-h EtOH Access (4 weeks) and Abstinence (2 weeks). Findings showed that both male and female HAD-1 rats spontaneously emitted a large number of 22–28 kHz and 50–55 kHz FM USVs and that females drank significantly more alcohol compared to males over the entire course of the experiment. Analyses of USV acoustic characteristics (i.e. mean frequency, duration, bandwidth and power) revealed distinct sex-specific phenotypes in both 50–55 kHz FM and 22–28 kHz USV transmission that were modulated by ethanol exposure. Moreover, by using a linear combination of these acoustic characteristics, we were able to develop binomial logistic regression models able to discriminate between male and female HAD-1 rats with high accuracy. Together these results highlight unique emotional phenotypes in male and female HAD-1 rats that are differentially modulated by alcohol experience.

Ultrasonic vocalizations (USVs) have been established as an animal model of emotional status and are often utilized in drug abuse studies as motivational and emotional indices. Further USV functionality has been demonstrated in our recent work showing accurate identification of selectively-bred high versus low alcohol-consuming male rats ascertained exclusively from 22 to 28kHz and 50-55kHz FM USV acoustic parameters. With the hypothesis that alcohol-sensitive sex differences could be revealed through USV acoustic parameters, the present study examined USVs and alcohol consumption in male and female selectively bred high-alcohol drinking (HAD-1) rats. For the current study, we examined USV data collected during a 12-week experiment in male and female HAD-1 rats. Experimental phases included Baseline (2weeks), 4-h EtOH Access (4weeks), 24-h EtOH Access (4weeks) and Abstinence (2weeks). Findings showed that both male and female HAD-1 rats spontaneously emitted a large number of 22-28kHz and 50-55kHz FM USVs and that females drank significantly more alcohol compared to males over the entire course of the experiment. Analyses of USV acoustic characteristics (i.e. mean frequency, duration, bandwidth and power) revealed distinct sex-specific phenotypes in both 50-55kHz FM and 22-28kHz USV transmission that were modulated by ethanol exposure. Moreover, by using a linear combination of these acoustic characteristics, we were able to develop binomial logistic regression models able to discriminate between male and female HAD-1 rats with high accuracy. Together these results highlight unique emotional phenotypes in male and female HAD-1 rats that are differentially modulated by alcohol experience.

Chronic alcohol consumption alters the levels of microRNAs and mRNAs in the brain, but the specific microRNAs and processes that target mRNAs to affect cellular function and behavior are not known. We examined the in vivo manipulation of previously identified alcohol-responsive microRNAs as potential targets to reduce alcohol consumption. Silencing of miR-411 by infusing antagomiR-411 into the prefrontal cortex of female C57BL/6J mice reduced alcohol consumption and preference, without altering total fluid consumption, saccharin consumption, or anxiety-related behaviors. AntagomiR-411 reduced alcohol consumption when given to mice exposed to a chronic alcohol drinking paradigm but did not affect the acquisition of consumption in mice without a history of alcohol exposure, suggesting that antagomiR-411 has a neuroadaptive, alcohol-dependent effect. AntagomiR-411 decreased the levels of miR-411, as well as the association of immunoprecipitated miR-411 with Argonaute2; and, it increased levels of Faah and Ppard mRNAs. Moreover, antagomiR-411 increased the neuronal expression of glutamate receptor AMPA-2 protein, a known alcohol target and a predicted target of miR-411. These results suggest that alcohol and miR-411 function in a homeostatic manner to regulate synaptic mRNA and protein, thus reversing alcohol-related neuroadaptations and reducing chronic alcohol consumption.

Chronic alcohol consumption alters the levels of microRNAs and mRNAs in the brain, but the specific microRNAs and processes that target mRNAs to affect cellular function and behavior are not known. We examined the in vivo manipulation of previously identified alcohol‐responsive microRNAs as potential targets to reduce alcohol consumption. Silencing of miR‐411 by infusing antagomiR‐411 into the prefrontal cortex of female C57BL/6J mice reduced alcohol consumption and preference, without altering total fluid consumption, saccharin consumption, or anxiety‐related behaviors. AntagomiR‐411 reduced alcohol consumption when given to mice exposed to a chronic alcohol drinking paradigm but did not affect the acquisition of consumption in mice without a history of alcohol exposure, suggesting that antagomiR‐411 has a neuroadaptive, alcohol‐dependent effect. AntagomiR‐411 decreased the levels of miR‐411, as well as the association of immunoprecipitated miR‐411 with Argonaute2; and, it increased levels of Faah and Ppard mRNAs. Moreover, antagomiR‐411 increased the neuronal expression of glutamate receptor AMPA‐2 protein, a known alcohol target and a predicted target of miR‐411. These results suggest that alcohol and miR‐411 function in a homeostatic manner to regulate synaptic mRNA and protein, thus reversing alcohol‐related neuroadaptations and reducing chronic alcohol consumption.

Alcohol use disorder is a significant global burden. Stress has been identified as an etiological factor in the initiation and continuation of ethanol consumption. Understanding adaptations within stress circuitry is an important step toward novel treatment strategies. The effects of protracted abstinence following long-term ethanol self-administration on the central nucleus of the amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) were evaluated in male rhesus monkeys. Using whole-cell patch-clamp electrophysiology, inhibitory GABAergic transmission in the CeA and excitatory glutamatergic transmission in the PVN were measured. CeA neurons from abstinent drinkers displayed an elevated baseline spontaneous inhibitory postsynaptic current (sIPSC) frequency compared with controls, indicating increased presynaptic GABA release. Application of acute ethanol significantly increased the frequency of sIPSCs in controls, but not in abstinent drinkers, suggesting a tolerance to ethanol-enhanced GABA release in abstinent rhesus monkeys with a history of chronic ethanol self-administration and repeated abstinence. In the PVN, the frequency of spontaneous excitatory postsynaptic currents (sEPSC) was elevated in abstinent drinkers compared with controls, indicating increased presynaptic glutamate release. Notably, acute ethanol decreased presynaptic glutamate release onto parvocellular PVN neurons in both controls and abstinent drinkers, suggesting a lack of tolerance to acute ethanol among PVN neurons. These results are the first to demonstrate distinct synaptic adaptations and ethanol sensitivity in both the extrahypothalamic and hypothalamic stress circuits in abstinent rhesus males. Importantly, our findings describe adaptations in stress circuitry present in the brain at a state during abstinence, just prior to relapse to ethanol drinking.

Alcohol use disorder is a significant global burden. Stress has been identified as an etiological factor in the initiation and continuation of ethanol consumption. Understanding adaptations within stress circuitry is an important step toward novel treatment strategies. The effects of protracted abstinence following long-term ethanol self-administration on the central nucleus of the amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) were evaluated in male rhesus monkeys. Using whole-cell patch-clamp electrophysiology, inhibitory GABAergic transmission in the CeA and excitatory glutamatergic transmission in the PVN were measured. CeA neurons from abstinent drinkers displayed an elevated baseline spontaneous inhibitory postsynaptic current (sIPSC) frequency compared with controls, indicating increased presynaptic GABA release. Application of acute ethanol significantly increased the frequency of sIPSCs in controls, but not in abstinent drinkers, suggesting a tolerance to ethanol-enhanced GABA release in abstinent rhesus monkeys with a history of chronic ethanol self-administration and repeated abstinence. In the PVN, the frequency of spontaneous excitatory postsynaptic currents (sEPSC) was elevated in abstinent drinkers compared with controls, indicating increased presynaptic glutamate release. Notably, acute ethanol decreased presynaptic glutamate release onto parvocellular PVN neurons in both controls and abstinent drinkers, suggesting a lack of tolerance to acute ethanol among PVN neurons. These results are the first to demonstrate distinct synaptic adaptations and ethanol sensitivity in both the extrahypothalamic and hypothalamic stress circuits in abstinent rhesus males. Importantly, our findings describe adaptations in stress circuitry present in the brain at a state during abstinence, just prior to relapse to ethanol drinking.

 

Alcohol use disorder is a significant global burden. Stress has been identified as an etiological factor in the initiation and continuation of ethanol consumption. Understanding adaptations within stress circuitry is an important step toward novel treatment strategies. The effects of protracted abstinence following long-term ethanol self-administration on the central nucleus of the amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) were evaluated in male rhesus monkeys. Using whole-cell patch-clamp electrophysiology, inhibitory GABAergic transmission in the CeA and excitatory glutamatergic transmission in the PVN were measured. CeA neurons from abstinent drinkers displayed an elevated baseline spontaneous inhibitory postsynaptic current (sIPSC) frequency compared with controls, indicating increased presynaptic GABA release. Application of acute ethanol significantly increased the frequency of sIPSCs in controls, but not in abstinent drinkers, suggesting a tolerance to ethanol-enhanced GABA release in abstinent rhesus monkeys with a history of chronic ethanol self-administration and repeated abstinence. In the PVN, the frequency of spontaneous excitatory postsynaptic currents (sEPSC) was elevated in abstinent drinkers compared with controls, indicating increased presynaptic glutamate release. Notably, acute ethanol decreased presynaptic glutamate release onto parvocellular PVN neurons in both controls and abstinent drinkers, suggesting a lack of tolerance to acute ethanol among PVN neurons. These results are the first to demonstrate distinct synaptic adaptations and ethanol sensitivity in both the extrahypothalamic and hypothalamic stress circuits in abstinent rhesus males. Importantly, our findings describe adaptations in stress circuitry present in the brain at a state during abstinence, just prior to relapse to ethanol drinking.

Many genes differentially expressed in brain tissue from human alcoholics and animals that have consumed large amounts of alcohol are components of the innate immune toll-like receptor (TLR) pathway. TLRs initiate inflammatory responses via two branches: (1) MyD88-dependent or (2) TRIF-dependent. All TLRs signal through MyD88 except TLR3. Prior work demonstrated a direct role for MyD88-dependent signaling in regulation of alcohol consumption. However, the role of TLR3 as a potential regulator of excessive alcohol drinking has not previously been investigated. To test the possibility TLR3 activation regulates alcohol consumption, we injected mice with the TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) and tested alcohol consumption in an every-other-day two-bottle choice test. Poly(I:C) produced a persistent increase in alcohol intake that developed over several days. Repeated poly(I:C) and ethanol exposure altered innate immune transcript abundance; increased levels of TRIF-dependent pathway components correlated with increased alcohol consumption. Administration of poly(I:C) before exposure to alcohol did not alter alcohol intake, suggesting that poly(I:C) and ethanol must be present together to change drinking behavior. To determine which branch of TLR signaling mediates poly(I:C)-induced changes in drinking behavior, we tested either mice lacking MyD88 or mice administered a TLR3/dsRNA complex inhibitor. MyD88 null mutants showed poly(I:C)-induced increases in alcohol intake. In contrast, mice pretreated with a TLR3/dsRNA complex inhibitor reduced their alcohol intake, suggesting poly(I:C)-induced escalations in alcohol intake are, at least partially, dependent on TLR3. Together, these results strongly suggest that TLR3-dependent signaling drives excessive alcohol drinking behavior.