Kappa Opioid Receptors Negatively Regulate Real Time Spontaneous Dopamine Signals by Reducing Release and Increasing Uptake.

The role of the dynorphin/kappa opioid receptor (KOR) system in dopamine (DA) regulation has been extensively investigated. KOR activation reduces extracellular DA concentrations and increases DA transporter (DAT) activity and trafficking to the membrane. To explore KOR influences on real-time DA fluctuations, we used the photosensor dLight1.2 with fiber photometry in the nucleus accumbens (NAc) core of freely moving male and female C57BL/6 mice. First, we established that the rise and fall of spontaneous DA signals were due to DA release and reuptake, respectively. Then mice were systemically administered the KOR agonist U50,488H (U50), with or without pretreatment with the KOR antagonist aticaprant (ATIC). U50 reduced both the amplitude and width of spontaneous signals in males, but only reduced width in females. Further, the slope of the correlation between amplitude and width was increased in both sexes, suggesting that DA uptake rates were increased. U50 also reduced the frequency of signals in both males and females. All effects of KOR activation were stronger in males. Overall, KORs exerted significant inhibitory control over spontaneous DA signaling, acting through at least three mechanisms - inhibiting DA release, promoting DAT-mediated uptake, and reducing the frequency of signals.

PiRATeMC: A highly flexible, scalable, and low-cost system for obtaining high quality video recordings for behavioral neuroscience.

With the rapidly accelerating adoption of machine-learning based rodent behavioral tracking tools, there is an unmet need for a method of acquiring high quality video data that is scalable, flexible, and relatively low-cost. Many experimenters use webcams, GoPros, or other commercially available cameras that can be expensive, offer minimal flexibility of recording parameters, and not optimized for recording rodent behavior, leading to suboptimal and inconsistent video quality. Furthermore, commercially available products are not conducive for synchronizing multiple cameras, or interfacing with third-party equipment to allow time-locking of video to other equipment such as microcontrollers for closed-loop experiments. We present a low-cost, customizable ecosystem of behavioral recording equipment, PiRATeMC (Pi-based Remote Acquisition Technology for Motion Capture) based on Raspberry Pi Camera Boards with the ability to acquire high quality recordings in bright/low light, or dark conditions under infrared light. PiRATeMC offers users control over nearly every recording parameter, and can be fine-tuned to produce optimal videos in any behavioral apparatus. This setup can be scaled up for synchronous control of any number of cameras via a self-contained network without burdening institutional network infrastructure. The Raspberry Pi is an excellent platform with a large online community designed for novice and inexperienced programmers interested in using an open-source recording system. Importantly, PiRATeMC supports TTL and serial communication, allowing for synchronization and interfacing of video recording with behavioral or other third-party equipment. In sum, PiRATeMC minimizes the cost-prohibitive nature of conducting and analyzing high quality behavioral neuroscience studies, thereby increasing accessibility to behavioral neuroscience.

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.

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.

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.

Better living through understanding the insula: Why subregions can make all the difference.

Insula function is considered critical for many motivated behaviors, with proposed functions ranging from attention, behavioral control, emotional regulation, goal-directed and aversion-resistant responding. Further, the insula is implicated in many neuropsychiatric conditions including substance abuse. More recently, multiple insula subregions have been distinguished based on anatomy, connectivity, and functional contributions. Generally, posterior insula is thought to encode more somatosensory inputs, which integrate with limbic/emotional information in middle insula, that in turn integrate with cognitive processes in anterior insula. Together, these regions provide rapid interoceptive information about the current or predicted situation, facilitating autonomic recruitment and quick, flexible action. Here, we seek to create a robust foundation from which to understand potential subregion differences, and provide direction for future studies. We address subregion differences across humans and rodents, so that the latter's mechanistic interventions can best mesh with clinical relevance of human conditions. We first consider the insula's suggested roles in humans, then compare subregional studies, and finally describe rodent work. One primary goal is to encourage precision in describing insula subregions, since imprecision (e.g. including both posterior and anterior studies when describing insula work) does a disservice to a larger understanding of insula contributions. Additionally, we note that specific task details can greatly impact recruitment of various subregions, requiring care and nuance in design and interpretation of studies. Nonetheless, the central ethological importance of the insula makes continued research to uncover mechanistic, mood, and behavioral contributions of paramount importance and interest. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

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.

Driving the Downward Spiral: Alcohol-Induced Dysregulation of Extended Amygdala Circuits and Negative Affect.

Alcohol use disorder (AUD) afflicts a large number of individuals, families, and communities globally. Affective disturbances, including stress, depression, and anxiety, are highly comorbid with AUD, contributing in some cases to initial alcohol use and continued use. Negative affect has a particularly strong influence on the withdrawal/abstinence stage of addiction as individuals with AUD frequently report stressful events, depression, and anxiety as key factors for relapse. Treatment options for negative affect associated with AUD are limited and often ineffective, highlighting the pressing need for preclinical studies examining the underlying neural circuitry driving AUD-associated negative affect. The extended amygdala (EA) is a set of brain areas collectively involved in generating and regulating affect, and extensive research has defined a critical role for the EA in all facets of substance use disorder. Here, we review the expansive historical literature examining the effects of ethanol exposure on the EA, with an emphasis on the complex EA neural circuitry driving negative affect in all phases of the alcohol addiction cycle. Specifically, this review focuses on the effects of alcohol exposure on the neural circuitry in 2 key components of the EA, the central nucleus of the amygdala and the bed nucleus of the stria terminalis. Additionally, future directions are proposed to advance our understanding of the relationship between AUD-associated negative affect and neural circuitry in the EA, with the long-term goal of developing better diagnostic tools and new pharmacological targets aimed at treating negative affect in AUD. The concepts detailed here will serve as the foundation for a companion review focusing on the potential for the endogenous cannabinoid system in the EA as a novel target for treating the stress, anxiety, and negative emotional state driving AUD.

Endocannabinoid Signaling in the Central Amygdala and Bed Nucleus of the Stria Terminalis: Implications for the Pathophysiology and Treatment of Alcohol Use Disorder.

High rates of relapse are a chronic and debilitating obstacle to effective treatment of alcohol use disorder (AUD); however, no effective treatments are available to treat symptoms induced by protracted abstinence. In the first part of this 2-part review series, we examine the literature supporting the effects of alcohol exposure within the extended amygdala (EA) neural circuitry. In Part 2, we focus on a potential way to combat negative affect associated with AUD, by exploring the therapeutic potential of the endogenous cannabinoid (eCB) system. The eCB system is a potent modulator of neural activity in the brain, and its ability to mitigate stress and negative affect has long been an area of interest for developing novel therapeutics. This review details the recent advances in our understanding of eCB signaling in 2 key regions of the EA, the central nucleus of the amygdala and the bed nucleus of the stria terminalis (BNST), and their role in regulating negative affect. Despite an established role for EA eCB signaling in reducing negative affect, few studies have examined the potential for eCB-based therapies to treat AUD-associated negative affect. In this review, we present an overview of studies focusing on eCB signaling in EA and cannabinoid modulation on EA synaptic activity. We further discuss studies suggesting dysregulation of eCB signaling in models of AUD and propose that pharmacological augmentation of eCB could be a novel approach to treat aspects of AUD. Lastly, future directions are proposed to advance our understanding of the relationship between AUD-associated negative affect and the EA eCB system that could yield new pharmacotherapies targeting negative affective symptoms associated with AUD.

Endocannabinoid control of the insular-bed nucleus of the stria terminalis circuit regulates negative affective behavior associated with alcohol abstinence.

Negative affect is a core symptom domain associated with an array of neurological and psychiatric disorders and is only partially targeted by current therapies, highlighting the need for better, more targeted treatment options. This study focuses on negative affective symptoms associated with prolonged alcohol abstinence, one of the leading causes of relapse. Using a mouse model of chronic alcohol consumption followed by forced abstinence (CDFA), prolonged alcohol abstinence increased c-fos expression and spontaneous glutamatergic neurotransmission in the dorsal bed nucleus of the stria terminalis (dBNST), a region heavily implicated in negative affect in both humans and rodents. Further, pharmacologically enhancing endogenous cannabinoids (eCB) with JZL184 prevents abstinence-induced increases in dBNST neuronal activity, underscoring the therapeutic potential of drugs targeting the brain's eCB system. Next, we used a channelrhodopsin-assisted mapping strategy to identify excitatory inputs to the dBNST that could contribute to CDFA-induced negative affect. We identified the insular cortex (insula), a region involved in regulating interoception, as a dense, functional, eCB-sensitive input to the dBNST. Using a chemogenetic strategy to locally mimic eCB signaling, we demonstrate that the insula strongly influences the CDFA behavioral phenotype and dBNST neuronal activity. Lastly, we used an anterograde strategy for transynaptic targeting of Cre expression in combination with a Gq-DREADD to selectively recruit dBNST neurons receiving insula projections. Chemogenetic recruitment of these neurons mimicked behavioral and c-fos responses observed in CDFA. Collectively, this study supports a role for the insula-BNST neural circuit in negative affective disturbances and highlights the therapeutic potential of the eCB system for treating negative affective disorders.

α2A-Adrenergic Receptor Activation Decreases Parabrachial Nucleus Excitatory Drive onto BNST CRF Neurons and Reduces Their Activity In Vivo.

Stress contributes to numerous psychiatric disorders. Corticotropin releasing factor (CRF) signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of α2A-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that α- and ß-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that α2A-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the α2A-AR is a Gi-linked GPCR, we assessed the impact of activating the Gi-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced Fos in BNST Crh neurons. Further, using Prkcd as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the coexpression of Prkcd/Crh in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that α2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.SIGNIFICANCE STATEMENT Stress is a major variable contributing to mood disorders. Here, we show that stress increases activation of BNST CRF neurons that drive negative affective behavior. We find that the clinically well tolerated α2A-AR agonist guanfacine reduces activity of these cells in vivo, and reduces excitatory PBN inputs onto these cells ex vivo Additionally, we uncover a novel sex-dependent coexpression of Prkcd with Crh in female BNST neurons after stress, an effect abolished by ovariectomy. These results demonstrate input-specific interactions between norepinephrine and CRF, and point to an action by which guanfacine may reduce negative affective responses.

Noradrenergic Transmission at Alpha1-Adrenergic Receptors in the Ventral Periaqueductal Gray Modulates Arousal.

BACKGROUND: Dysregulation of arousal is symptomatic of numerous psychiatric disorders. Previous research has shown that the activity of dopamine (DA) neurons in the ventral periaqueductal gray (vPAG) tracks with arousal state, and lesions of vPAGDA cells increase sleep. However, the circuitry controlling these wake-promoting DA neurons is unknown. METHODS: This study combined designer receptors exclusively activated by designer drugs (DREADDs), behavioral pharmacology, electrophysiology, and immunoelectron microscopy in male and female mice to elucidate mechanisms in the vPAG that promote arousal. RESULTS: Activation of locus coeruleus projections to the vPAG or vPAGDA neurons induced by DREADDs promoted arousal. Similarly, agonist stimulation of vPAG alpha1-adrenergic receptors (α1ARs) increased latency to fall asleep, whereas α1AR blockade had the opposite effect. α1AR stimulation drove vPAGDA activity in a glutamate-dependent, action potential-independent manner. Compared with other dopaminergic brain regions, α1ARs were enriched on astrocytes in the vPAG, and mimicking α1AR transmission specifically in vPAG astrocytes via Gq-DREADDS was sufficient to increase arousal. In general, the wake-promoting effects observed were not accompanied by hyperactivity. CONCLUSIONS: These experiments revealed that vPAG α1ARs increase arousal, promote glutamatergic input onto vPAGDA neurons, and are abundantly expressed on astrocytes. Activation of locus coeruleus inputs, vPAG astrocytes, or vPAGDA neurons increase sleep latency but do not produce hyperactivity. Together, these results support an arousal circuit whereby noradrenergic transmission at astrocytic α1ARs activates wake-promoting vPAGDA neurons via glutamate transmission.

Dorsal BNST α2A-Adrenergic Receptors Produce HCN-Dependent Excitatory Actions That Initiate Anxiogenic Behaviors.

Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress, yet minimally affects relapse, potentially due to competing actions in the brain. Here, we show that heteroceptor α2A-ARs postsynaptically enhance dorsal bed nucleus of the stria terminalis (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels because inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons and its activation elicits anxiety-like behavior in the elevated plus maze. Together, these data provide a framework for elucidating cell-specific actions of GPCR signaling and provide a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENT Stress affects the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here, we show that guanfacine increases dorsal BNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons and that these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting that care must be taken regarding interpretation of data obtained with these tools.

Metabotropic Glutamate Receptors in Alcohol Use Disorder: Physiology, Plasticity, and Promising Pharmacotherapies.

Developing efficacious treatments for alcohol use disorder (AUD) has proven difficult. The insidious nature of the disease necessitates a deep understanding of its underlying biology as well as innovative approaches to ameliorate ethanol-related pathophysiology. Excessive ethanol seeking and relapse are generated by long-term changes to membrane properties, synaptic physiology, and plasticity throughout the limbic system and associated brain structures. Each of these factors can be modulated by metabotropic glutamate (mGlu) receptors, a diverse set of G protein-coupled receptors highly expressed throughout the central nervous system. Here, we discuss how different components of the mGlu receptor family modulate neurotransmission in the limbic system and other brain regions involved in AUD etiology. We then describe how these processes are dysregulated following ethanol exposure and speculate about how mGlu receptor modulation might restore such pathophysiological changes. To that end, we detail the current understanding of the behavioral pharmacology of mGlu receptor-directed drug-like molecules in animal models of AUD. Together, this review highlights the prominent position of the mGlu receptor system in the pathophysiology of AUD and provides encouragement that several classes of mGlu receptor modulators may be translated as viable treatment options.

Role of Striatal Direct Pathway 2-Arachidonoylglycerol Signaling in Sociability and Repetitive Behavior.

BACKGROUND: Endocannabinoid signaling plays an important role in regulating synaptic transmission in the striatum, a brain region implicated as a central node of dysfunction in autism spectrum disorder. Deficits in signaling mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG) have been reported in mouse models of autism spectrum disorder, but a causal role for striatal 2-AG deficiency in phenotypes relevant to autism spectrum disorder has not been explored. METHODS: Using conditional knockout mice, we examined the electrophysiological, biochemical, and behavioral effects of 2-AG deficiency by deleting its primary synthetic enzyme, diacylglycerol lipase α (DGLα), from dopamine D1 receptor-expressing or adenosine A2a receptor-expressing medium spiny neurons (MSNs) to determine the role of 2-AG signaling in striatal direct or indirect pathways, respectively. We then used viral-mediated deletion of DGLα to study the effects of 2-AG deficiency in the ventral and dorsal striatum. RESULTS: Targeted deletion of DGLα from direct-pathway MSNs caused deficits in social interaction, excessive grooming, and decreased exploration of a novel environment. In contrast, deletion from indirect-pathway MSNs had no effect on any measure of behavior examined. Loss of 2-AG in direct-pathway MSNs also led to increased glutamatergic drive, which is consistent with a loss of retrograde feedback inhibition. Subregional DGLα deletion from the dorsal striatum produced deficits in social interaction, whereas deletion from the ventral striatum resulted in repetitive grooming. CONCLUSIONS: These data suggest a role for 2-AG deficiency in social deficits and repetitive behavior, and they demonstrate a key role for 2-AG in regulating striatal direct-pathway MSNs.

Loss of δ-GABAA receptor-mediated tonic currents in the adult prelimbic cortex following adolescent alcohol exposure.

Delayed maturation of the adolescent prefrontal cortex may render it particularly vulnerable to insults, including those associated with drugs of abuse. Using a rat model of binge alcohol exposure, the present study examined the effect of adolescent intermittent ethanol (AIE) exposure during postnatal days 28-42 on γ-aminobutyric acid (GABA)ergic neurotransmission in the prelimbic cortex. In control rats, patch-clamp electrophysiology in acute slices obtained at different postnatal ages revealed a developmental increase in the GABAA receptor-mediated tonic current in layer V pyramidal neurons but no change in layers II/III when measured in the adult. In slices from AIE-exposed rats, the amplitude of the tonic current was significantly reduced compared with controls when tested at postnatal days 45, 60 and 90-120. This AIE-induced reduction in tonic current was found to reflect attenuation of currents mediated by δ-subunit containing receptors. Consistent with this, facilitation of the tonic current by bath application of either ethanol or allopregnanolone was attenuated in slices from AIE-exposed adult rats compared with control rats. However, expression of this facilitation as a percent of the amplitude of the total current mediated by δ-GABAA receptors revealed that AIE did not alter their sensitivity to either agonist. Lastly, immunohistochemistry and Western blot analysis revealed no change in the expression of δ-GABAA subunits or their surface expression. Taken together, these studies reveal that AIE exposure results in persistent deficits in δ-GABAA tonic currents in the adult prelimbic cortex that may contribute to deficits in decision-making and behavioral control in adulthood.

Binge-Like Alcohol Exposure During Adolescence Disrupts Dopaminergic Neurotransmission in the Adult Prelimbic Cortex.

Repeated binge-like exposure to alcohol during adolescence has been reported to perturb prefrontal cortical development, yet the mechanisms underlying these effects are unknown. Here we report that adolescent intermittent ethanol exposure induces cellular and dopaminergic abnormalities in the adult prelimbic cortex (PrL-C). Exposing rats to alcohol during early-mid adolescence (PD28-42) increased the density of long/thin dendritic spines of layer 5 pyramidal neurons in the adult PrL-C. Interestingly, although AIE exposure did not alter the expression of glutamatergic proteins in the adult PrL-C, there was a pronounced reduction in dopamine (DA) D1 receptor modulation of both intrinsic firing and evoked NMDA currents in pyramidal cells, whereas D2 receptor function was unaltered. Recordings from fast-spiking interneurons also revealed that AIE reduced intrinsic excitability, glutamatergic signaling, and D1 receptor modulation of these cells. Analysis of PrL-C tissue of AIE-exposed rats further revealed persistent changes in the expression of DA-related proteins, including reductions in the expression of tyrosine hydroxylase and catechol-O-methyltransferase (COMT). AIE exposure was associated with hypermethylation of the COMT promoter at a conserved CpG site in exon II. Taken together, these findings demonstrate that AIE exposure disrupts DA and GABAergic transmission in the adult medial prefrontal cortex (mPFC). As DA and GABA work in concert to shape and synchronize neuronal ensembles in the PFC, these alterations could contribute to deficits in behavioral control and decision-making in adults who abused alcohol during adolescence.