Acute and chronic alcohol modulation of extended amygdala calcium dynamics.

The central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) are reciprocally connected nodes of the extended amygdala thought to play an important role in alcohol consumption. Studies of immediate-early genes indicate that BNST and CeA are acutely activated following alcohol drinking and may signal alcohol reward in nondependent drinkers, while stress signaling in the extended amygdala following chronic alcohol exposure drives increased drinking via negative reinforcement. However, the temporal dynamics of neuronal activation in these regions during drinking behavior are poorly understood. In this study, we used fiber photometry and the genetically encoded calcium sensor GCaMP6s to assess acute changes in neuronal activity during alcohol consumption in BNST and CeA before and after a chronic drinking paradigm. Activity was examined in the pan-neuronal population and separately in dynorphinergic neurons. BNST and CeA showed increased pan-neuronal activity during acute consumption of alcohol and other fluid tastants of positive and negative valence, as well as highly palatable chow. Responses were greatest during initial consummatory bouts and decreased in amplitude with repeated consumption of the same tastant, suggesting modulation by stimulus novelty. Dynorphin neurons showed similar consumption-associated calcium increases in both regions. Following three weeks of continuous alcohol access (CA), calcium increases in dynorphin neurons during drinking were maintained, but pan-neuronal activity and BNST-CeA coherence were altered in a sex-specific manner. These results indicate that BNST and CeA, and dynorphin neurons specifically, are engaged during drinking behavior, and activity dynamics are influenced by stimulus novelty and chronic alcohol.

Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2c receptor modulate operant alcohol self-administration behavior in mice.

The serotonin 5HT2c receptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2c receptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT2c in the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT2c-containing neurons (BNST5HT2c) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 µM quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2c project to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2c neurons, and to a small extent the BNST 5HT2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

The Influence of Arsenic Co-Exposure in a Model of Alcohol-Induced Neurodegeneration in C57BL/6J Mice.

Both excessive alcohol consumption and exposure to high levels of arsenic can lead to neurodegeneration, especially in the hippocampus. Co-exposure to arsenic and alcohol can occur because an individual with an Alcohol Use Disorder (AUD) is exposed to arsenic in their drinking water or food or because of arsenic found directly in alcoholic beverages. This study aims to determine if co-exposure to alcohol and arsenic leads to worse outcomes in neurodegeneration and associated mechanisms that could lead to cell death. To study this, mice were exposed to a 10-day gavage model of alcohol-induced neurodegeneration with varying doses of arsenic (0, 0.005, 2.5, or 10 mg/kg). The following were examined after the last dose of ethanol: (1) microglia activation assessed via immunohistochemical detection of Iba-1, (2) reactive oxygen and nitrogen species (ROS/RNS) using a colorimetric assay, (3) neurodegeneration using Fluoro-Jade® C staining (FJC), and 4) arsenic absorption using ICP-MS. After exposure, there was an additive effect of the highest dose of arsenic (10 mg/kg) in the dentate gyrus of alcohol-induced FJC+ cells. This additional cell loss may have been due to the observed increase in microglial reactivity or increased arsenic absorption following co-exposure to ethanol and arsenic. The data also showed that arsenic caused an increase in CYP2E1 expression and ROS/RNS production in the hippocampus which could have independently contributed to increased neurodegeneration. Altogether, these findings suggest a potential cyclical impact of co-exposure to arsenic and ethanol as ethanol increases arsenic absorption but arsenic also enhances alcohol's deleterious effects in the CNS.

Acute and chronic alcohol modulation of extended amygdala calcium dynamics.

The central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) are reciprocally connected nodes of the extended amygdala thought to play an important role in alcohol consumption. Studies of immediate-early genes indicate that BNST and CeA are acutely activated following alcohol drinking and may signal alcohol reward in nondependent drinkers, while increased stress signaling in the extended amygdala following chronic alcohol exposure drives increased drinking via negative reinforcement. However, the temporal dynamics of neuronal activation in these regions during drinking behavior are poorly understood. In this study, we used fiber photometry and the genetically encoded calcium sensor GCaMP6s to assess acute changes in neuronal activity during alcohol consumption in BNST and CeA before and after a chronic drinking paradigm. Activity was examined in the pan-neuronal population and separately in dynorphinergic neurons. BNST and CeA showed increased pan-neuronal activity during acute consumption of alcohol and other fluid tastants of positive and negative valence, as well as highly palatable chow. Responses were greatest during initial consummatory bouts and decreased in amplitude with repeated consumption of the same tastant, suggesting modulation by stimulus novelty. Dynorphin neurons showed similar consumption-associated calcium increases in both regions. Following three weeks of continuous alcohol access (CA), calcium increases in dynorphin neurons during drinking were maintained, but pan-neuronal activity and BNST-CeA coherence were altered in a sex-specific manner. These results indicate that BNST and CeA, and dynorphin neurons specifically, are engaged during drinking behavior, and activity dynamics are influenced by stimulus novelty and chronic alcohol.

Impact and Role of Hypothalamic Corticotropin Releasing Hormone Neurons in Withdrawal from Chronic Alcohol Consumption in Female and Male Mice.

Worldwide, alcohol use and abuse are a leading risk of mortality, causing 5.3% of all deaths (World Health Organization, 2022). The endocrine stress system, initiated by the peripheral release of corticotropin releasing hormone (CRH) from primarily glutamatergic neurons in the paraventricular nucleus of the hypothalamus (PVN), is profoundly linked with alcohol use, abuse, and relapse (Blaine and Sinha, 2017). These PVN CRH-releasing (PVNCRH) neurons are essential for peripheral and central stress responses (Rasiah et al., 2023), but little is known about how alcohol affects these neurons. Here, we show that two-bottle choice alcohol consumption blunts the endocrine-mediated corticosterone response to stress during acute withdrawal in female mice. Conversely, using slice electrophysiology, we demonstrate that acute withdrawal engenders a hyperexcitable phenotype of PVNCRH neurons in females that is accompanied by increased glutamatergic transmission in both male and female mice. GABAergic synaptic transmission was unaffected by alcohol history. We then tested whether chemogenetic inhibition of PVNCRH neurons would restore stress response in female mice with a history of alcohol drinking in the looming disk test, which mimics an approaching predator threat. Accordingly, inhibition of PVNCRH neurons reduced active escape in hM4Di alcohol history mice only. This study indicates that stress-responsive PVNCRH neurons in females are particularly affected by a history of alcohol consumption. Interestingly, women have indicated an increase in heavy alcohol use to cope with stress (Rodriguez et al., 2020), perhaps pointing to a potential underlying mechanism in alcohol-mediated changes to PVNCRH neurons that alter stress response.SIGNIFICANCE STATEMENT Paraventricular nucleus of the hypothalamus neurons that release corticotropin releasing hormone (PVNCRH) are vital for stress response. These neurons have been understudied in relation to alcohol and withdrawal despite profound relations between stress, alcohol use disorders (AUD), and relapse. In this study, we use a variety of techniques to show that acute withdrawal from a history of alcohol impacts peripheral stress response, PVNCRH neurons, and behavior. Specifically, PVNCRH are in a hyperactive state during withdrawal, which drives an increase in active stress coping behaviors in female mice only. Understanding how alcohol use and withdrawal affects stress responding PVNCRH neurons may contribute to finding new potential targets for the treatment of alcohol use disorder.

Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2c receptor modulate operant alcohol self-administration behavior in mice.

The serotonin 5HT2c receptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2c receptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT2c in the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT2c-containing neurons (BNST5HT2c) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 µM quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2c project to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2c neurons, and to a small extent the BNST 5HT2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

Dopamine D2 receptors in the bed nucleus of the stria terminalis modulate alcohol-related behaviors.

Dysregulation of the dopamine (DA) system is a hallmark of substance abuse disorders, including alcohol use disorder (AUD). Of the DA receptor subtypes, the DA D2 receptors (D2Rs) play a key role in the reinforcing effects of alcohol. D2Rs are expressed in numerous brain regions associated with the regulation of appetitive behaviors. One such region is the bed nucleus of the stria terminalis (BNST), which has been linked to the development and maintenance of AUD. Recently, we identified alcohol withdrawal-related neuroadaptations in the periaqueductal gray/dorsal raphe to BNST DA circuit in male mice. However, the role of D2R-expressing BNST neurons in voluntary alcohol consumption is not well characterized. In this study, we used a CRISPR-Cas9-based viral approach, to selectively reduce the expression of D2Rs in BNST VGAT neurons and interrogated the impact of BNST D2Rs in alcohol-related behaviors. In male mice, reduced D2R expression potentiated the stimulatory effects of alcohol and increased voluntary consumption of 20% w/v alcohol in a two-bottle choice intermittent access paradigm. This effect was not specific to alcohol, as D2R deletion also increased sucrose intake in male mice. Interestingly, cell-specific deletion of BNST D2Rs in female mice did not alter alcohol-related behaviors but lowered the threshold for mechanical pain sensitivity. Collectively, our findings suggest a role for postsynaptic BNST D2Rs in the modulation of sex-specific behavioral responses to alcohol and sucrose.

Chemogenetic manipulation of astrocytic signaling in the basolateral amygdala reduces binge-like alcohol consumption in male mice.

Binge drinking is a common occurrence in the United States, but a high concentration of alcohol in the blood has been shown to have reinforcing and reciprocal effects on the neuroimmune system in both dependent and non-dependent scenarios. The first part of this study examined alcohol's effects on the astrocytic response in the central amygdala and basolateral amygdala (BLA) in a non-dependent model. C57BL/6J mice were given access to either ethanol, water, or sucrose during a "drinking in the dark" paradigm, and astrocyte number and astrogliosis were measured using immunohistochemistry. Results indicate that non-dependent consumption increased glial fibrillary acidic protein (GFAP) density but not the number of GFAP+ cells, suggesting that non-dependent ethanol is sufficient to elicit astrocyte activation. The second part of this study examined how astrocytes impacted behaviors and the neurochemistry related to alcohol using the chemogenetic tool, DREADDs (designer receptors exclusively activated by designer drugs). Transgenic GFAP-hM3Dq mice were administered clozapine N-oxide both peripherally, affecting the entire central nervous system (CNS), or directly into the BLA. In both instances, GFAP-Gq-signaling activation significantly reduced ethanol consumption and correlating blood ethanol concentrations. However, GFAP-Gq-DREADD activation throughout the CNS had more broad effects resulting in decreased locomotor activity and sucrose consumption. More targeted GFAP-Gq-signaling activation in the BLA only impacted ethanol consumption. Finally, a glutamate assay revealed that after GFAP-Gq-signaling activation glutamate concentrations in the amygdala were partially normalized to control levels. Altogether, these studies support the theory that astrocytes represent a viable target for alcohol use disorder therapies.