Adenosine 2A Receptors Link Astrocytic Alpha-1 Adrenergic Signaling to Wake-Promoting Dopamine Neurons.

BACKGROUND: Sleep and arousal disorders are common, but the underlying physiology of wakefulness is not fully understood. The locus coeruleus promotes arousal via alpha-1 adrenergic receptor (α1AR) driven recruitment of wake-promoting dopamine (DA) neurons in the ventral periaqueductal gray (vPAGDA neurons). α1AR expression is enriched on vPAG astrocytes, and chemogenetic activation of astrocytic Gq signaling promotes wakefulness. Astrocytes can release extracellular "gliotransmitters," such as ATP and adenosine, but the mechanism underlying how vPAG astrocytic α1ARs influence sleep/wake behavior and vPAGDA neuron physiology is unknown. METHODS: In this study, we utilized genetic manipulations with ex vivo calcium imaging in vPAGDA neurons and astrocytes, patch-clamp electrophysiology, and behavioral experiments in mice to probe our hypothesis that astrocytic α1ARs mediate noradrenergic modulation of wake-promoting vPAGDA neurons via adenosine signaling. RESULTS: Activation of α1ARs with phenylephrine increased calcium transients in vPAGDA neurons and vPAG astrocytes, and increased vPAGDA neuron excitability ex vivo. Chemogenetic Gq-DREADD activation of vPAG astrocytes similarly increased vPAGDA neuron calcium activity and intrinsic excitability. Conversely, shRNA knockdown of vPAG astrocytic α1ARs reduced the excitatory effect of phenylephrine on vPAGDA neurons and blunted arousal during the wake phase. Pharmacological blockade of adenosine 2A (A2A) receptors precludes the α1AR-induced increase in vPAGDA calcium activity and excitability in brain slices, as well as the wake-promoting effects of vPAG α1AR activation in vivo. CONCLUSIONS: We have identified a crucial role for vPAG astrocytic α1AR receptors in sustaining arousal through heightened excitability and activity of vPAGDA neurons mediated by local A2A receptors.

Operant ethanol self-administration behaviors do not predict sex differences in continuous access home cage drinking.

Understanding sex differences in disease prevalence is critical to public health, particularly in the context of alcohol use disorder (AUD). The goal of this study was to understand sex differences in ethanol drinking behavior and define the precise conditions under which sex differences emerge. Consistent with prior work, C57BL/6J females drank more than males under continuous access two-bottle choice conditions. However, using ethanol self-administration - where an operant response results in access to an ethanol sipper for a fixed time period - we found no sex differences in operant response rates or ethanol consumption (volume per body weight consumed, as well as lick behavior). This remained true across a wide range of parameters including acquisition, when the ethanol sipper access period was manipulated, and when the concentration of the ethanol available was scaled. The only sex differences observed were in total ethanol consumption, which was explained by differences in body weight between males and females, rather than by sex differences in motivation to drink. Using dimensionality reduction approaches, we found that drinking behavior in the operant context did not cluster by sex, but rather clustered by high and low drinking phenotypes. Interestingly, these high and low drinking phenotypes in the operant context showed no correlation with those same categorizations in the home cage context within the same animals. These data underscore the complexity of sex differences in ethanol consumption, highlighting the important role that drinking conditions/context plays in the expression of these differences.

A Novel Mouse Home Cage Lickometer System Reveals Sex- and Housing-Based Influences on Alcohol Drinking.

Alcohol use disorder (AUD) is a significant global health issue. Despite historically higher rates among men, AUD prevalence and negative alcohol-related outcomes in women are rising. Loneliness in humans has been associated with increased alcohol use, and traditional rodent drinking models involve single housing, presenting challenges for studying social enrichment. We developed LIQ PARTI (Lick Instance Quantifier with Poly-Animal RFID Tracking Integration), an open-source tool to examine home cage continuous access two-bottle choice drinking behavior in a group-housed setting, investigating the influence of sex and social isolation on ethanol consumption and bout microstructure in C57Bl/6J mice. LIQ PARTI, based on our previously developed single-housed LIQ HD system, accurately tracks drinking behavior using capacitive-based sensors and RFID technology. Group-housed female mice exhibited higher ethanol preference than males, while males displayed a unique undulating pattern of ethanol preference linked to cage changes, suggesting a potential stress or novelty-related response. Chronic ethanol intake distinctly altered bout microstructure between male and female mice, highlighting sex and social environmental influences on drinking behavior. Social isolation with the LIQ HD system amplified fluid intake and ethanol preference in both sexes, accompanied by sex- and fluid-dependent changes in bout microstructure. However, these effects largely reversed upon resocialization, indicating the plasticity of these behaviors in response to social context. Utilizing a novel group-housed home cage lickometer device, our findings illustrate the critical interplay of sex and housing conditions in voluntary alcohol drinking behaviors in C57Bl/6J mice, facilitating nuanced insights into the potential contributions to AUD etiology.

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

BACKGROUND: 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 play a key role in chronic ethanol-induced increases in volitional intake, hypothesized to be driven by emergent negative affective behaviors. Excitatory N-methyl-d-aspartate receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. Specifically, GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. METHODS: Male and female mice underwent a home cage chronic drinking forced abstinence model (CDFA) to assess the impact of 1 day or 2 weeks of ethanol abstinence on BNST synaptic transmission and BNST Grin gene expression. Constitutive and conditional BNST GluN2D knockout mice were used to assess the impact of GluN2D deletion on continuous access ethanol intake as well as negative affect behaviors, using the open field, elevated zero maze, and forced swim tasks. RESULTS: We report here that excitatory transmission undergoes time-dependent upregulation in BNST Crh+ cells. Further, 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 the dBNST or dBNST Crh+ neurons. Finally, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. CONCLUSIONS: These data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking but not ethanol abstinence, highlighting sex differences and behavioral specificity. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.

Bed nucleus of the stria terminalis network responses to unpredictable threat in early alcohol abstinence.

BACKGROUND: Anxiety during early alcohol abstinence, likely resulting from neural changes caused by chronic alcohol use, contributes to high relapse rates. Studies in rodents show heightened activation during early abstinence in the bed nucleus of the stria terminalis (BNST)-a neural hub for anxiety-and its extended anxiety-related corticolimbic network. Despite the clinical importance of early abstinence, few studies investigate the underlying neural mechanisms. METHODS: To address this gap, we investigated brain function in early alcohol abstinence (EA = 20, 9 women) relative to controls (HC = 20, 11 women) using an unpredictable threat task shown to engage the BNST and corticolimbic brain regions involved in anxiety and alcohol use disorder (AUD). Group, anxiety, and sex were predictors used to determine whole-brain activation and BNST functional connectivity. RESULTS: We found widespread interactions of group × anxiety and group × anxiety × sex for both activation and BNST connectivity during unpredictable threat. In the EA group, higher anxiety was correlated with activation in the BNST, rostral anterior cingulate cortex (ACC), insula (men only), and dorsal ACC (men only). In the HC group, higher anxiety was negatively correlated with activation in the BNST, nucleus accumbens, thalamus, and insula (men only). For connectivity, anxiety was positively correlated in EA and negatively correlated in HC, between the BNST and the amygdala, ventromedial prefrontal cortex (PFC), and dorsomedial PFC; EA men showed stronger BNST-vmPFC connectivity than HC men. CONCLUSIONS: These novel findings provide preliminary evidence for alterations in the BNST and anxiety-related corticolimbic brain regions in early alcohol abstinence, adding to growing literature in humans supporting the BNST's role in anxiety and sex-dependent effects of chronic alcohol use.

A Cocaine-Activated Ensemble Exerts Increased Control Over Behavior While Decreasing in Size.

BACKGROUND: Substance use disorder is characterized by long-lasting changes in reward-related brain regions, such as the nucleus accumbens. Previous work has shown that cocaine exposure induces plasticity in broad, genetically defined cell types in the nucleus accumbens; however, in response to a stimulus, only a small percentage of neurons are transcriptionally active-termed an ensemble. Here, we identify an Arc-expressing neuronal ensemble that has a unique trajectory of recruitment and causally controls drug self-administration after repeated, but not acute, cocaine exposure. METHODS: Using Arc-CreERT2 transgenic mice, we expressed transgenes in Arc+ ensembles activated by cocaine exposure (either acute [1 × 10 mg/kg intraperitoneally] or repeated [10 × 10 mg/kg intraperitoneally]). Using genetic, optical, and physiological recording and manipulation strategies, we assessed the contribution of these ensembles to behaviors associated with substance use disorder. RESULTS: Repeated cocaine exposure reduced the size of the ensemble while simultaneously increasing its control over behavior. Neurons within the repeated cocaine ensemble were hyperexcitable, and their optogenetic excitation was sufficient for reinforcement. Finally, lesioning the repeated cocaine, but not the acute cocaine, ensemble blunted cocaine self-administration. Thus, repeated cocaine exposure reduced the size of the ensemble while simultaneously increasing its contributions to drug reinforcement. CONCLUSIONS: We showed that repeated, but not acute, cocaine exposure induced a physiologically distinct ensemble characterized by the expression of the immediate early gene Arc, which was uniquely capable of modulating reinforcement behavior.

Thalamocortical mGlu8 Modulates Dorsal Thalamus Excitatory Transmission and Sensorimotor Activity.

Metabotropic glutamate receptor 8 (mGlu8) is a heterogeneously expressed and poorly understood glutamate receptor with potential pharmacological significance. The thalamic reticular nucleus (TRN) is a critical inhibitory modulator of the thalamocortical-corticothalamic (TC-CT) network and plays a crucial role in information processing throughout the brain, is implicated in a variety of psychiatric conditions, and is also a site of significant mGlu8 expression. Using both male and female mice, we determined via fluorescent in situ hybridization that parvalbumin-expressing cells in the TRN core and shell matrices (identified by spp1+ and ecel1+ expression, respectively), as well as the cortical layers involved in CT signaling, express grm8 mRNA. We then assayed the physiological and behavioral impacts of perturbing grm8 signaling in the TC circuit through conditional (adeno-associated virus-CRE mediated) and cell-type-specific constitutive deletion strategies. We show that constitutive parvalbumin grm8 knock-out (PV grm8 knock-out) mice exhibited (1) increased spontaneous excitatory drive onto dorsal thalamus relay cells and (2) impaired sensorimotor gating, measured via paired-pulse inhibition, but observed no differences in locomotion and thigmotaxis in repeated bouts of open field test (OFT). Conversely, we observed hyperlocomotive phenotypes and anxiolytic effects of AAV-mediated conditional knockdown of grm8 in the TRN (TRN grm8 knockdown) in repeated OFT. Our findings underscore a role for mGlu8 in regulating excitatory neurotransmission as well as anxiety-related locomotor behavior and sensorimotor gating, revealing potential therapeutic applications for various neuropsychiatric disorders and guiding future research endeavors into mGlu8 signaling and TRN function.

A master regulator of opioid reward in the ventral prefrontal cortex.

In addition to their intrinsic rewarding properties, opioids can also evoke aversive reactions that protect against misuse. Cellular mechanisms that govern the interplay between opioid reward and aversion are poorly understood. We used whole-brain activity mapping in mice to show that neurons in the dorsal peduncular nucleus (DPn) are highly responsive to the opioid oxycodone. Connectomic profiling revealed that DPn neurons innervate the parabrachial nucleus (PBn). Spatial and single-nuclei transcriptomics resolved a population of PBn-projecting pyramidal neurons in the DPn that express µ-opioid receptors (µORs). Disrupting µOR signaling in the DPn switched oxycodone from rewarding to aversive and exacerbated the severity of opioid withdrawal. These findings identify the DPn as a key substrate for the abuse liability of opioids.

A novel mouse home cage lickometer system reveals sex- and housing-based influences on alcohol drinking.

Alcohol use disorder (AUD) is a significant global health issue. Despite historically higher rates among men, AUD prevalence and negative alcohol-related outcomes in women are rising. Loneliness in humans has been associated with increased alcohol use, and traditional rodent drinking models involve single housing, presenting challenges for studying social enrichment. We developed LIQ PARTI (Lick Instance Quantifier with Poly-Animal RFID Tracking Integration), an open-source tool to examine home cage continuous access two-bottle choice drinking behavior in a group-housed setting, investigating the influence of sex and social isolation on ethanol consumption and bout microstructure in C57Bl/6J mice. LIQ PARTI, based on our previously developed single-housed LIQ HD system, accurately tracks drinking behavior using capacitive-based sensors and RFID technology. Group-housed female mice exhibited higher ethanol preference than males, while males displayed a unique undulating pattern of ethanol preference linked to cage changes, suggesting a potential stress-related response. Chronic ethanol intake distinctly altered bout microstructure between male and female mice, highlighting sex and social environmental influences on drinking behavior. Social isolation with the LIQ HD system amplified fluid intake and ethanol preference in both sexes, accompanied by sex- and fluid-dependent changes in bout microstructure. However, these effects largely reversed upon resocialization, indicating the plasticity of these behaviors in response to social context. Utilizing a novel group-housed home cage lickometer device, our findings illustrate the critical interplay of sex and housing conditions in voluntary alcohol drinking behaviors in C57Bl/6J mice, facilitating nuanced insights into the potential contributions to AUD etiology.

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.