Chemogenetic inhibition of central amygdala CRF-expressing neurons decreases alcohol intake but not trauma-related behaviors in a rat model of post-traumatic stress and alcohol use disorder.

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are often comorbid. Few treatments exist to reduce comorbid PTSD/AUD. Elucidating the mechanisms underlying their comorbidity could reveal new avenues for therapy. Here, we employed a model of comorbid PTSD/AUD, in which rats were subjected to a stressful shock in a familiar context followed by alcohol drinking. We then examined fear overgeneralization and irritability in these rats. Familiar context stress elevated drinking, increased fear overgeneralization, increased alcohol-related aggressive signs, and elevated peripheral stress hormones. We then examined transcripts of stress- and fear-relevant genes in the central amygdala (CeA), a locus that regulates stress-mediated alcohol drinking. Compared with unstressed rats, stressed rats exhibited increases in CeA transcripts for Crh and Fkbp5 and decreases in transcripts for Bdnf and Il18. Levels of Nr3c1 mRNA, which encodes the glucocorticoid receptor, increased in stressed males but decreased in stressed females. Transcripts of Il18 binding protein (Il18bp), Glp-1r, and genes associated with calcitonin gene-related peptide signaling (Calca, Ramp1, Crlr-1, and Iapp) were unaltered. Crh, but not Crhr1, mRNA was increased by stress; thus, we tested whether inhibiting CeA neurons that express corticotropin-releasing factor (CRF) suppress PTSD/AUD-like behaviors. We used Crh-Cre rats that had received a Cre-dependent vector encoding hM4D(Gi), an inhibitory Designer Receptors Exclusively Activated by Designer Drugs. Chemogenetic inhibition of CeA CRF neurons reduced alcohol intake but not fear overgeneralization or irritability-like behaviors. Our findings suggest that CeA CRF modulates PTSD/AUD comorbidity, and inhibiting CRF neural activity is primarily associated with reducing alcohol drinking but not trauma-related behaviors that are associated with PTSD/AUD.

Ethanol's interaction with BK channel α subunit residue K361 does not mediate behavioral responses to alcohol in mice.

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol's direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary ß subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

Withdrawal from chronic alcohol impairs the serotonin-mediated modulation of GABAergic transmission in the infralimbic cortex in male rats.

The infralimbic cortex (IL) is part of the medial prefrontal cortex (mPFC), exerting top-down control over structures that are critically involved in the development of alcohol use disorder (AUD). Activity of the IL is tightly controlled by γ-aminobutyric acid (GABA) transmission, which is susceptible to chronic alcohol exposure and withdrawal. This inhibitory control is regulated by various neuromodulators, including 5-hydroxytryptamine (5-HT; serotonin). We used chronic intermittent ethanol vapor inhalation exposure, a model of AUD, in male Sprague-Dawley rats to induce alcohol dependence (Dep) followed by protracted withdrawal (WD; 2 weeks) and performed ex vivo electrophysiology using whole-cell patch clamp to study GABAergic transmission in layer V of IL pyramidal neurons. We found that WD increased frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs), whereas miniature IPSCs (mIPSCs; recorded in the presence of tetrodotoxin) were unaffected by either Dep or WD. The application of 5-HT (50 µM) increased sIPSC frequencies and amplitudes in naive and Dep rats but reduced sIPSC frequencies in WD rats. Additionally, 5-HT2A receptor antagonist M100907 and 5-HT2C receptor antagonist SB242084 reduced basal GABA release in all groups to a similar extent. The blockage of either 5-HT2A or 5-HT2C receptors in WD rats restored the impaired response to 5-HT, which then resembled responses in naive rats. Our findings expand our understanding of synaptic inhibition in the IL in AUD, indicating that antagonism of 5-HT2A and 5-HT2C receptors may restore GABAergic control over IL pyramidal neurons. SIGNIFICANCE STATEMENT: Impairment in the serotonergic modulation of GABAergic inhibition in the medial prefrontal cortex contributes to alcohol use disorder (AUD). We used a well-established rat model of AUD and ex vivo whole-cell patch-clamp electrophysiology to characterize the serotonin modulation of GABAergic transmission in layer V infralimbic (IL) pyramidal neurons in ethanol-naive, ethanol-dependent (Dep), and ethanol-withdrawn (WD) male rats. We found increased basal inhibition following WD from chronic alcohol and altered serotonin modulation. Exogenous serotonin enhanced GABAergic transmission in naive and Dep rats but reduced it in WD rats. 5-HT2A and 5-HT2C receptor blockage in WD rats restored the typical serotonin-mediated enhancement of GABAergic inhibition. Our findings expand our understanding of synaptic inhibition in the infralimbic neurons in AUD.

Brain-specific serine/threonine-protein kinase 1 is a substrate of protein kinase C epsilon involved in sex-specific ethanol and anxiety phenotypes.

Protein kinase C epsilon (PKCε) regulates behavioural responses to ethanol and plays a role in anxiety-like behaviour, but knowledge is limited on downstream substrates of PKCε that contribute to these behaviours. We recently identified brain-specific serine/threonine-protein kinase 1 (BRSK1) as a substrate of PKCε. Here, we test the hypothesis that BRSK1 mediates responses to ethanol and anxiety-like behaviours that are also PKCε dependent. We used in vitro kinase assays to further validate BRSK1 as a substrate of PKCε and used Brsk1-/- mice to assess the role of BRSK1 in ethanol- and anxiety-related behaviours and in physiological responses to ethanol. We found that BRSK1 is phosphorylated by PKCε at a residue identified in a chemical genetic screen of PKCε substrates in mouse brain. Like Prkce-/- mice, male and female Brsk1-/- mice were more sensitive than wild-type to the acute sedative-hypnotic effect of alcohol. Unlike Prkce-/- mice, Brsk1-/- mice responded like wild-type to ataxic doses of ethanol. Although in Prkce-/- mice ethanol consumption and reward are reduced in both sexes, they were reduced only in female Brsk1-/- mice. Ex vivo slice electrophysiology revealed that ethanol-induced facilitation of GABA release in the central amygdala was absent in male Brsk1-/- mice similar to findings in male Prkce-/- mice. Collectively, these results indicate that BRSK1 is a target of PKCε that mediates some PKCε-dependent responses to ethanol in a sex-specific manner and plays a role distinct from PKCε in anxiety-like behaviour.

Ethanol withdrawal-induced adaptations in prefrontal corticotropin releasing factor receptor 1-expressing neurons regulate anxiety and conditioned rewarding effects of ethanol.

Prefrontal circuits are thought to underlie aberrant emotion contributing to relapse in abstinence; however, the discrete cell-types and mechanisms remain largely unknown. Corticotropin-releasing factor and its cognate type-1 receptor, a prominent brain stress system, is implicated in anxiety and alcohol use disorder (AUD). Here, we tested the hypothesis that medial prefrontal cortex CRF1-expressing (mPFCCRF1+) neurons comprise a distinct population that exhibits neuroadaptations following withdrawal from chronic ethanol underlying AUD-related behavior. We found that mPFCCRF1+ neurons comprise a glutamatergic population with distinct electrophysiological properties and regulate anxiety and conditioned rewarding effects of ethanol. Notably, mPFCCRF1+ neurons undergo unique neuroadaptations compared to neighboring neurons including a remarkable decrease in excitability and glutamatergic signaling selectively in withdrawal, which is driven in part by the basolateral amygdala. To gain mechanistic insight into these electrophysiological adaptations, we sequenced the transcriptome of mPFCCRF1+ neurons and found that withdrawal leads to an increase in colony-stimulating factor 1 (CSF1) in this population. We found that selective overexpression of CSF1 in mPFCCRF1+ neurons is sufficient to decrease glutamate transmission, heighten anxiety, and abolish ethanol reinforcement, providing mechanistic insight into the observed mPFCCRF1+ synaptic adaptations in withdrawal that drive these behavioral phenotypes. Together, these findings highlight mPFCCRF1+ neurons as a critical site of enduring adaptations that may contribute to the persistent vulnerability to ethanol misuse in abstinence, and CSF1 as a novel target for therapeutic intervention for withdrawal-related negative affect.

Alcohol dependence and withdrawal increase sensitivity of central amygdalar GABAergic synapses to the glucocorticoid receptor antagonist mifepristone in male rats.

Aberrant glucocorticoid signaling via glucocorticoid receptors (GR) plays a critical role in alcohol use disorder (AUD). Acute alcohol withdrawal and protracted abstinence in dependent rats are associated with increased GR signaling and changes in GR-mediated transcriptional activity in the rat central nucleus of the amygdala (CeA). The GR antagonist mifepristone decreases alcohol consumption in dependent rats during acute withdrawal and protracted abstinence. Regulation of CeA synaptic activity by GR is currently unknown. Here, we utilized mifepristone and the selective GR antagonist CORT118335 (both at 10 µM) as pharmacological tools to dissect the role of GR on GABA transmission in male, adult Sprague-Dawley rats using slice electrophysiology. We subjected rats to chronic intermittent alcohol vapor exposure for 5-7 weeks to induce alcohol dependence. A subset of dependent rats subsequently underwent protracted alcohol withdrawal for 2 weeks, and air-exposed rats served as controls. Mifepristone reduced the frequency of pharmacologically-isolated spontaneous inhibitory postsynaptic currents (sIPSC) in the CeA (medial subdivision) without affecting postsynaptic measures in all groups, suggesting decreased GABA release with the largest effect in dependent rats. CORT118335 did not significantly alter GABA transmission in naïve, but decreased sIPSC frequency in dependent rats. Similarly, mifepristone decreased amplitudes of evoked inhibitory postsynaptic potentials only in dependent rats and during protracted withdrawal. Collectively, our study provides insight into regulation of CeA GABAergic synapses by GR. Chronic ethanol enhances the efficiency of mifepristone and CORT118335, thus highlighting the potential of drugs targeting GR as a promising pharmacological avenue for the treatment of AUD.

Importance of sex and trauma context on circulating cytokines and amygdalar GABAergic signaling in a comorbid model of posttraumatic stress and alcohol use disorders.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share mechanisms that could be therapeutic targets. To facilitate mechanistic studies, we adapted an inhibitory avoidance-based "2-hit" rat model of posttraumatic stress disorder (PTSD) and identified predictors and biomarkers of comorbid alcohol (ethanol)/PTSD-like symptoms in these animals. Stressed Wistar rats received a single footshock on two occasions. The first footshock occurred when rats crossed into the dark chamber of a shuttle box. Forty-eight hours later, rats received the second footshock in a familiar (FAM) or novel (NOV) context. Rats then received 4 weeks of two-bottle choice (2BC) ethanol access. During subsequent abstinence, PTSD-like behavior responses, GABAergic synaptic transmission in the central amygdala (CeA), and circulating cytokine levels were measured. FAM and NOV stress more effectively increased 2BC drinking in males and females, respectively. Stressed male rats, especially drinking-vulnerable individuals (≥0.8 g/kg average 2-h ethanol intake with >50% ethanol preference), showed higher fear overgeneralization in novel contexts, increased GABAergic transmission in the CeA, and a profile of increased G-CSF, GM-CSF, IL-13, IL-6, IL-17a, leptin, and IL-4 that discriminated between stress context (NOV > FAM > Control). However, drinking-resilient males showed the highest G-CSF, IL-13, and leptin levels. Stressed females showed increased acoustic startle and decreased sleep maintenance, indicative of hyperarousal, with increased CeA GABAergic transmission in NOV females. This paradigm promotes key features of PTSD, including hyperarousal, fear generalization, avoidance, and sleep disturbance, with comorbid ethanol intake, in a sex-specific fashion that approximates clinical comorbidities better than existing models, and identifies increased CeA GABAergic signaling and a distinct pro-hematopoietic, proinflammatory, and pro-atopic cytokine profile that may aid in treatment.

Alcohol Dependence Induces CRF Sensitivity in Female Central Amygdala GABA Synapses.

Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by the recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin-releasing factor (CRF)-CRF1 receptor and Gamma-Aminobutyric Acid (GABA)-ergic signaling dysregulation occurring in alcohol-dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch-clamp electrophysiology to examine the effects of alcohol dependence on the CRF modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared to males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had fewer CeA neurons containing mRNA for the CRF1 receptor (Crhr1) than males, but in dependence, the percentage of Crhr1-expressing neurons in females increased, unlike in males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.

Alcohol Dependence and Withdrawal Impair Serotonergic Regulation of GABA Transmission in the Rat Central Nucleus of the Amygdala.

Excessive serotonin (5-HT) signaling plays a critical role in the etiology of alcohol use disorder. The central nucleus of the amygdala (CeA) is a key player in alcohol-dependence associated behaviors. The CeA receives dense innervation from the dorsal raphe nucleus, the major source of 5-HT, and expresses 5-HT receptor subtypes (e.g., 5-HT2C and 5-HT1A) critically linked to alcohol use disorder. Notably, the role of 5-HT regulating rat CeA activity in alcohol dependence is poorly investigated. Here, we examined neuroadaptations of CeA 5-HT signaling in adult, male Sprague Dawley rats using an established model of alcohol dependence (chronic intermittent alcohol vapor exposure), ex vivo slice electrophysiology and ISH. 5-HT increased frequency of sIPSCs without affecting postsynaptic measures, suggesting increased CeA GABA release in naive rats. In dependent rats, this 5-HT-induced increase of GABA release was attenuated, suggesting blunted CeA 5-HT sensitivity, which partially recovered in protracted withdrawal (2 weeks). 5-HT increased vesicular GABA release in naive and dependent rats but had split effects (increase and decrease) after protracted withdrawal indicative of neuroadaptations of presynaptic 5-HT receptors. Accordingly, 5-HT abolished spontaneous neuronal firing in naive and dependent rats but had bidirectional effects in withdrawn. Alcohol dependence and protracted withdrawal did not alter either 5-HT1A-mediated decrease of CeA GABA release or Htr1a expression but disrupted 5-HT2C-signaling without affecting Htr2c expression. Collectively, our study provides detailed insights into modulation of CeA activity by the 5-HT system and unravels the vulnerability of the CeA 5-HT system to chronic alcohol and protracted withdrawal.SIGNIFICANCE STATEMENT Elevated GABA signaling in the central nucleus of the amygdala (CeA) underlies key behaviors associated with alcohol dependence. The CeA is reciprocally connected with the dorsal raphe nucleus, the main source of serotonin (5-HT) in the mammalian brain, and excessive 5-HT signaling is critically implicated in the etiology of alcohol use disorder. Our study, using a well-established rat model of alcohol dependence, ex vivo electrophysiology and ISH, provides mechanistic insights into how both chronic alcohol exposure and protracted withdrawal dysregulate 5-HT signaling in the CeA. Thus, our study further expands our understanding of CeA cellular mechanisms involved in the pathophysiology of alcohol dependence and withdrawal.

Sex and context differences in the effects of trauma on comorbid alcohol use and post-traumatic stress phenotypes in actively drinking rats.

Alcohol use disorder (AUD) and affective disorders are frequently comorbid and share underlying mechanisms that could be targets for comprehensive treatment. Post-traumatic stress disorder (PTSD) has high comorbidity with AUD, but comprehensive models of this overlap are nascent. We recently characterized a model of comorbid AUD and PTSD-like symptoms, wherein stressed rats receive an inhibitory avoidance (IA)-related footshock on two occasions followed by two-bottle choice (2BC) voluntary alcohol drinking. Stressed rats received the second footshock in a familiar (FAM, same IA box as the first footshock) or novel context (NOV, single-chambered apparatus); the FAM paradigm more effectively increased alcohol drinking in males and the NOV paradigm in females. During abstinence, stressed males displayed avoidance-like PTSD symptoms, and females showed hyperarousal-like PTSD symptoms. Rats in the model had altered spontaneous action potential-independent GABAergic transmission in the central amygdala (CeA), a brain region key in alcohol dependence and stress-related signaling. However, PTSD sufferers may have alcohol experience prior to their trauma. Here, we therefore modified our AUD/PTSD comorbidity model to provide 3 weeks of intermittent extended alcohol access before footshock and then studied the effects of NOV and FAM stress on drinking and PTSD phenotypes. NOV stress suppressed the escalation of alcohol intake and preference seen in male controls, but no stress effects were seen on drinking in females. Additionally, NOV males had decreased action potential-independent presynaptic GABA release and delayed postsynaptic GABAA receptor kinetics in the CeA compared to control and FAM males. Despite these changes to alcohol intake and CeA GABA signaling, stressed rats showed broadly similar anxiogenic-like behaviors to our previous comorbid model, suggesting decoupling of the PTSD symptoms from the AUD vulnerability for some of these animals. The collective results show the importance of alcohol history and trauma context in vulnerability to comorbid AUD/PTSD-like symptoms.

Sex Differences in Acute Alcohol Sensitivity of Naïve and Alcohol Dependent Central Amygdala GABA Synapses.

AIMS: Alcohol use disorder (AUD) is linked to hyperactivity of brain stress systems, leading to withdrawal states which drive relapse. AUD differs among the sexes, as men are more likely to have AUD than women, but women progress from casual use to binge and heavy alcohol use more quickly and are more likely to relapse into repetitive episodes of heavy drinking. In alcohol dependence animal models of AUD, the central amygdala (CeA) functions as a hub of stress and anxiety processing and gamma-Aminobutyric acid (GABA)ergic signaling within the CeA is involved in dependence-induced increases in alcohol consumption. We have shown dysregulation of CeA GABAergic synaptic signaling in alcohol dependence animal models, but previous studies have exclusively used males. METHODS: Here, we used whole-cell patch clamp electrophysiology to examine basal CeA GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) and the effects of acute alcohol in both naïve and alcohol dependent rats of both sexes. RESULTS: We found that sIPSC kinetics differ between females and males, as well as between naïve and alcohol-dependent animals, with naïve females having the fastest current kinetics. Additionally, we find differences in baseline current kinetics across estrous cycle stages. In contrast to the increase in sIPSC frequency routinely found in males, acute alcohol (11-88 mM) had no effect on sIPSCs in naïve females, however the highest concentration of alcohol increased sIPSC frequency in dependent females. CONCLUSION: These results provide important insight into sex differences in CeA neuronal function and dysregulation with alcohol dependence and highlight the need for sex-specific considerations in the development of effective AUD treatment.

Cessation of fluoxetine treatment increases alcohol seeking during relapse and dysregulates endocannabinoid and glutamatergic signaling in the central amygdala.

Administration of selective serotonin reuptake inhibitors (SSRIs), typically used as antidepressants, induces long-lasting behavioral changes associated with alcohol use disorder (AUD). However, the contribution of SSRI (fluoxetine)-induced alterations in neurobiological processes underlying alcohol relapse such as endocannabinoid and glutamate signaling in the central amygdala (CeA) remains largely unknown. We utilized an integrative approach to study the effects of repeated fluoxetine administration during abstinence on ethanol drinking. Gene expression and biochemical and electrophysiological studies explored the hypothesis that dysregulation in glutamatergic and endocannabinoid mechanisms in the CeA underlie the susceptibility to alcohol relapse. Cessation of daily treatment with fluoxetine (10 mg/kg) during abstinence resulted in a marked increase in ethanol seeking during re-exposure periods. The increase in ethanol self-administration was associated with (a) reductions in levels of the endocannabinoids N-arachidonoylethanolomine and 2-arachidonoylglycerol in the CeA, (b) increased amygdalar gene expression of cannabinoid type-1 receptor (CB1), N-acyl phosphatidylethanolamine phospholipase D (Nape-pld), fatty acid amid hydrolase (Faah), (c) decreased amygdalar gene expression of ionotropic AMPA (GluA2 and GluA4) and metabotropic (mGlu3) glutamate receptors, and (d) increased glutamatergic receptor function. Overall, our data suggest that the administration of the antidepressant fluoxetine during abstinence dysregulates endocannabinoid signaling and glutamatergic receptor function in the amygdala, facts that likely facilitate alcohol drinking behavior during relapse.

Increased IL-6 expression in astrocytes is associated with emotionality, alterations in central amygdala GABAergic transmission, and excitability during alcohol withdrawal.

Accumulating evidence from preclinical and clinical studies has implicated a role for the cytokine IL-6 in a variety of CNS diseases including anxiety-like and depressive-like behaviors, as well as alcohol use disorder. Here we use homozygous and heterozygous transgenic mice expressing elevated levels of IL-6 in the CNS due to increased astrocyte expression and non-transgenic littermates to examine a role for astrocyte-produced IL-6 in emotionality (response to novelty, anxiety-like, and depressive-like behaviors). Our results from homozygous IL-6 mice in a variety of behavioral tests (light/dark transfer, open field, digging, tail suspension, and forced swim tests) support a role for IL-6 in stress-coping behaviors. Ex vivo electrophysiological studies of neuronal excitability and inhibitory GABAergic synaptic transmission in the central nucleus of the amygdala (CeA) of the homozygous transgenic mice revealed increased inhibitory GABAergic signaling and increased excitability of CeA neurons, suggesting a role for astrocyte produced IL-6 in the amygdala in exploratory drive and depressive-like behavior. Furthermore, studies in the hippocampus of activation/expression of proteins associated with IL-6 signal transduction and inhibitory GABAergic mechanisms support a role for astrocyte produced IL-6 in depressive-like behaviors. Our studies indicate a complex and dose-dependent relationship between IL-6 and behavior and implicate IL-6 induced neuroadaptive changes in neuronal excitability and the inhibitory GABAergic system as important contributors to altered behavior associated with IL-6 expression in the CNS.

IL-1ß expression is increased and regulates GABA transmission following chronic ethanol in mouse central amygdala.

The interleukin-1 system (IL-1) is a prominent pro-inflammatory pathway responsible for the initiation and regulation of immune responses. Human genetic and preclinical studies suggest a critical role for IL-1ß signaling in ethanol drinking and dependence, but little is known about the effects of chronic ethanol on the IL-1 system in addiction-related brain regions such as the central amygdala (CeA). In this study, we generated naïve, non-dependent (Non-Dep) and dependent (Dep) male mice using a paradigm of chronic-intermittent ethanol vapor exposure interspersed with two-bottle choice to examine 1) the expression of IL-1ß, 2) the role of the IL-1 system on GABAergic transmission, and 3) the potential interaction with the acute effects of ethanol in the CeA. Immunohistochemistry with confocal microscopy was used to assess expression of IL-1ß in microglia and neurons in the CeA, and whole-cell patch clamp recordings were obtained from CeA neurons to measure the effects of IL-1ß (50 ng/ml) or the endogenous IL-1 receptor antagonist (IL-1ra; 100 ng/ml) on action potential-dependent spontaneous inhibitory postsynaptic currents (sIPSCs). Overall, we found that IL-1ß expression is significantly increased in microglia and neurons of Dep compared to Non-Dep and naïve mice, IL-1ß and IL-1ra bi-directionally modulate GABA transmission through both pre- and postsynaptic mechanisms in all three groups, and IL-1ß and IL-1ra do not alter the facilitation of GABA release induced by acute ethanol. These data suggest that while ethanol dependence induces a neuroimmune response in the CeA, as indicated by increased IL-1ß expression, this does not significantly alter the neuromodulatory role of IL-1ß on synaptic transmission.

Genetic and Pharmacologic Manipulation of TLR4 Has Minimal Impact on Ethanol Consumption in Rodents.

Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target. SIGNIFICANCE STATEMENT: Toll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human alcoholics. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium participated in the first comprehensive study across multiple laboratories to test the hypothesis that TLR4 regulates excessive alcohol consumption in different species and different models of chronic, dependence-driven, and binge-like drinking. Although TLR4 was not a critical determinant of excessive drinking, it was important in the acute sedative effects of alcohol. Current research efforts are directed at determining which neuroimmune pathways mediate excessive alcohol drinking and these findings will help to prioritize relevant pathways and potential therapeutic targets.

Ethanol and Cytokines in the Central Nervous System.

The innate immune system plays a critical role in the ethanol-induced neuroimmune response in the brain. Ethanol initiates the innate immune response via activation of the innate immune receptors Toll-like receptors (TLRs, e.g., TLR4, TLR3, TLR7) and NOD-like receptors (inflammasome NLRs) leading to a release of a plethora of chemokines and cytokines and development of the innate immune response. Cytokines and chemokines can have pro- or anti-inflammatory properties through which they regulate the immune response. In this chapter, we will focus on key cytokines (e.g., IL-1, IL-6, TNF-α) and chemokines (e.g., MCP-1/CCL2) that mediate the ethanol-induced neuroimmune responses. In this regard, we will use IL-1ß, as an example cytokine, to discuss the neuromodulatory properties of cytokines on cellular properties and synaptic transmission. We will discuss their involvement through a set of evidence: (1) changes in gene and protein expression following ethanol exposure, (2) association of gene polymorphisms (humans) and alterations in gene expression (animal models) with increased alcohol intake, and (3) modulation of alcohol-related behaviors by transgenic or pharmacological manipulations of chemokine and cytokine systems. Over the last years, our understanding of the molecular mechanisms mediating cytokine- and chemokine-dependent regulation of immune responses has advanced tremendously, and we review evidence pointing to cytokines and chemokines serving as neuromodulators and regulators of neurotransmission.

Chronic alcohol exposure disrupts CB1 regulation of GABAergic transmission in the rat basolateral amygdala.

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 GABAergic transmission 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 GABAergic transmission similarly in naïve and chronic ethanol exposed rats, via both presynaptic and postsynaptic mechanisms. Notably, CB1 activation impaired ethanol's facilitation of GABAergic transmission 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 GABAergic transmission that is dysregulated by chronic ethanol exposure and, thus, may contribute to the alcohol-dependent state.

Chronic ethanol exposure decreases CB1 receptor function at GABAergic synapses in the rat central amygdala.

The endogenous cannabinoids (eCBs) influence the acute response to ethanol and the development of tolerance, dependence and relapse. Chronic alcohol exposure alters eCB levels and Type 1 cannabinoid receptor (CB1 ) expression and function in brain regions associated with addiction. CB1 inhibits GABA release, and GABAergic dysregulation in the central nucleus of the amygdala (CeA) is critical in the transition to alcohol dependence. We investigated possible disruptions in CB1 signaling of rat CeA GABAergic transmission following intermittent ethanol exposure. In the CeA of alcohol-naive rats, CB1 agonist WIN 55,212-2 (WIN) decreased the frequency of spontaneous and miniature GABAA receptor-mediated inhibitory postsynaptic currents (s/mIPSCs). This effect was prevented by CB1 antagonism, but not Type 2 cannabinoid receptor (CB2 ) antagonism. After 2-3 weeks of intermittent ethanol exposure, these WIN inhibitory effects were attenuated, suggesting ethanol-induced impairments in CB1 function. The CB1 antagonist AM251 revealed a tonic eCB/CB1 control of GABAergic transmission in the alcohol-naive CeA that was occluded by calcium chelation in the postsynaptic cell. Chronic ethanol exposure abolished this tonic CB1 influence on mIPSC, but not sIPSC, frequency. Finally, acute ethanol increased CeA GABA release in both naive and ethanol-exposed rats. Although CB1 activation prevented this effect, the AM251- and ethanol-induced GABA release were additive, ruling out a direct participation of CB1 signaling in the ethanol effect. Collectively, these observations demonstrate an important CB1 influence on CeA GABAergic transmission and indicate that the CeA is particularly sensitive to alcohol-induced disruptions of CB1 signaling.

Innate immune factors modulate ethanol interaction with GABAergic transmission in mouse central amygdala.

Excessive ethanol drinking in rodent models may involve activation of the innate immune system, especially toll-like receptor 4 (TLR4) signaling pathways. We used intracellular recording of evoked GABAergic inhibitory postsynaptic potentials (eIPSPs) in central amygdala (CeA) neurons to examine the role of TLR4 activation by lipopolysaccharide (LPS) and deletion of its adapter protein CD14 in acute ethanol effects on the GABAergic system. Ethanol (44, 66 or 100mM) and LPS (25 and 50µg/ml) both augmented eIPSPs in CeA of wild type (WT) mice. Ethanol (44mM) decreased paired-pulse facilitation (PPF), suggesting a presynaptic mechanism of action. Acute LPS (25µg/ml) had no effect on PPF and significantly increased the mean miniature IPSC amplitude, indicating a postsynaptic mechanism of action. Acute LPS pre-treatment potentiated ethanol (44mM) effects on eIPSPs in WT mice and restored ethanol's augmenting effects on the eIPSP amplitude in CD14 knockout (CD14 KO) mice. Both the LPS and ethanol (44-66mM) augmentation of eIPSPs was diminished significantly in most CeA neurons of CD14 KO mice; however, ethanol at the highest concentration tested (100mM) still increased eIPSP amplitudes. By contrast, ethanol pre-treatment occluded LPS augmentation of eIPSPs in WT mice and had no significant effect in CD14 KO mice. Furthermore, (+)-naloxone, a TLR4-MD-2 complex inhibitor, blocked LPS effects on eIPSPs in WT mice and delayed the ethanol-induced potentiation of GABAergic transmission. In CeA neurons of CD14 KO mice, (+)-naloxone alone diminished eIPSPs, and subsequent co-application of 100mM ethanol restored the eIPSPs to baseline levels. In summary, our results indicate that TLR4 and CD14 signaling play an important role in the acute ethanol effects on GABAergic transmission in the CeA and support the idea that CD14 and TLR4 may be therapeutic targets for treatment of alcohol abuse.

Functional and morphological adaptation of medial prefrontal corticotropin releasing factor receptor 1-expressing neurons in male mice following chronic ethanol exposure.

Chronic ethanol dependence and withdrawal activate corticotropin releasing factor (CRF)-containing GABAergic neurons in the medial prefrontal cortex (mPFC), which tightly regulate glutamatergic pyramidal neurons. Using male CRF1:GFP reporter mice, we recently reported that CRF1-expressing (mPFCCRF1+) neurons predominantly comprise mPFC prelimbic layer 2/3 pyramidal neurons, undergo profound adaptations following chronic ethanol exposure, and regulate anxiety and conditioned rewarding effects of ethanol. To explore the effects of acute and chronic ethanol exposure on glutamate transmission, the impact of chronic alcohol on spine density and morphology, as well as persistent changes in dendritic-related gene expression, we employed whole-cell patch-clamp electrophysiology, diOlistic labeling for dendritic spine analysis, and dendritic gene expression analysis to further characterize mPFCCRF1+ and mPFCCRF1- prelimbic layer 2/3 pyramidal neurons. We found increased glutamate release in mPFCCRF1+ neurons with ethanol dependence, which recovered following withdrawal. In contrast, we did not observe significant changes in glutamate transmission in neighboring mPFCCRF1- neurons. Acute application of 44 mM ethanol significantly reduced glutamate release onto mPFCCRF1+ neurons, which was observed across all treatment groups. However, this sensitivity to acute ethanol was only evident in mPFCCRF1- neurons during withdrawal. In line with alterations in glutamate transmission, we observed a decrease in total spine density in mPFCCRF1+ neurons during dependence, which recovered following withdrawal, while again no changes were observed in mPFCCRF- neurons. Given the observed decreases in mPFCCRF1+ stubby spines during withdrawal, we then identified persistent changes at the dendritic gene expression level in mPFCCRF1+ neurons following withdrawal that may underlie these structural adaptations. Together, these findings highlight the varying responses of mPFCCRF1+ and mPFCCRF1- cell-types to acute and chronic ethanol exposure, as well as withdrawal, revealing specific functional, morphological, and molecular adaptations that may underlie vulnerability to ethanol and the lasting effects of ethanol dependence.