BACKGROUND:Alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD) are highly comorbid, and prior alcohol use may increase susceptibility to PTSD. We recently showed that prior chronic ethanol consumption and withdrawal potentiate future stress-enhanced fear learning. However, the neurobiological mechanism underlying this behavior is unknown. Given the role of astrocytes in the neuroimmune system, we investigated the relationship between the pro-inflammatory cytokine interleukin-1β (IL-1β) and dorsal hippocampal astrocytes in this behavior. METHODS:Experiments 1 and 2 assessed the expression of IL-1β and glial fibrillary acidic protein (GFAP) and their relationship through colocalization in the dentate gyrus (DG) of the dorsal hippocampus (DH) following chronic ethanol consumption and withdrawal and severe stress exposure. Experiments 3 and 4 used an AAV-based clustered regularly interspaced short palindromic repeats (CRISPR) approach to knock down Il1b selectively in dorsal hippocampal astrocytes to test the role of astrocyte-derived IL-1β in ethanol- and stress-enhanced fear learning. RESULTS:We found that severe stress increased IL-1β expression, whereas ethanol consumption and withdrawal increased GFAP expression and the colocalization of IL-1β with GFAP. Knockdown of Il1b in dorsal hippocampal astrocytes was achievable and partially reduced enhanced fear learning following the combination of ethanol withdrawal and severe stress. CONCLUSIONS:Collectively, these results suggest that a complex relationship between IL-1β, astrocyte reactivity, and IL-1β produced by astrocytes may occur in the DH, where ethanol and stress differentially impact these neuroimmune components that are partially involved in the augmentation of stress-enhanced fear learning following prior ethanol dependence and withdrawal.

Ethanol comprises innately aversive properties that may act as a deterrent to overconsumption. We have previously found that chemogenetic activation or inhibition of a noradrenergic locus coeruleus (LC) to rostromedial tegmental nucleus (RMTg) pathway bidirectionally modulates binge-like ethanol intake and aversive reactivity in male and female TH-ires-Cre mice. We previously hypothesized that noradrenergic RMTg circuitry may modulate ethanol intake and aversion through inhibitory inputs to the ventral tegmental area (VTA), a region that is both densely innervated by the RMTg and proposed to coordinate the balance between the rewarding and aversive properties of ethanol. Here, we build upon this work by providing evidence for a direct role of noradrenergic signaling in the VTA in the modulation of binge-like ethanol intake and unconditioned aversive responses. Using "drinking-in-the-dark" procedures, we reveal that site-directed administration of an α-1 adrenergic receptor (AR) agonist into the VTA blunts binge-like ethanol intake and associated blood ethanol concentrations (BECs) without altering sucrose consumption or locomotion. Next, we demonstrate that chemogenetic activation of noradrenergic LC to VTA projection neurons blunts binge-like ethanol intake and BECs in male and female mice without altering sucrose intake or locomotion. Further, we demonstrate that chemogenetic activation of LC to VTA projection neurons induces mid-frequency vocalizations consistent with an aversion- or malaise-like state in mice. The present findings indicate an important involvement of noradrenergic LC to VTA circuitry in the modulation of ethanol intake and aversion.

BACKGROUND:Binge drinking is a risky pattern of alcohol (ethanol) consumption associated with a variety of negative outcomes, including the development of alcohol use disorder (AUD). Many neuropeptide systems are thought to become dysregulated in AUD; however, whether repeated cycles of binge-like ethanol consumption and abstinence following binge-like drinking alter neuropeptide mRNA in key brain regions, such as the medial prefrontal cortex (mPFC), insular cortex (IC), amygdala, and lateral hypothalamus (LH), remains unknown. METHODS:Male and female mice underwent 0, 3, or 6 cycles of binge-like ethanol consumption using the "Drinking in the Dark" (DID) paradigm. Brain tissue was collected either immediately following the final session of DID or after a 24-h period of abstinence, and quantitative polymerase chain reaction (qPCR) was performed to assess how repeated cycles of binge-like ethanol intake and abstinence alter relative mRNA expression for 22 neuropeptide-related targets. RESULTS:We observed that repeated cycles of binge-like ethanol consumption and abstinence altered relative mRNA expression for 11 targets in the mPFC, five targets in the IC, eight targets in the amygdala, and two targets in the LH. Two of these alterations were specific to female mice, while one was specific to male mice. CONCLUSIONS:These data suggest that neuropeptide mRNA is altered by repeated cycles of binge-like ethanol intake and abstinence in a brain region and sex-dependent manner. The current findings provide a useful foundation from which to explore potential targets to decrease binge-like ethanol consumption and prevent the development of AUD.

Background:Binge alcohol drinking is a dangerous behavior that can contribute to the development of more severe alcohol use disorder. Importantly, the rate and severity of alcohol use disorder has historically differed between men and women, suggesting that there may be sex differences in the central mechanisms that modulate alcohol (ethanol) consumption. Corticotropin-releasing factor (CRF) is a centrally expressed neuropeptide that has been implicated in the modulation of binge-like ethanol intake, and emerging data highlight sex differences in CRF systems. Methods:In the current report, we characterized CRF+ neurocircuitry arising from the central nucleus of the amygdala (CeA) and innervating the lateral hypothalamus (LH) in the modulation of binge-like ethanol intake in male and female mice. Results:Using chemogenetic tools, we found that silencing the CRF+ CeA to LH circuit significantly blunted binge-like ethanol intake in male but not female mice. Consistently, genetic deletion of CRF from neurons of the CeA blunted ethanol intake exclusively in male mice. Furthermore, pharmacological blockade of the CRF1 receptor in the LH significantly reduced binge-like ethanol intake in male mice only, while CRF2 receptor activation in the LH failed to alter ethanol intake in either sex. Finally, a history of binge-like ethanol drinking reduced C rf messenger RNA levels in the CeA regardless of sex. Conclusions:These observations provide novel evidence that CRF+ CeA to LH neurocircuitry is more sensitive for modulating binge-like ethanol intake in male mice, which may provide insight into the mechanisms that guide known sex differences in binge-like ethanol intake.

BACKGROUND:Alcohol use disorder is characterized by maladaptive patterns of alcohol consumption, with emerging evidence suggesting that neuropeptide Y (NPY) signaling through Y1 and Y2 receptors (Y1R and Y2R) within the central amygdala (CeA) plays a critical role in modulating ethanol intake. The current experiments investigate the neural mechanisms underlying binge-like ethanol drinking, focusing on the involvement of Y1R+ CeA-to-lateral hypothalamus (LH) projections, dynamic interactions between Y1R and Y2R within the CeA, and the impact of chronic ethanol exposure on Y1R protein expression. METHODS:NPY1R-ires-cre mice received LH cannulation, were infused with cre-dependent inhibitory (Gi) Designer Receptor Exclusively Activated by Designer Drug (DREADD) or control virus into the CeA, and went through drinking in the dark (DID). Other animals were treated intra-CeA with an NPY overexpression vector (FIB-NPY) or control, and went through DID, intermittent access to ethanol (IAE), and open-field testing. Viral placements and receptor targets were assessed via qPCR. Finally, mice went through six cycles of DID, and Y1R immunohistochemical (IHC) labeling on neurons was assessed for animals sacrificed after the final DID session or after a 24-h period of abstinence. RESULTS:Chemogenetic inhibition of Y1R+ CeA-LH projections selectively reduced binge-like ethanol drinking in male mice without affecting female mice. Viral NPY overexpression revealed behavioral effects and predictive relationships between receptor mRNA expression and intake patterns. Although no significant differences were found in Y1R/NeuN colocalization across sex and treatment groups, correlational analyses revealed that Y1R expression varied with individual ethanol consumption. CONCLUSIONS:Collectively, these results support a model wherein Y1R signaling within the CeA regulates ethanol consumption through circuit-specific mechanisms and broader neuroadaptive changes influenced by sex and individual drinking patterns. This research advances our understanding of the neurobiological mechanisms underlying binge-like ethanol intake and highlights the complex, sex-dependent roles of NPY-Y1R and Y2R signaling in the CeA.

While there is strong evidence that the reinforcing effects of ethanol motivate seeking and consumption, ethanol produces aversive effects that limit consumption. We have previously found that in doses that support conditioned taste aversion (CTA) learning ethanol induces activity of noradrenergic (NE+) neurons of the A2 subregion of the nucleus of the solitary tract (NTS) as well as neurons within the lateral parabrachial nucleus (L-PBN), regions that have been implicated in integrating aversive responses. Here we provide evidence of a NE + circuit arising from the A2 and innervating the L-PBN in tyrosine hydroxylase (TH)-ires-cre mice. Next, we used male and female TH-ires-cre mice in tandem with an intersectional chemogenetic approach to assess the role of the NE + A2 to L-BPN circuit in modulating binge-like ethanol intake as well as unconditioned aversive behavior. Using "drinking in the dark" (DID) procedures we found that activating this circuit significantly blunted binge-like ethanol intake and associated blood ethanol concentrations (BECs) without altering sucrose solution intake. Furthermore, silencing this pathway during light cycle drinking revealed a trend of increased ethanol intake and an associated significant increase of BECs with no changes in sucrose intake. Additionally, activation of this circuit, as well as peripheral administration of the emetic agent LiCl, significantly increased the emission of mid-frequency vocalizations (MFVs) in mice, a phenotype reflecting aversive reactivity. The present findings provide novel evidence of a NE + A2 to L-PBN circuit in the modulation of binge-like ethanol intake and aversive responses.

Preclinical and clinical findings support the potential for GLP-1 receptor activation to reduce alcohol consumption and reward. Human experimental studies are needed to clarify whether acute changes in endogenous GLP-1 influence alcohol craving and responses to alcohol. Dietary stimulation may present a method to study the effects of endogenous GLP-1 on responses to alcohol. Assess the effects of a dietary manipulation on acute changes in GLP-1 and laboratory responses to alcohol. Healthy young adult heavy drinkers (N = 40) were recruited to participate in two study visits where they received a dietary supplement designed to increase endogenous GLP-1 or a calorically matched placebo in counterbalanced order. Blood was sampled before and 40 min after supplement or placebo administration to measure changes in plasma GLP-1. Subjective effects, craving, and alcohol attentional bias were measured in response to a priming drink of alcohol (target BAC 30 mg

Neuropeptide Y (NPY) signaling regulation of corticolimbic communication is known to modulate binge-like ethanol consumption in rodents. In this work we sought to assess the impact of intra-BLA NPY system modulation on binge-like ethanol intake and to assess the role of the NPY1R+ projection from the BLA to the mPFC in this behavior. We used “drinking-in-the-dark” (DID) procedures in C57BL6J mice to address these questions. First, the impact of intra-BLA administration of NPY on binge-like ethanol intake was assessed. Next, the impact of repeated cycles of DID intake on NPY1R expression in the BLA was assessed with use of immunohistochemistry (IHC). Finally, chemogenetic inhibition of BLA→mPFC NPY1R+ projections was assessed to determine if limbic communication with the mPFC was specifically involved in binge-like ethanol intake. Importantly, as both the BLA and NPY system are sexually dimorphic, both sexes were assessed in these studies. Intra-BLA NPY dose-dependently decreased binge-like ethanol intake in males only. Repeated DID reduced NPY1R expression in the BLA of both sexes. Silencing of BLA→mPFC NPY1R+ neurons significantly reduced binge-like ethanol intake in both sexes in a dose-dependent manner. We provide novel evidence that (1) intra-BLA NPY reduces binge-like ethanol intake in males; (2) binge-like ethanol intake reduces NPY1R levels in the BLA; and (3) chemogenetic inhibition of BLA→mPFC NPY1R+ neurons blunts binge-like drinking in male and female mice. These observations provide the first direct evidence that NPY signaling in the BLA, and specifically BLA communication with the mPFC, modulates binge-like ethanol consumption.

While there are numerous brain regions that have been shown to play a role in this AUD in humans and animal models, the central nucleus of the amygdala (CeA) has emerged as a critically important locus mediating binge alcohol consumption. In this study, we sought to understand how relative gene expression of key signaling molecules in the CeA changes during different periods of abstinence following bouts of binge drinking. To test this, we performed drinking in the dark (DID) on two separate cohorts of C57BL/6J mice and collected CeA brain tissue at 1 day (acute) and 7 days (protracted) abstinence after DID. We used qRTPCR to evaluate relative gene expression changes of 25 distinct genes of interest related to G protein-coupled receptors (GPCRs), neuropeptides, ion channel subunits, and enzymes that have been previously implicated in AUD. Our findings show that during acute abstinence CeA punches collected from female mice had upregulated relative mRNA expression of the gamma-aminobutyric acid receptor subunit alpha 2 (Gabra2), and the peptidase, angiotensinase c (Prcp). CeA punches from male mice at the same time point in abstinence had upregulated relative mRNA encoding for neuropeptide-related molecules, neuropeptide Y (Npy) and somatostatin (Sst), as well as the neuropeptide Y receptor Y2 (Npyr2), but downregulated Glutamate ionotropic receptor NMDA type subunit 1 (Grin1). After protracted abstinence, CeA punches collected from female mice had increased mRNA expression of corticotropin releasing hormone (Crh) and Npy. CeA punches collected from male mice at the same timepoint had upregulated relative mRNA expression of Npy2r, Npy, and Sst. Our findings support that there are differences in how the CeA of male and female mice respond to binge-alcohol exposure, highlighting the need to understand the implications of such differences in the context of AUD and binge drinking behavior.

Orexin in both the lateral hypothalamus (LH) and medial septum (MS) is involved in sleep- and consciousness-related conditions. Since orexin modulates the intoxicating as well as rewarding effects of ethanol, this study focused on the role of orexin-projecting neurons from the LH to the MS, and this neurocircuit's role in mediating the sedative effects of alcohol. Drinking-in-the-Dark (DID) behavior was also assessed as a measure of the role of the LH-MS pathway in modulating binge-like ethanol intake, with a particular focus on sex differences in both behavioral paradigms. Male and female Hcrt-ires-cre mice received cannulation in the MS, while the LH was injected bilaterally with cre-dependent excitatory (Gq) Designer Receptor Exclusively Activated by Designer Drug (DREADD), inhibitory (Gi) DREADD or control virus. All subjects received a 3.75 g/kg dose of 20 % ethanol intraperitoneally and the sedative effect was assessed by the loss of righting reflex (LORR). After behavioral testing, brains were used for cFos immunohistochemistry analyses. A separate cohort of mice was used for a 2-week DID protocol using excitatory (Gq) DREADD and control virus. Gq DREADD-induced activation of the orexin neurocircuitry from the LH to the MS significantly reduced sedation time in both female and male mice. Furthermore, CNO treatment failed to alter ethanol sedation times in both animals expressing Gi DREADDs and control virus. There were no significant differences in blood ethanol concentrations (BECs) in any experimental group, suggesting that changes in sedation were not due to treatment-induced alterations of ethanol metabolism. Interestingly, in the DID study, only male mice decreased their ethanol consumption when Gq DREADDs were activated. These results provide novel evidence on the role played by this orexinergic LH to MS circuit on the sedative effects of ethanol and ethanol consumption in a sex-dependent manner. Thus, the MS should be considered further as a novel sexually dimorphic target. (c) 2023 Elsevier Inc. All rights reserved.

ABSTRACTBackgroundBinge alcohol drinking is a dangerous pattern of consumption that can contribute to the development of more severe alcohol use disorders (AUDs). Importantly, the rate and severity of AUDs has historically differed between men and women, suggesting that there may be sex differences in the central mechanisms that modulate alcohol (ethanol) consumption. Corticotropin releasing factor (CRF) is a centrally expressed neuropeptide that has been implicated in the modulation of binge-like ethanol intake, and emerging data highlight sex differences in central CRF systems.MethodsIn the present report we characterized CRF+ neurocircuitry arising from the central nucleus of the amygdala (CeA) and innervating the lateral hypothalamus (LH) in the modulation of binge-like ethanol intake in male and female mice.ResultsUsing chemogenetic tools we found that silencing the CRF+ CeA to LH circuit significantly blunted binge-like ethanol intake in male, but not female, mice. Consistently, genetic deletion of CRF from neurons of the CeA blunted ethanol intake exclusively in male mice. Furthermore, pharmacological blockade of the CRF type-1 receptor (CRF1R) in the LH significantly reduced binge-like ethanol intake in male mice only, while CRF2R activation in the LH failed to alter ethanol intake in either sex. Finally, a history of binge-like ethanol drinking blunted CRF mRNA in the CeA regardless of sex.ConclusionsThese observations provide novel evidence that CRF+ CeA to LH neurocircuitry modulates binge-like ethanol intake in male, but not female mice, which may provide insight into the mechanisms that guide known sex differences in binge-like ethanol intake.

We recently showed that chemogenetic activation of the locus coeruleus (LC) to the rostromedial tegmental nucleus (RMTg) noradrenergic (NE) pathway significantly blunted binge-like ethanol drinking and induced aversive-like behaviors in mice. The aim of the present study is to determine if silencing this TH + LC → RMTg noradrenergic pathway promotes increased levels of binge-like ethanol intake and reduced ethanol-induced conditioned taste aversion (CTA). To this end, both male and female TH-ires-cre mice on a C57BL/6 J background were cannulated in the RMTg and injected in the LC with rAVV viruses that encode cre-dependent Gi-expressing designer receptor exclusively activated by designer drugs (DREADDs), or its control, to directly control the activity of NE neurons. Inhibition of the LC to RMTg pathway had no effect on the binge-ethanol drinking in a “drinking-in-the-dark” (DID) paradigm. However, when using this paradigm during the light cycle, silencing of this circuit significantly increased ethanol intake without altering sucrose drinking. Moreover, we found that inhibition of this circuit significantly attenuated an ethanol-induced CTA. In addition, when compared to control animals, pairing RMTg-directed Clozapine N-oxide (CNO) with an i.p. injection of 1.5 g/kg ethanol reduced c-Fos activation in the LC, and increased c-Fos expression in the ventral tegmental area (VTA) in Gi-expressing mice. Our data show that inhibition of the TH + LC to the RMTg pathway significantly increased ethanol drinking as well as attenuated ethanol-induced CTA, supporting the involvement of the LC to RMTg noradrenergic circuit as an important protective mechanism against excessive ethanol consumption.

Somatostatin (SST) is a neuropeptide widely expressed in the central nervous system with dense expression in limbic regions such as the extended amygdala. It has recently gained attention for playing a role in modulating alcohol use disorders and co-morbid neuropsychiatric disorders. However, the role of SST in the central nucleus of the amygdala (CeA), a key region for neuropeptide regulation of alcohol and anxiety related behaviors, in alcohol consumption has not been assessed. In this work we perform an initial examination of the interaction between the CeA SST system and binge ethanol intake. Binge intake is a dangerous pattern of excessive ethanol consumption associated with health complications and the transition into alcohol dependence. We use the Drinking in the Dark (DID) model of binge intake in C57BL/6J male and female mice to examine: 1) the impact of 3 DID cycles on CeA SST expression; 2) the effect of intra-CeA SST injection on binge-like ethanol consumption; and 3) if the SST receptor 2 or 4 (SST2R or SST4R) mediate any effect on consumption. Our results show binge-like ethanol intake decreases SST expression in the CeA, but not neighboring basolateral amygdala. We further found intra-SST CeA administration reduces binge ethanol intake. This decrease was replicated by the administration of an SST4R agonist. These effects were not sex-dependent. Overall, this work lends further support for SST playing a role in alcohol related behaviors and as a potential therapeutic target.

Excessive ethanol drinking is a major problem within the United States, causing alterations in brain plasticity and neurocognitive function. Astrocytes are glial cells that regulate neurosynaptic plasticity, modulate neurochemicals, and monitor other homeostatic roles. Astrocytes have been found to play a part in modulating excessive ethanol consumption. The basolateral amygdala (BLA), central amygdala (CeA), and bed nucleus of the stria terminalis (BNST) are brain regions that process stress, anxiety, and reward; they are also implicated in modulating ethanol intake. Little is understood, however, about how astrocyte expression in each region is modulated by chronic and binge-like ethanol drinking patterns. In the present report, we utilized two separate animal models of excessive drinking: chronic intermittent ethanol (CIE) and "Drinking-in-the-dark" (DID). Following these paradigms, animal brains were processed through immunohistochemistry (IHC) and stained for glial fibrillary acidic protein (GFAP). Collected data illustrated a sex-dependent relationship between ethanol intake and GFAP immunoreactivity (IR) in the BLA and BNST, but not in the CeA. Specifically, CIE and DID ethanol drinking resulted in blunted GFAP-IR (specifically via GFAP-positive cell count) in the BLA and BNST, particularly in males. These findings may implicate sex-dependent ethanol-induced changes in BLA and BNST astrocytes, providing a potential therapeutic target for anxiety and stress disorders.