BNST GluN2D-containing NMDARs contribute to ethanol intake but not negative affective behaviors in female mice.

Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST), and Crh + neurons in this region are thought to play a key role in chronic ethanol-induced increases in volitional ethanol intake. This role has been hypothesized to be driven by emergent BNST-dependent negative affective behaviors. Indeed, we report here that in female mice undergoing a home cage chronic drinking forced abstinence model (CDFA), excitatory transmission undergoes time-dependent upregulation in BNST Crh + cells. Excitatory NMDA receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. We find that knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in dBNST or specifically in dBNST Crh + neurons. Finally, to determine the impact of GluN2D expression on negative affective behaviors, open field, elevated zero maze, and forced swim tasks were used to measure anxiety- and depressive-like behaviors in constitutive and conditional BNST GluN2D knockout mice. Surprisingly, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. Together, these data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking behaviors but not abstinence from ethanol, highlighting potential sex differences and behavioral specificity in the context of AUD behaviors. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.

Forced Abstinence from Volitional Ethanol Intake Drives a Vulnerable Period of Hyperexcitability in BNST-Projecting Insular Cortex Neurons.

The insular cortex (IC) integrates sensory and interoceptive cues to inform downstream circuitry executing adaptive behavioral responses. The IC communicates with areas involved canonically in stress and motivation. IC projections govern stress and ethanol recruitment of bed nucleus of the stria terminalis (BNST) activity necessary for the emergence of negative affective behaviors during alcohol abstinence. Here, we assess the impact of the chronic drinking forced abstinence (CDFA) volitional home cage ethanol intake paradigm on synaptic and excitable properties of IC neurons that project to the BNST (IC→BNST). Using whole-cell patch-clamp electrophysiology, we investigated IC→BNST circuitry 24 h or 2 weeks following forced abstinence (FA) in female C57BL6/J mice. We find that IC→BNST cells are transiently more excitable following acute ethanol withdrawal. In contrast, in vivo ethanol exposure via intraperitoneal injection, ex vivo via ethanol wash, and acute FA from a natural reward (sucrose) all failed to alter excitability. In situ hybridization studies revealed that at 24 h post FA BK channel mRNA expression is reduced in IC. Further, pharmacological inhibition of BK channels mimicked the 24 h FA phenotype, while BK activation was able to decrease AP firing in control and 24 h FA subjects. All together these data suggest a novel mechanism of homeostatic plasticity that occurs in the IC→BNST circuitry following chronic drinking.

Neural Circuitries and Alcohol Use Disorder: Cutting Corners in the Cycle.

An implicit tenet of the alcohol use disorder (AUD) research field is that knowledge of how alcohol interacts with the brain is critical to the development of an understanding of vulnerability to AUD and treatment approaches. Gaining this understanding requires the mapping of brain function critical to specific components of this heterogeneous disorder. Early approaches in humans and animal models focused on the determination of specific brain regions sensitive to alcohol action and their participation in AUD-relevant behaviors. Broadly speaking, this research has focused on three domains, Binge/Intoxication, Negative Affect/Withdrawal, and Preoccupation/Anticipation, with a number of regions identified as participating in each. With the generational advances in technologies that the field of neuroscience has undergone over the last two decades, this focus has shifted to a circuit-based analysis. A wealth of new data has sharpened the field's focus on the specific roles of the interconnectivity of multiple brain regions in AUD and AUD-relevant behaviors, as well as demonstrating that the three major domains described above have much fuzzier edges than originally thought.In this chapter, we very briefly review brain regions previously implicated in aspects of AUD-relevant behavior from animal model research. Next, we move to a more in-depth overview of circuit-based approaches, and the utilization of these approaches in current AUD research.

Synaptic and cellular endocannabinoid signaling mechanisms regulate stress-induced plasticity of nucleus accumbens somatostatin neurons.

Interneuron populations within the nucleus accumbens (NAc) orchestrate excitatory-inhibitory balance, undergo experience-dependent plasticity, and gate-motivated behavior, all biobehavioral processes heavily modulated by endogenous cannabinoid (eCB) signaling. While eCBs are well known to regulate synaptic plasticity onto NAc medium spiny neurons and modulate NAc function at the behavioral level, how eCBs regulate NAc interneuron function is less well understood. Here, we show that eCB signaling differentially regulates glutamatergic and feedforward GABAergic transmission onto NAc somatostatin-expressing interneurons (NAcSOM+) in an input-specific manner, while simultaneously increasing postsynaptic excitability of NAcSOM+ neurons, ultimately biasing toward vHPC (ventral hippocampal), and away from BLA (basolateral amygdalalar), activation of NAcSOM+ neurons. We further demonstrate that NAcSOM+ are activated by stress in vivo and undergo stress-dependent plasticity, evident as a global increase in intrinsic excitability and an increase in excitation-inhibition balance specifically at vHPC, but not BLA, inputs onto NAcSOM+ neurons. Importantly, both forms of stress-induced plasticity are dependent on eCB signaling at cannabinoid type 1 receptors. These findings reveal eCB-dependent mechanisms that sculpt afferent input and excitability of NAcSOM+ neurons and demonstrate a key role for eCB signaling in stress-induced plasticity of NAcSOM+-associated circuits.

LIQ HD (Lick Instance Quantifier Home Cage Device): An Open-Source Tool for Recording Undisturbed Two-Bottle Drinking Behavior in a Home Cage Environment.

Investigation of rodent drinking behavior has provided insight into drivers of thirst, circadian rhythms, anhedonia, and drug and ethanol consumption. Traditional methods of recording fluid intake involve weighing bottles, which is cumbersome and lacks temporal resolution. Several open-source devices have been designed to improve drink monitoring, particularly for two-bottle choice tasks. However, beam-break sensors lack the ability to detect individual licks for bout microstructure analysis. Thus, we designed LIQ HD (Lick Instance Quantifier Home cage Device) with the goal of using capacitive sensors to increase accuracy and analyze lick microstructure, building a device compatible with ventilated home cages, increasing scale with prolonged undisturbed recordings, and creating a design that is easy to build and use with an intuitive touchscreen graphical user interface. The system tracks two-bottle choice licking behavior in up to 18 rodent cages, or 36 single bottles, on a minute-to-minute timescale controlled by a single Arduino microcontroller. The data are logged to a single SD card, allowing for efficient downstream analysis. LIQ HD accuracy was validated with sucrose, quinine, and ethanol two-bottle choice tasks. The system measures preference over time and changes in bout microstructure, with undisturbed recordings tested up to 7 d. All designs and software are open-source to allow other researchers to build on the system and adapt LIQ HD to their animal home cages.

BNST PKCδ neurons are activated by specific aversive conditions to promote anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a critical mediator of stress responses and anxiety-like behaviors. Neurons expressing protein kinase C delta (BNSTPKCδ) are an abundant but understudied subpopulation implicated in inhibiting feeding, but which have conflicting reports about their role in anxiety-like behaviors. We have previously shown that expression of PKCδ is dynamically regulated by stress and that BNSTPKCδ cells are recruited during bouts of active stress coping. Here, we first show that in vivo activation of this population is mildly aversive. This aversion was insensitive to prior restraint stress exposure. Further investigation revealed that unlike other BNST subpopulations, BNSTPKCδ cells do not exhibit increased cfos expression following restraint stress. Ex vivo current clamp recordings also indicate they are resistant to firing. To elucidate their afferent control, we next used rabies tracing with whole-brain imaging and channelrhodopsin-assisted circuit mapping, finding that BNSTPKCδ cells receive abundant input from affective, arousal, and sensory regions including the basolateral amygdala (BLA) paraventricular thalamus (PVT) and central amygdala PKCδ-expressing cells (CeAPKCδ). Given these findings, we used in vivo optogenetics and fiber photometry to further examine BNSTPKCδ cells in the context of stress and anxiety-like behavior. We found that BNSTPKCδ cell activity is associated with increased anxiety-like behavior in the elevated plus maze, increases following footshock, and unlike other BNST subpopulations, does not desensitize to repeated stress exposure. Taken together, we propose a model in which BNSTPKCδ cells may serve as threat detectors, integrating exteroceptive and interoceptive information to inform stress coping behaviors.

Alterations in BNST Intrinsic Functional Connectivity in Early Abstinence from Alcohol Use Disorder.

AIMS: Maintaining abstinence from alcohol use disorder (AUD) is extremely challenging, partially due to increased symptoms of anxiety and stress that trigger relapse. Rodent models of AUD have identified that the bed nucleus of the stria terminalis (BNST) contributes to symptoms of anxiety-like behavior and drug-seeking during abstinence. In humans, however, the BNST's role in abstinence remains poorly understood. The aims of this study were to assess BNST network intrinsic functional connectivity in individuals during abstinence from AUD compared to healthy controls and examine associations between BNST intrinsic functional connectivity, anxiety and alcohol use severity during abstinence. METHODS: The study included resting state fMRI scans from participants aged 21-40 years: 20 participants with AUD in abstinence and 20 healthy controls. Analyses were restricted to five pre-selected brain regions with known BNST structural connections. Linear mixed models were used to test for group differences, with sex as a fixed factor given previously shown sex differences. RESULTS: BNST-hypothalamus intrinsic connectivity was lower in the abstinent group relative to the control group. There were also pronounced sex differences in both the group and individual analyses; many of the findings were specific to men. Within the abstinent group, anxiety was positively associated with BNST-amygdala and BNST-hypothalamus connectivity, and men, not women, showed a negative relationship between alcohol use severity and BNST-hypothalamus connectivity. CONCLUSIONS: Understanding differences in connectivity during abstinence may help explain the clinically observed anxiety and depression symptoms during abstinence and may inform the development of individualized treatments.

GluN2D expression is regulated by restraint stress and supports active stress coping bouts.

Stress coping strategies represent critical responses to environmental challenges, and active coping has been linked to stress resilience in humans. Understanding the neuroadaptations that support these strategies may provide insights into adaptive and maladaptive stress responses. NMDA receptors (NMDARs) play key roles in neuroadaptation, and NMDARs have been specifically implicated in stress responsiveness. Constitutive knockout mice have been used to implicate the GluN2D NMDAR subunit in regulation of stress-sensitive and affective behavior, but the brain regions in which GluN2D expression changes drive these effects remain unknown. Here we report that following an acute restraint stressor, GluN2D subunit expression is specifically decreased in the bed nucleus of the stria terminalis (BNST), a key region involved in stress processing, in male but not female mice, with no differences found in the thalamus or ventral hippocampus in either sex. Rodents engage in active struggling events during restraint stress that may represent active coping strategies to stress. Thus, we assessed active coping bouts during acute and chronic restraint stress sessions in GluN2D knockout mice. During the first restraint session, GluN2D knockout mice exhibited a pronounced decrease in struggling bouts during restraint stress relative to wild-type littermates, consistent with a role of GluN2D in active coping responses to stress. Repeated, daily restraint sessions revealed a sex-specific role of GluN2D expression on certain aspects of active coping behaviors, with male GluN2D KO mice exhibiting a decrease in total coping bouts measured across five sessions. However, BNST-specific knockdown of GluN2D in male mice did not alter active coping bouts, suggesting either a multi-synaptic role of GluN2D and/or a developmental role of GluN2D in this behavior. Altogether, these data are consistent with a growing literature suggesting that exploration of GluN2D control of stress circuit actions may lead to a novel therapeutic target to consider for stress-related mood disorders.

An ensemble recruited by α2a-adrenergic receptors is engaged in a stressor-specific manner in mice.

α2a-adrenergic receptor (α2a-AR) agonists are candidate substance use disorder therapeutics due to their ability to recruit noradrenergic autoreceptors to dampen stress system engagement. However, we recently found that postsynaptic α2a-ARs are required for stress-induced reinstatement of cocaine-conditioned behavior. Understanding the ensembles recruited by these postsynaptic receptors (heteroceptors) is necessary to understand noradrenergic circuit control. We utilized a variety of approaches in FosTRAP (Targeted Recombination in Active Populations) mice to define an ensemble of cells activated by the α2a-AR partial agonist guanfacine ("Guansembles") in the bed nucleus of the stria terminalis (BST/BNST), a region key to stress-induced reinstatement of drug seeking. We define BNST "Guansembles" and show they differ from restraint stress-activated cells. Guanfacine produced inhibition of cAMP-dependent signaling in Guansembles, while chronic restraint stress increased cAMP-dependent signaling. Guanfacine both excited and inhibited aspects of Guansemble neuronal activity. Further, while some stressors produced overall reductions in Guansemble activity, active coping events during restraint stress and exposure to unexpected shocks were both associated with Guansemble recruitment. Using viral tracing, we define a BNST Guansemble afferent network that includes regions involved in the interplay of stress and homeostatic functions. Finally, we show that activation of Guansembles produces alterations in behavior on the elevated plus maze consistent with task-specific anxiety-like behavior. Overall, we define a population of BNST neurons recruited by α2a-AR signaling that opposes the behavioral action of canonical autoreceptor α2a-AR populations and which are differentially recruited by distinct stressors. Moreover, we demonstrate stressor-specific physiological responses in a specific neuronal population.

High fat diet blunts stress-induced hypophagia and activation of Glp1r dorsal lateral septum neurons in male but not in female mice.

OBJECTIVE: While stress typically reduces caloric intake (hypophagia) in chow-fed rodents, presentation of palatable, high calorie substances during stress can increase caloric consumption (i.e. "comfort feeding") and promote obesity. However, little is known about how obesity itself affects feeding behavior in response to stress and the mechanisms that can influence stress-associated feeding in the context of obesity. METHODS: We assessed food intake and other metabolic parameters in lean and obese male and female mice following acute restraint stress. We also measured real-time activity of glucagon-like peptide-1 (Glp1) receptor (Glp1r)-expressing neurons in the dorsal lateral septum (dLS) during stress in lean and obese mice using fiber photometry. Glp1r activation in various brain regions, including the dLS, promotes hypophagia in response to stress. Finally, we used inhibitory Designer Receptors Activated Exclusively by Designer Drugs (DREADDs) to test whether activation of Glp1r-expressing neurons in the LS is required for stress-induced hypophagia. RESULTS: Lean male mice display the expected hypophagic response following acute restraint stress, but obese male mice are resistant to this acute stress-induced hypophagia. Glp1r-positive neurons in the dLS are robustly activated during acute restraint stress in lean but not in obese male mice. This raises the possibility that activation of dLS Glp1r neurons during restraint stress contributes to subsequent hypophagia. Supporting this, we show that chemogenetic inhibition of LS Glp1r neurons attenuates acute restraint stress hypophagia in male mice. Surprisingly, we show that both lean and obese female mice are resistant to acute restraint stress-induced hypophagia and activation of dLS Glp1r neurons. CONCLUSIONS: These results suggest that dLS Glp1r neurons contribute to the hypophagic response to acute restraint stress in male mice, but not in female mice, and that obesity disrupts this response in male mice. Broadly, these findings show sexually dimorphic mechanisms and feeding behaviors in lean vs. obese mice in response to acute stress.

Prefrontal cortex parvalbumin interneurons exhibit decreased excitability and potentiated synaptic strength after ethanol reward learning.

The prefrontal cortex (PFC) is intimately associated with behavioral characteristics of alcohol use disorders, including high motivation to drink and difficulty with moderation. Thus, continued mechanistic research investigating PFC cells and targets altered by ethanol experiences should inform translational efforts to craft new, efficacious treatments. Inhibitory interneurons expressing parvalbumin (PV-INs) comprise only a minor fraction of cells within the PFC, yet these cells are indispensable for coordinating PFC ensemble function, oscillatory activity, and subcortical output. Based on this, PV-INs represent an exciting target for the rational design of breakthrough treatments for alcohol use disorders. Here, we assessed experience-dependent physiological adaptations via ethanol place conditioning. By manipulating the timing of administration relative to conditioning sessions, equivalent ethanol exposure can form either rewarding or aversive memories in different individuals. Here, we found that female mice and male mice on a C57BL/6J background display conditioned place preference (CPP) or aversion (CPA) to an intoxicating dose of ethanol (2 g/kg, intraperitoneal [i.p.]) without overt differences between sexes. Ethanol reward learning was associated with decreased PV-IN excitability in deep layer prelimbic PFC, whereas PV-INs from CPA mice were not different from controls. Furthermore, PV-INs from mice in the CPP group, but not the CPA group, displayed potentiated excitatory synaptic strength that emerged during 1 week of abstinence. Taken together, these findings illustrate that synaptic and intrinsic adaptations associated with ethanol can depend on an individual's experience. These studies provide further context and support for PFC PV-INs as intriguing targets for modulating alcohol associations.

Acute restraint stress redirects prefrontal cortex circuit function through mGlu5 receptor plasticity on somatostatin-expressing interneurons.

Inhibitory interneurons orchestrate prefrontal cortex (PFC) activity, but we have a limited understanding of the molecular and experience-dependent mechanisms that regulate synaptic plasticity across PFC microcircuits. We discovered that mGlu5 receptor activation facilitates long-term potentiation at synapses from the basolateral amygdala (BLA) onto somatostatin-expressing interneurons (SST-INs) in mice. This plasticity appeared to be recruited during acute restraint stress, which induced intracellular calcium mobilization within SST-INs and rapidly potentiated postsynaptic strength onto SST-INs. Restraint stress and mGlu5 receptor activation each augmented BLA recruitment of SST-IN phasic feedforward inhibition, shunting information from other excitatory inputs, including the mediodorsal thalamus. Finally, studies using cell-type-specific mGlu5 receptor knockout mice revealed that mGlu5 receptor function in SST-expressing cells is necessary for restraint stress-induced changes to PFC physiology and related behaviors. These findings provide new insights into interneuron-specific synaptic plasticity mechanisms and suggest that SST-IN microcircuits may be promising targets for treating stress-induced psychiatric diseases.

The impact of intermittent exercise on mouse ethanol drinking and abstinence-associated affective behavior and physiology.

BACKGROUND: Negative emotional states are associated with the initiation and maintenance of alcohol use and drive relapse to drinking during withdrawal and protracted abstinence. Physical exercise is correlated with decreased negative affective symptoms, although a direct relationship between drinking patterns and exercise level has not been fully elucidated. METHODS: We incorporated intermittent running wheel access into a chronic continuous access, two-bottle choice alcohol drinking model in female C57BL/6J mice. Wheel access was granted intermittently once mice established a preference for alcohol over water. After 6 weeks, alcohol was removed (forced abstinence) and mice were given continuous access to unlocked or locked wheels. Negative affect-like behavior, home cage behavior, and metabolic activity were measured during protracted abstinence. RESULTS: Wheel access shifted drinking patterns in the mice, increasing drinking when the wheel was locked, and decreasing drinking when unlocked. Moreover, alcohol preference and consumption were strongly negatively correlated with the amount of running. An assessment of negative affect-like behavior in abstinence via the novelty suppressed feeding and saccharin preference tests (SPT) showed that unlimited wheel access mitigated abstinence-induced latency increases. Mice in abstinence also spent more time sleeping during the active dark cycle than control mice, providing additional evidence for abstinence-induced anhedonia- and depression-like behavior. Furthermore, running wheel access in abstinence decreased dark cycle sleep to comparable alcohol- and wheel-naïve mice. Given the positive impact of exercise and the negative impact of alcohol on metabolic health, we compared metabolic phenotypes of alcohol-abstinent mice with and without wheel access. Wheel access increased energy expenditure, carbon dioxide production, and oxygen consumption, providing a potential metabolic mechanism through which wheel access improves affective state. CONCLUSIONS: This study suggests that including exercise in AUD treatment regimens has the potential to reduce drinking, improve affective state during abstinence and could serve as a non-pharmacological approach to prevent the development of an AUD in high-risk individuals.

Targeting diacylglycerol lipase reduces alcohol consumption in preclinical models.

Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options for AUD are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing and alcohol intake is positively correlated with release of the eCB ligand 2-Arachidonoylglycerol (2-AG) within reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical models ranging from a voluntary free-access model to aversion resistant-drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure in mice. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal was devoid of anxiogenic and depressive-like behavioral effects. Lastly, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reduce alcohol consumption across the spectrum of AUD severity.

Delineation of an insula-BNST circuit engaged by struggling behavior that regulates avoidance in mice.

Active responses to stressors involve motor planning, execution, and feedback. Here we identify an insular cortex to BNST (insula→BNST) circuit recruited during restraint stress-induced active struggling that modulates affective behavior. We demonstrate that activity in this circuit tightly follows struggling behavioral events and that the size of the fluorescent sensor transient reports the duration of the struggle event, an effect that fades with repeated exposure to the homotypic stressor. Struggle events are associated with enhanced glutamatergic- and decreased GABAergic signaling in the insular cortex, indicating the involvement of a larger circuit. We delineate the afferent network for this pathway, identifying substantial input from motor- and premotor cortex, somatosensory cortex, and the amygdala. To begin to dissect these incoming signals, we examine the motor cortex input, and show that the cells projecting from motor regions to insular cortex are engaged shortly before struggle event onset. This study thus demonstrates a role for the insula→BNST pathway in monitoring struggling activity and regulating affective behavior.

Alterations in connectivity of the bed nucleus of the stria terminalis during early abstinence in individuals with alcohol use disorder.

BACKGROUND: For individuals with Alcohol Use Disorder (AUD), long-term recovery is difficult in part due to symptoms of anxiety that occur during early abstinence and can trigger relapse. Research in rodent models of AUD has identified the bed nucleus of the stria terminalis (BNST), a small, sexually dimorphic, subcortical region, as critical for regulating anxiety-like behaviors during abstinence, particularly in female mice. Furthermore, prolonged alcohol use and subsequent abstinence alter BNST afferent and efferent connections to other brain regions. To our knowledge, however, no studies of early abstinence have investigated BNST structural connectivity in humans during abstinence; this study addresses that gap. METHODS: Nineteen participants with AUD currently in early abstinence and 20 healthy controls completed a diffusion tensor imaging (DTI) scan. BNST structural connectivity was evaluated using probabilistic tractography. A linear mixed model was used to test between-groups differences in BNST network connectivity. Exploratory analyses were conducted to test for correlations between BNST connectivity and alcohol use severity and anxiety within the abstinence group. Sex was included as a factor for all analyses. RESULTS: The BNST showed stronger structural connectivity with the BNST network in early abstinence women than in control women, which was not seen in men. Women also showed region-specific differences, with stronger BNST-hypothalamus structural connectivity but weaker vmPFC-BNST structural connectivity than men. Exploratory analyses also demonstrated a relationship between alcohol use severity and vmPFC-BNST structural connectivity that was moderated by sex. CONCLUSIONS: This study is the first to demonstrate BNST structural connectivity differences in early abstinence and revealed key sex differences. The sex-specific differences in BNST structural connectivity during early abstinence could underlie known sex differences in abstinence symptoms and relapse risk and help to inform potential sex-specific treatments.

Increased Synaptic Strength and mGlu2/3 Receptor Plasticity on Mouse Prefrontal Cortex Intratelencephalic Pyramidal Cells Following Intermittent Access to Ethanol.

BACKGROUND: The medial prefrontal cortex (PFC) is crucial for regulating craving and alcohol seeking in alcohol use disorder (AUD) patients and alcohol seeking in animal models. Maladaptive changes in volitional ethanol (EtOH) intake have been associated with PFC function, yet synaptic adaptations within PFC have not been consistently detected in voluntary drinking rodent models. At least 80% of the neurons in PFC are glutamatergic pyramidal cells. Pyramidal cells provide the predominant cortical output to several brain regions relevant to AUD, including structures within the telencephalon (IT: e.g., basal ganglia, amygdala, other neocortical regions) and outside the telencephalon (ET: e.g., lateral hypothalamus, midbrain monoaminergic structures, thalamus). METHODS: In addition to their anatomical distinctions, studies from several laboratories have revealed that prefrontal cortical IT and ET pyramidal cells may be differentiated by specific electrophysiological parameters. These distinguishable parameters make it possible to readily classify pyramidal cells into separable subtypes. Here, we employed and validated the hyperpolarization sag ratio as a diagnostic proxy for separating ET (type A) and IT (type B) neurons. We recorded from deep-layer prelimbic PFC pyramidal cells of mice 1 day after 4 to 5 weeks of intermittent access (IA) EtOH exposure. RESULTS: Membrane properties were not altered by IA EtOH, but excitatory postsynaptic strength onto IT type B neurons was selectively enhanced in slices from IA EtOH mice. The increased excitatory drive was accompanied by enhanced mGlu2/3 receptor plasticity on IT type B neurons, providing a potential translational approach to mitigate cognitive and motivational changes to PFC function related to binge drinking. CONCLUSIONS: Together, these studies provide insight into the specific PFC neurocircuits altered by voluntary drinking. In addition, the findings provide an additional rationale for developing compounds that potentiate mGlu2 and/or mGlu3 receptor function as potential treatments for AUD.

Anxiety during abstinence from alcohol: A systematic review of rodent and human evidence for the anterior insula's role in the abstinence network.

Alcohol Use Disorder (AUD) is a chronic, relapsing disease that impacts almost a third of Americans. Despite effective treatments for attaining sobriety, the majority of patients relapse within a year, making relapse a substantial barrier to long-term treatment success. A major factor contributing to relapse is heightened negative affect that results from the combination of abstinence-related increases in stress-reactivity and decreases in reward sensitivity. Substantial research has contributed to the understanding of reward-related changes in AUD. However, less is known about anxiety during abstinence, a critical component of understanding addiction as anxiety during abstinence can trigger relapse. Most of what we know about abstinence-related negative affect comes from rodent studies which have identified key brain regions responsible for abstinence-related behaviors. This abstinence network is composed of brain regions that make up the extended amygdala: the nucleus accumbens (NAcc), the central nucleus of the amygdala (CeA), and the bed nucleus of the stria terminalis (BNST). More recently, emerging evidence from rodent and human studies suggests a fourth brain region, the anterior insula, might be part of the abstinence network. Here, we review current rodent and human literature on the extended amygdala's role in alcohol abstinence and anxiety, present evidence for the anterior insula's role in the abstinence network, and provide future directions for research to further elucidate the neural underpinnings of abstinence in humans. A better understanding of the abstinence network is critical toward understanding and possibly preventing relapse in AUD.

Contrasting sex-dependent adaptations to synaptic physiology and membrane properties of prefrontal cortex interneuron subtypes in a mouse model of binge drinking.

Alcohol use disorder (AUD) affects all sexes, however women who develop AUD may be particularly susceptible to cravings and other components of the disease. While many brain regions are involved in AUD etiology, proper prefrontal cortex (PFC) function is particularly important for top-down craving management and the moderation of drinking behaviors. Essential regulation of PFC output is provided by local inhibitory interneurons, yet how drinking affects interneuron physiology remains poorly understood, particularly in female individuals. To address this gap, we generated fluorescent reporter transgenic mice to label the two major classes of interneuron in deep layer prelimbic PFC, based on expression of parvalbumin (PV-IN) or somatostatin (SST-IN). We then interrogated PV-IN and SST-IN membrane and synaptic physiology in a rodent model of binge drinking. Beginning in late adolescence, mice received 3-4 weeks of intermittent access (IA) ethanol. We prepared acute brain slices one day after the last drinking session. PV-INs but not SST-INs from IA ethanol mice displayed increased excitability relative to controls, regardless of sex. On the contrary, synaptic adaptations to PV-INs differed based on sex. While drinking decreased excitatory synaptic strength onto PV-INs from female mice, PV-INs from IA ethanol male mice exhibited potentiated excitatory transmission relative to controls. In contrast, decreased synaptic strength onto SST-INs was observed following IA ethanol in all groups of mice. Together, these findings illustrate novel sex differences in drinking-related PFC pathophysiology. Discovering means to restore PV-IN and SST-IN dysfunction following extended drinking provides opportunities for developing new treatments for all AUD patients.

BNST GluN2D-Containing NMDA Receptors Influence Anxiety- and Depressive-like Behaviors and ModulateCell-Specific Excitatory/Inhibitory Synaptic Balance.

Excitatory signaling mediated by NMDARs has been shown to regulate mood disorders. However, current treatments targeting NMDAR subtypes have shown limited success in treating patients, highlighting a need for alternative therapeutic targets. Here, we identify a role for GluN2D-containing NMDARs in modulating emotional behaviors and neural activity in the bed nucleus of the stria terminalis (BNST). Using a GluN2D KO mouse line (GluN2D-/-), we assessed behavioral phenotypes across tasks modeling emotional behavior. We then used a combination of ex vivo electrophysiology and in vivo fiber photometry to assess changes in BNST plasticity, cell-specific physiology, and cellular activity profiles. GluN2D-/- male mice exhibit evidence of exacerbated negative emotional behavior, and a deficit in BNST synaptic potentiation. We also found that GluN2D is functionally expressed on corticotropin-releasing factor (CRF)-positive BNST cells implicated in driving negative emotional states, and recordings in mice of both sexes revealed increased excitatory and reduced inhibitory drive onto GluN2D-/- BNST-CRF cells ex vivo and increased activity in vivo Using a GluN2D conditional KO line (GluN2Dflx/flx) to selectively delete the subunit from the BNST, we find that BNST-GluN2Dflx/flx male mice exhibit increased depressive-like behaviors, as well as altered NMDAR function and increased excitatory drive onto BNST-CRF neurons. Together, this study supports a role for GluN2D-NMDARs in regulating emotional behavior through their influence on excitatory signaling in a region-specific manner, and suggests that these NMDARs may serve as a novel target for selectively modulating glutamate signaling in stress-responsive structures and cell populations.SIGNIFICANCE STATEMENT Excitatory signaling mediated through NMDARs plays an important role in shaping emotional behavior; however, the receptor subtypes/brain regions through which this occurs are poorly understood. Here, we demonstrate that loss of GluN2D-containing NMDARs produces an increase in anxiety- and depressive-like behaviors in mice, deficits in BNST synaptic potentiation, and increased activity in BNST-CRF neurons known to drive negative emotional behavior. Further, we determine that deleting GluN2D in the BNST leads to increased depressive-like behaviors and increased excitatory drive onto BNST-CRF cells. Collectively, these results demonstrate a role for GluN2D-NMDARs in regulating the activity of stress-responsive structures and neuronal populations in the adult brain, suggesting them as a potential target for treating negative emotional states in mood-related disorders.