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Chronic ethanol exposure results in numerous neurobiological adaptations that promote deficits in medial prefrontal cortical (mPFC) function associated with blunted inhibitory control and elevated anxiety during withdrawal. Studies exploring alcohol dependence-related changes in this region have largely investigated adaptations in glutamatergic signaling, with inhibitory neurotransmission remaining relatively understudied. To address this, we used biochemical and electrophysiological methods to evaluate the effects of ethanol on the activity of deep-layer prelimbic mPFC Fast-Spiking (FS) and Martinotti interneurons after chronic ethanol exposure in male and female rats. We report that chronic alcohol exposure significantly impairs FS neuron excitability in both males and females. Interestingly, we observed a marked sex difference in the baseline activity of Martinotti cells that furthermore displayed differential sex-specific responses to alcohol exposure. In addition, alcohol effects on Martinotti neuron excitability negatively correlated with hyperpolarization-activated currents mediated by hyperpolarization-activated cyclic nucleotide gated (HCN) channels, indicative of a causal relationship. Analysis of HCN1 protein expression also revealed a substantial sex difference, although no effect of ethanol on HCN1 protein expression was observed. Taken together, these findings further elucidate the complex adaptations that occur in the mPFC after chronic ethanol exposure and reveal fundamental differences in interneuron activity between sexes. Furthermore, this disparity may reflect innate differences in intracortical microcircuit function between male and female rats, and offers a tenable circuit-level explanation for sex-dependent behavioral responses to alcohol.

One of the hallmarks of alcohol dependence is the presence of a withdrawal syndrome during abstinence, which manifests as physical craving for alcohol accompanied by subjective feelings of anxiety. Using a model of chronic intermittent ethanol (CIE) vapor in mice, we investigated the role of serotonin2c signaling in the BNST as a neural substrate underlying ethanol-induced anxiety during withdrawal. Mice were subjected to a 5-day CIE regimen of 16 hours of ethanol vapor exposure followed by an 8 hour “withdrawal” period between exposures. After the 5th and final exposure, mice were withdrawn for 24 hours or 1 week before experiments began. Anxiety-like behavior was assessed in the social approach, light dark, and open field test with mice showing deficits in social, but not general anxiety-like behavior that was alleviated by pretreatment with the 5HT2c-R antagonist SB 242,084 (3 mg/kg, i.p.) 24 hours and 1 week post-CIE. Using immunohistochemistry and whole cell patch clamp electrophysiology, we also found that CIE increased FOS-IR and enhanced neuronal excitability in the ventral BNST (vBNST) 24 hrs into withdrawal in a 5HT2c-R dependent manner. This enhanced excitability persisted for 1 week post-CIE. We also found that mCPP, a 5HT2c/b agonist, induced a more robust depolarization in cells of the vBNST in CIE mice, confirming that 5HT2c-R signaling is upregulated in the vBNST following CIE. Taken together, these results suggest that CIE upregulates 5HT2c-R signaling in the vBNST, leading to increased excitability. This enhanced excitability of the vBNST may drive increased anxiety-like behavior during ethanol withdrawal.

Interoception refers to the perception of the internal state of the body and is increasingly being recognized as an important factor in mental health disorders. Drugs of abuse produce powerful interoceptive states that are upstream of behaviors that drive and influence drug intake, and addiction pathology is impacted by interoceptive processes. The goal of the present review is to discuss interoceptive processes related to alcohol. We will cover physiological responses to alcohol, how interoceptive states can impact drinking, and the recruitment of brain networks as informed by clinical research. We also review the molecular and brain circuitry mechanisms of alcohol interoceptive effects as informed by preclinical studies. Finally, we will discuss emerging treatments with consideration of interoception processes. As our understanding of the role of interoception in drug and alcohol use grows, we suggest that the convergence of information provided by clinical and preclinical studies will be increasingly important. Given the complexity of interoceptive processing and the multitude of brain regions involved, an overarching network-based framework can provide context for how focused manipulations modulate interoceptive processing as a whole. In turn, preclinical studies can systematically determine the roles of individual nodes and their molecular underpinnings in a given network, potentially suggesting new therapeutic targets and directions. As interoceptive processing drives and influences motivation, emotion, and subsequent behavior, consideration of interoception is important for our understanding of processes that drive ongoing drinking and relapse.

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.

Stressful life events and chronic stressors have been associated with escalations in alcohol drinking. Stress exposure leads to the secretion of glucocorticoids (cortisol in the human; corticosterone (CORT) in the rodent). To model a period of heightened elevations in CORT, the present work assessed the effects of chronic exposure to the stress hormone CORT on alcohol self-administration. Male Long Evans rats were trained to self-administer a sweetened alcohol solution (2% sucrose/15% alcohol) resulting in moderate levels of daily alcohol intake (0.5–0.7 g/kg). Following stable baseline operant self-administration, rats received CORT in the drinking water for 7 days. A transient increase in alcohol self-administration was observed on the first self-administration session following CORT exposure, and behavior returned to control levels by the second session. Control experiments determined that this increase in alcohol self-administration was specific to alcohol, unrelated to general motor activation, and functionally dissociated from decreased CORT levels at the time of testing. These results indicate that repeated exposure to heightened levels of stress hormone (e.g., as may be experienced during stressful episodes) has the potential to lead to exacerbated alcohol intake in low to moderate drinkers. Given that maladaptive drinking patterns, such as escalated alcohol drinking following stressful episodes, have the potential to put an individual at risk for future drinking disorders, utilization of this model will be important for examination of neuroadaptations that occur as a consequence of CORT exposure in order to better understand escalated drinking following stressful episodes in nondependent individuals.

Alcohol is a commonly used drug that can produce alcohol use disorders (AUDs). Few individuals with AUDs receive treatment and treatment options are complicated by issues with effectiveness and compliance. Alcohol has been shown to differentially affect specific brain regions and an improved understanding of circuit-specific dysregulation caused by alcohol is warranted. Previous work has implicated both the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) in alcohol-associated plasticity, however studies directly examining the impact of alcohol exposure on this circuit are lacking. The current study employed an optogenetic strategy to investigate the prelimbic mPFC to BLA circuit and changes in circuit activity following chronic intragastric ethanol exposure in male Sprague Dawley rats. We observed monosynaptic connections with light-evoked stimulation of mPFC terminals in the BLA with efficacy and short latency. We also found that mPFC-BLA projections are primarily glutamatergic under basal inhibitory control, with a lesser population of GABAergic projections. We examined optically-evoked glutamate currents in the BLA using repeated trains of stimulation that displayed accommodation, or a reduction in evoked current amplitude over repeated stimulations. We found that following chronic ethanol exposure mPFC-BLA glutamatergic connections were dysregulated such that there were decreases in overall function, notably in synaptic strength and accommodation, with no change in probability of evoked glutamate release. The lesser GABAergic component of the mPFC-BLA circuit was not altered by chronic ethanol exposure. Collectively these data indicate that mPFC-BLA circuitry is a significant target of alcohol-associated plasticity, which may contribute to pathological behavior associated with AUDs.

Chronic alcohol consumption and withdrawal leads to anxiety, escalated alcohol drinking behavior, and alcohol dependence. Alterations in the function of key structures within the cortico-limbic neural circuit have been implicated in underlying the negative behavioral consequences of chronic alcohol exposure in both humans and rodents. Here, we used chronic intermittent ethanol vapor exposure (CIE) in male C57BL/6J mice to evaluate the effects of chronic alcohol exposure and withdrawal on anxiety-like behavior and basal synaptic function and neuronal excitability in prefrontal cortical and extended amygdala brain regions. Forty-eight hours after four cycles of CIE, mice were either assayed in the marble burying test (MBT) or their brains were harvested and whole-cell electrophysiological recordings were performed in the prelimbic and infralimbic medial prefrontal cortex (PLC and ILC), the lateral and medial central nucleus of the amygdala (lCeA and mCeA), and the dorsal and ventral bed nucleus of the stria terminalis (dBNST and vBNST). Ethanol-exposed mice displayed increased anxiety in the MBT compared to air-exposed controls, and alterations in neuronal function were observed in all brain structures examined, including several distinct differences between subregions within each structure. Chronic ethanol exposure induced hyperexcitability of the ILC, as well as a shift toward excitation in synaptic drive and hyperexcitability of vBNST neurons; in contrast, there was a net inhibition of the CeA. This study reveals extensive effects of chronic ethanol exposure on the basal function of cortico-limbic brain regions, suggests that there may be complex interactions between these regions in the regulation of ethanol-dependent alterations in anxiety state, and highlights the need for future examination of projection-specific effects of ethanol in cortico-limbic circuitry.

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.