Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase recently implicated in biochemical, physiological, and behavioral responses to ethanol. Thus, manipulation of ALK signaling may represent a novel approach to treating alcohol use disorder (AUD). Ethanol induces adaptations in glutamatergic synapses onto nucleus accumbens shell (NAcSh) medium spiny neurons (MSNs), and putative targets for treating AUD may be validated for further development by assessing how their manipulation modulates accumbal glutamatergic synaptic transmission and plasticity. Here, we report that Alk knockout (AlkKO) mice consumed greater doses of ethanol, relative to wild-type (AlkWT) mice, in an operant self-administration model. Using ex vivo electrophysiology to examine excitatory synaptic transmission and plasticity at NAcSh MSNs that express dopamine D1 receptors (D1MSNs), we found that the amplitude of spontaneous excitatory post-synaptic currents (EPSCs) in NAcSh D1MSNs was elevated in AlkKO mice and in the presence of an ALK inhibitor, TAE684. Furthermore, when ALK was absent or inhibited, glutamatergic synaptic plasticity - long-term depression of evoked EPSCs - in D1MSNs was attenuated. Thus, loss of ALK activity in mice is associated with elevated ethanol consumption and enhanced excitatory transmission in NAcSh D1MSNs. These findings add to the mounting evidence of a relationship between excitatory synaptic transmission onto NAcSh D1MSNs and ethanol consumption, point toward ALK as one important molecular mediator of this interaction, and further validate ALK as a target for therapeutic intervention in the treatment of AUD.
Alcohol misuse is the fifth leading risk factor for premature death and disability worldwide. Fewer than 10% of afflicted Americans receive pharmacological treatment for alcohol use disorder. Gabapentin is a calcium channel GABAergic modulator that is widely used for pain. Studies showing reduced drinking and decreased craving and alcohol-related disturbances in sleep and affect in the months following alcohol cessation suggest therapeutic potential for alcohol use disorder. Areas covered: Human laboratory and clinical studies assessing gabapentin for alcohol use disorder are reviewed. Data were obtained by searching for English peer-reviewed articles on PubMed, reference lists of identified articles, and trials registered on clinicaltrials.gov. Additionally, the mechanism of action of gabapentin specific to alcohol use disorder, and studies of gabapentin for alcohol withdrawal and non-alcohol substance use disorders are summarized. Expert opinion: Alcohol use disorder represents a challenge and large, unmet medical need. Evidence from single-site studies lend support to the safety and efficacy of gabapentin as a novel treatment for alcohol use disorder, with unique benefits for alcohol-related insomnia and negative affect, relative to available treatments. Proprietary gabapentin delivery systems may open a path to pivotal trials and registration of gabapentin as a novel treatment for alcohol use disorder.
Chronic alcohol exposure produces widespread neuroadaptations and alterations in gene expression in human alcoholics and animal models. Technological advances in the past decade have increasingly highlighted the role of non-protein-coding RNAs (ncRNAs) in the regulation of gene expression and function. These recently characterized molecules were discovered to mediate diverse processes in the central nervous system, from normal development and physiology to regulation of disease, including alcoholism and other psychiatric disorders. This review will investigate the recent studies in human alcoholics and rodent models that have profiled different classes of ncRNAs and their dynamic alcohol-dependent regulation in brain.
Background: Innate immune signaling in the brain has emerged as a contributor to many central nervous system (CNS) pathologies, including mood disorders, neurodegenerative disorders, neurodevelopmental disorders, and addiction. Toll-like receptors (TLRs), a key component of the innate immune response, are particularly implicated in neuroimmune dysfunction. However, most of our understanding about TLRsignaling comes from the peripheral immune response, and it is becoming clear that the CNS immune response is unique. One controversial aspect of neuroimmune signaling is which CNS cell types are involved. While microglia are the CNScell-type derived from a myeloid lineage, studies suggest that other glial cell types and even neurons express TLRs, although this idea is controversial. Furthermore, recent work suggests a discrepancy between RNA and protein expression within the CNS. Methods: To elucidate the CNScell-typelocalization of TLRs and their downstream signaling molecules, we isolated microglia and astrocytes from the brain of adult mice treated with saline or the TLR4 ligand lipopolysaccharide (LPS). Glial mRNA and protein expression was compared to a cellular-admixture to determine cell-type enrichment. Results: Enrichment analysis revealed that most of the TLR pathway genes are localized in microglia and changed in microglia following immune challenge. However, expression of Tlr3 was enriched in astrocytes, where it increased in response to LPS. Furthermore, attempts to determine protein cell-typelocalization revealed that many antibodies are non-specific and that antibody differences are contributing to conflicting localization results. Conclusions: Together these results highlight the cell types that should be looked at when studying TLRsignaling gene expression and suggest that non-antibody approaches need to be used to accurately evaluate protein expression
Microglia are fundamentally important immune cells within the central nervous system (CNS) that respond to environmental challenges to maintain normal physiological processes. Alterations in steady-state cellular function and over-activation of microglia can facilitate the initiation and progression of neuropathological conditions such as Alzheimer's disease, Multiple Sclerosis, and Major Depressive Disorder. Alcoholconsumption disrupts signaling pathways including both innate and adaptive immune responses that are necessary for CNS homeostasis. Coordinate expression of these genes is not ascertained from an admixture of CNS cell-types, underscoring the importance of examining isolated cellular populations to reveal systematic gene expression changes arising from mature microglia. Unbiased RNA-Seq profiling was used to identify gene expression changes in isolated prefrontal cortical microglia in response to recurring bouts of voluntary alcohol drinking behavior. The voluntary ethanol paradigm utilizes long-term consumption ethanol that results in escalated alcohol intake and altered cortical plasticity that is seen in humans. Gene coexpression analysis identified a coordinately regulated group of genes, unique to microglia, that collectively are associated with alcoholconsumption. Genes within this group are involved in toll-like receptor signaling and transforming growth factor beta signaling. Network connectivity of this group identified Siglech as a putative hub gene and highlighted the potential importance of proteases in the microglial response to chronic ethanol. In conclusion, we identified a distinctive microglial gene expression signature for neuroimmune responses related to alcoholconsumption that provides valuable insight into microglia-specific changes underlying the development of substance abuse, and possibly other CNS disorders.
Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including decreased hippocampal and prefrontalcortex volume and deficits in memory. We used RNA sequencing to assess the effects of adolescent binge drinking on geneexpression in these regions. Male adolescentalcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-h sessions/day during the dark/cycle, 5 days/week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5-3 g/kg/session). Ethanol significantly altered the expression of 416 of 11,727 genes expressed in the ventralhippocampus. Genes and pathways involved in neurogenesis, long-term potentiation, and axonal guidance were decreased, which could relate to the impaired memory function found in subjects with adolescentalcoholbinge-like exposure. The decreased expression of myelin and cholesterol genes and apparent decrease in oligodendrocytes in Prats could result in decreased myelination. In the medialprefrontalcortex, 638 of 11,579 genes were altered; genes in cellular stress and inflammatory pathways were increased, as were genes involved in oxidative phosphorylation. Overall, the results of this study suggest that adolescentbinge-likealcoholdrinking may alter the development of the ventralhippocampus and medialprefrontalcortex and produce long-term consequences on learning and memory, and on control of impulsive behaviors.
Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition through the activation of heteromeric glycine receptors (GlyRs) composed primarily of α1 and β subunits. Inhibitory GlyRs are also found throughout the brain, where GlyR α2 and α3 subunit expression exceeds that of α1, particularly in forebrain structures, and coassembly of these α subunits with the β subunit appears to occur to a lesser extent than in spinal cord. Here, we analyzed GlyR currents in several regions of the adolescent mouse forebrain (striatum, prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis). Our results show ubiquitous expression of GlyRs that mediate large-amplitude currents in response to exogenously applied glycine in these forebrain structures. Additionally, tonic inward currents were also detected, but only in the striatum, hippocampus, and prefrontal cortex (PFC). These toniccurrents were sensitive to both strychnine and picrotoxin, indicating that they are mediated by extrasynaptic homomeric GlyRs. Recordings from mice deficient in the GlyR α3 subunit (Glra3-/-) revealed a lack of tonic GlyR currents in the striatum and the PFC. In Glra2-/Y animals, GlyR toniccurrents were preserved; however, the amplitudes of current responses to exogenousglycine were significantly reduced. We conclude that functional α2 and α3 GlyRs are present in various regions of the forebrain and that α3 GlyRs specifically participate in tonic inhibition in the striatum and PFC. Our findings suggest roles for glycine in regulating neuronal excitability in the forebrain.
Despite acceptance that risk for alcohol-use disorder (AUD) has a large genetic component, the identification of genes underlying various components of risk for AUD has been hampered in humans, in part by the heterogeneity of expression of the phenotype. One aspect of AUD is physical dependence. Alcoholwithdrawal is a serious consequence of alcohol dependence with multiplesymptoms, many of which are seen in multiple species, and can be experienced over a wide-ranging time course. In the present three studies, we developed a battery of withdrawal tests in mice, examining behavioralsymptoms from multiple domains that could be measured over time. To permit eventual use of the battery in different strains of mice, we used male and female mice of a genetically heterogeneous stock developed from intercrossing eight inbred strains. Withdrawalsymptoms were assessed using commonly used tests after administration of ethanol in vapor for 72 continuous hours. We found significant effects of ethanol withdrawal versus air-breathing controls on nearly all symptoms, spanning 4 days following ethanol vapor inhalation. Withdrawal produced hypothermia, greater neurohyperexcitability (seizures and tremor), anxiety-like behaviors using an apparatus (such as reduced transitions between light and dark compartments), anhedonia (reduced sucrose preference), Straub tail, backward walking, and reductions in activity; however, there were no changes in thermal pain sensitivity, hyper-reactivity to handling, or anxiety-like emergence behaviors in other apparatus. Using these data, we constructed a refined battery of withdrawal tests. Individual differences in severity of withdrawal among different tests were weakly correlated at best. This battery should be useful for identifying genetic influences on particular withdrawal behaviors, which should reflect the influences of different constellations of genes.
Corticotropin releasing factor (CRF) is a neuropeptide that plays a key role in behavioral and physiological responses to stress. A large body of animal literature implicates CRF acting at type 1 CRF receptors (CRFR1) in consumption by alcohol‐dependent subjects, stress‐induced reinstatement of alcohol seeking, and possibly binge alcohol consumption. These studies have encouraged recent pilot studies of CRFR1 antagonists in humans with alcohol use disorder (AUD). It was a great disappointment to many in the field that these studies failed to show an effect of these compounds on stress‐induced alcohol craving. Here, we examine these studies to explore potential limitations and discuss preclinical and human literature to ask whether CRFR1 is still a valid drug target to pursue for the treatment of AUD.
There is growing evidence that small-molecule inhibitors of epigenetic modulators, such as histone deacetylases (HDAC) and DNA methyltransferases (DNMT), can reduce voluntary ethanolconsumption in animal models, but molecular and cellular processes underlying this behavioral effect are poorly understood. We used C57BL/6J male mice to investigate the effects of two FDA-approved drugs, decitabine (a DNMT inhibitor) and SAHA (an HDAC inhibitor), on ethanolconsumption using two tests: binge-like drinking in the dark (DID) and chronic intermittent every other day (EOD) drinking. Decitabine but not SAHA reduced ethanolconsumption in both tests. We further investigated decitabine's effects on the brain's reward pathway by gene expression profiling in the ventral tegmental area (VTA), using RNA sequencing and electrophysiological recordings from VTA dopaminergic neurons. Decitabine-induced decreases in EOD drinking were associated with global changes in gene expression, implicating regulation of cerebral blood flow, extracellular matrix organization, and neuroimmune functions in decitabine actions. In addition, an in vivo administration of decitabine shortened ethanol-induced excitation of VTA dopaminergic neurons in vitro, suggesting that decitabine reduces ethanol drinking via changes in the reward pathway. Taken together, our data suggest a contribution of both neuronal and non-neuronal mechanisms in the VTA in the regulation of ethanolconsumption. Decitabine and other epigenetic compounds have been approved for cancer treatment, and understanding their mechanisms of actions in the brain may assist in repurposing these drugs and developing novel therapies for central disorders, including drug addiction.
BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking.
Negative emotional status and adverse emotional events increase vulnerability to alcohol abuse. Ultrasonic vocalizations (USVs) emitted by rats are a well-established model of emotional status that can reflect positive or negative affective responses in real time. Most USV studies assess counts, yet each USV is a multidimensional data point characterized by several acousticcharacteristics that may provide insight into the neurocircuitry underlying emotional response.
USVs emitted from selectively bred alcohol-naïve and alcohol-experienced alcohol-preferring and nonpreferring rats (P and NPrats) were recorded during 4-hour sessions on alternating days over 4 weeks. Linear mixed modeling (LMM) and linear discriminant analysis (LDA) were applied to USVacousticcharacteristics (e.g., frequency, duration, power, and bandwidth) of negative affect (22 to 28 kilohertz [kHz])- and positive (50 to 55 kHz) affect-related USVs.
Hundred percent separation between alcohol-naïve P and NPrats was achieved through a linear combination (produced by LDA) of USVacousticcharacteristics of 22- to 28-kHz USVs, whereas poor separation (36.5%) was observed for 50- to 55-kHz USVs. 22- to 28-kHz LDA separation was high (87%) between alcohol-experienced P and NPrats, but was poor for 50- to 55-kHz USVs (57.3%). USV mean frequency and duration were the highest weighted characteristics in both the naïve and experienced 22- to 28-kHz LDA representations suggesting that alcohol experience does not alter the representations. LMM analyses of 22- to 28-kHz USVacousticcharacteristics matched the LDA results. Poor LDA separation was observed between alcohol-naïve and alcohol-experienced Prats for both 22- to 28-kHz and 50- to 55-kHz USVs.
Advanced statistical analysis of negativeaffect-associatedUSV data predicts future behaviors of excessivealcoholdrinking and alcoholavoidance in selectively bred rats. USVcharacteristics across rat lines reveal affect-related motivation to consume alcohol and may predict neural pathways mediating emotional response. Further characterization of these differences could delineate particular neurocircuitry and methods to ameliorate dysregulated emotional states often observed in human alcohol abusers.
Alcohol acts on numerous cellular and molecular targets to regulate neuronal communication within the brain. Chronic alcohol exposure and acute withdrawal generate prominent neuroadaptations at synapses, including compensatory effects on the expression, localization and function of synaptic proteins, channels and receptors. The present article reviews the literature describing the synaptic effects of chronic alcohol exposure and their relevance for synaptic transmission in the central nervous system. This review is not meant to be comprehensive, but rather to highlight the effects that have been observed most consistently and that are thought to contribute to the development of alcohol dependence and the negative aspects of withdrawal. Specifically, we will focus on the major excitatory and inhibitory neurotransmitters in the brain, glutamate and GABA, respectively, and how their neuroadaptations after chronic alcohol exposure contributes to alcohol reinforcement, dependence and withdrawal. This article is part of the Special Issue entitled "Alcoholism".
Drug addiction is a complex disorder that is characterized by compulsivity to seek and take the drug, loss of control in limiting intake of the drug, and emergence of a withdrawal syndrome in the absence of the drug. The transition from casual drug use to dependence is mediated by changes in reward and brain stress functions and has been linked to a shift from positive reinforcement to negative reinforcement. The recruitment of brain stress systems mediates the negative emotional state produced by dependence that drives drug seeking through negative reinforcement mechanisms, defined as the "dark side" of addiction. In this chapter we focus on behavioral and cellular neuropharmacological studies that have implicated brain stress systems (i.e., corticotropin-releasing factor [CRF]) in the transition to addiction and the predominant brain regions involved. We also discuss the implication of CRF recruitment in compulsive eating disorders.
The identification of new and more effective treatments for alcohol abuse remains a priority. Alcohol intake activates glucocorticoids, which have a key role in alcohol's reinforcing properties. Glucocorticoid effects are modulated in part by the activity of 11β-hydroxysteroid dehydrogenases (11β-HSD) acting as pre-receptors. Here, we tested the effects on alcohol intake of the 11β-HSD inhibitor carbenoxolone (CBX, 18β-glycyrrhetinic acid 3β-O-hemisuccinate), which has been extensively used in the clinic for the treatment of gastritis and peptic ulcer and is active on both 11β-HSD1 and 11β-HSD2 isoforms. We observed that CBX reduces both baseline and excessive drinking in rats and mice. The CBX diastereomer 18α-glycyrrhetinic acid 3β-O-hemisuccinate (αCBX), which we found to be selective for 11β-HSD2, was also effective in reducing alcohol drinking in mice. Thus, 11β-HSD inhibitors may be a promising new class of candidate alcohol abuse medications, and existing 11β-HSD inhibitor drugs may be potentially re-purposed for alcohol abuse treatment.
Proinflammatory pathways in neuronal and non-neuronal cells are implicated in the acute and chronic effects of alcohol exposure in animal models and humans. The nuclear factor-κB (NF-κB) family of DNA transcription factors plays important roles in inflammatory diseases. The kinase IKKβ mediates the phosphorylation and subsequent proteasomal degradation of cytosolic protein inhibitors of NF-κB, leading to activation of NF-κB. The role of IKKβ as a potential regulator of excessive alcohol drinking had not previously been investigated. Based on previous findings that the overactivation of innate immune/inflammatory signaling promotes ethanol consumption, we hypothesized that inhibiting IKKβ would limit/decrease drinking by preventing the activation of NF-κB. We studied the systemic effects of two pharmacological inhibitors of IKKβ, TPCA-1 and sulfasalazine, on ethanol intake using continuous- and limited-access, two-bottle choice drinking tests in C57BL/6J mice. In both tests, TPCA-1 and sulfasalazine reduced ethanol intake and preference without changing total fluid intake or sweet taste preference. A virus expressing Cre recombinase was injected into the nucleus accumbens and central amygdala to selectively knock down IKKβ in mice genetically engineered with a conditional Ikkb deletion (IkkbF/F ). Although IKKβ was inhibited to some extent in astrocytes and microglia, neurons were a primary cellular target. Deletion of IKKβ in either brain region reduced ethanol intake and preference in the continuous access two-bottle choice test without altering the preference for sucrose. Pharmacological and genetic inhibition of IKKβ decreased voluntary ethanol consumption, providing initial support for IKKβ as a potential therapeutic target for alcohol abuse.
Chronic alcohol use and abuse result in widespread changes to gene expression, some of which contribute to the development of alcohol-use disorders (AUD). Gene expression is controlled, in part, by a group of regulatory systems often referred to as epigenetic factors, which includes, among other mechanisms, chemical marks made on the histone proteins around which genomic DNA is wound to form chromatin, and on nucleotides of the DNA itself. In particular, alcohol has been shown to perturb the epigenetic machinery, leading to changes in gene expression and cellular functions characteristic of AUD and, ultimately, to altered behavior. DNAmodifications in particular are seeing increasing research in the context of alcohol use and abuse. To date, studies of DNAmodifications in AUD have primarily looked at global methylation profiles in human brain and blood, gene-specific methylation profiles in animal models, methylation changes associated with prenatal ethanol exposure, and the potential therapeutic abilities of DNA methyltransferase inhibitors. Future studies may be aimed at identifying changes to more recently discovered DNAmodifications, utilizing new methods to discriminate methylation profiles between cell types, thus clarifying how alcohol influences the methylomes of cell-type populations and how this may affect downstream processes. These studies and more in-depth probing of DNA methylation will be key to determining whether DNA-level epigenetic regulation plays a causative role in AUD and can thus be targeted for treatment of the disorder.
Chronic alcohol use and abuse result in widespread changes to gene expression, some of which contribute to the development of alcohol-use disorders (AUD). Gene expression is controlled, in part, by a group of regulatory systems often referred to as epigenetic factors, which includes, among other mechanisms, chemical marks made on the histone proteins around which genomic DNA is wound to form chromatin, and on nucleotides of the DNA itself. In particular, alcohol has been shown to perturb the epigenetic machinery, leading to changes in gene expression and cellular functions characteristic of AUD and, ultimately, to altered behavior. DNA modifications in particular are seeing increasing research in the context of alcohol use and abuse. To date, studies of DNA modifications in AUD have primarily looked at global methylation profiles in human brain and blood, gene-specific methylation profiles in animal models, methylation changes associated with prenatal ethanol exposure, and the potential therapeutic abilities of DNA methyltransferase inhibitors. Future studies may be aimed at identifying changes to more recently discovered DNA modifications, utilizing new methods to discriminate methylation profiles between cell types, thus clarifying how alcohol influences the methylomes of cell-type populations and how this may affect downstream processes. These studies and more in-depth probing of DNA methylation will be key to determining whether DNA-level epigenetic regulation plays a causative role in AUD and can thus be targeted for treatment of the disorder.
Gender differences in psychiatric disorders such as addiction may be modulated by the steroid hormone estrogen. For instance, 17β-estradiol (E2), the predominant form of circulating estrogen in pre-menopausal females, increases ethanol consumption, suggesting that E2 may affect the rewarding properties of ethanol and thus the development of alcohol use disorder in females. The ventral tegmental area (VTA) is critically involved in the rewarding and reinforcing effects of ethanol. In order to determine the role of E2 in VTA physiology, gonadally intact female mice were sacrificed during diestrus II (high E2) or estrus (low E2) for electrophysiology recordings. We measured the excitation by ethanol and inhibition by dopamine (DA) of VTA DA neurons and found that both excitation by ethanol and inhibition by dopamine were greater in diestrus II compared with estrus. Treatment of VTA slices from mice in diestrus II with an estrogen receptor antagonist (ICI 182,780) reduced ethanol-stimulated neuronal firing, but had no effect on ethanol-stimulated firing of neurons in slices from mice in estrus. Surprisingly, ICI 182,780 did not affect the inhibition by DA, indicating different mechanisms of action of estrogen receptors in altering ethanol and DA responses. We also examined the responses of VTA DA neurons to ethanol and DA in ovariectomized mice treated with E2 and found that E2 treatment enhanced the responses to ethanol and DA in a manner similar to what we observed in mice in diestrus II. Our data indicate that E2 modulates VTA neuron physiology, which may contribute to both the enhanced reinforcing and rewarding effects of alcohol and the development of other psychiatric disorders in females that involve alterations in DA neurotransmission.
The basolateral nucleus of the amygdala (BLA) is critical to the pathophysiology of anxiety-driven alcohol drinking and relapse. The endogenous cannabinoid/type 1 cannabinoid receptor (eCB/CB1 ) system curbs BLA-driven anxiety and stress responses via a retrograde negative feedback system that inhibits neurotransmitter release, and BLA CB1 activation reduces GABA release and drives anxiogenesis. Additionally, decreased amygdala CB1 is observed in abstinent alcoholic patients and ethanol withdrawn rats. Here, we investigated the potential disruption of eCB/CB1 signaling on GABAergictransmission in BLA pyramidal neurons of rats exposed to 2-3 weeks intermittent ethanol. In the naïve rat BLA, the CB1 agonist WIN 55,212-2 (WIN) decreased GABA release, and this effect was prevented by the CB1 antagonist AM251. AM251 alone increased GABA release via a mechanism requiring postsynaptic calcium-dependent activity. This retrograde tonic eCB/CB1 signaling was diminished in chronic ethanol exposed rats, suggesting a functional impairment of the eCB/CB1 system. In contrast, acute ethanol increased GABAergictransmission similarly in naïve and chronic ethanol exposed rats, via both presynaptic and postsynaptic mechanisms. Notably, CB1 activation impaired ethanol's facilitation of GABAergictransmission across both groups, but the AM251-induced and ethanol-induced facilitation of GABA release was additive, suggesting independent presynaptic sites of action. Collectively, the present findings highlight a critical CB1 influence on BLA GABAergictransmission that is dysregulated by chronic ethanol exposure and, thus, may contribute to the alcohol-dependent state