Alcoholism and alcohol use disorders are characterized by several months to decades of heavy and problematic drinking, interspersed with periods of abstinence and relapse to heavy drinking. This alcohol-drinking phenotype was modeled using macaque monkeys to explore neuronal adaptations in the striatum, a brain region controlling habitual behaviors. Prolonged drinking with repeated abstinence narrowed the variability in daily intake, increased the amount of ethanol consumed in bouts, and led to higher blood ethanol concentrations more than twice the legal intoxication limit. After the final abstinence period of this extensive drinking protocol, we found a selective increase in dendritic spine density and enhanced glutamatergic transmission in the putamen, but not in the caudate nucleus. Intrinsic excitability of medium-sized spiny neurons was also enhanced in the putamen of alcohol-drinking monkeys in comparison with non-drinkers, and GABAeric transmission was selectively suppressed in the putamen of heavy drinkers. These morphological and physiological changes indicate a shift in the balance of inhibitory/excitatory transmission that biases the circuit toward an enduring increase in synaptic activation of putamen output as a consequence of prolonged heavy drinking/relapse. The resultant potential for increased putamen activation may underlie an alcohol-drinking phenotype of regulated drinking and sustained intoxication.

Repeated cycles of binge alcohol drinking and abstinence are key components in the development of dependence. However, the precise behavioral mechanisms underlying binge-like drinking and its consequences on striatal synaptic physiology remain unclear. In the present study, ethanol and water drinking patterns were recorded with high temporal resolution over 6 weeks of binge-like ethanol drinking using the 'drinking in the dark' (DID) protocol. The bottle exchange occurring at the beginning of each session prompted a transient increase in the drinking rate that might facilitate the acquisition of ethanol binge-like drinking. Ethanol drinking mice also displayed a 'front-loading' behavior, in which the highest rate of drinking was recorded during the first 15 min. This rate increased over weeks and paralleled the mild escalation of blood ethanol concentrations. GABAergic and glutamatergic transmission in the dorsal striatum were examined following DID. Spontaneous glutamatergic transmission and the density of dendritic spines were unchanged after ethanol drinking. However, the frequency of GABAA receptor-mediated inhibitory postsynaptic currents was depressed in medium spiny neurons of ethanol drinking mice. A history of ethanol drinking also increased ethanol preference and altered the acute ethanol effects on GABAergic transmission differentially in dorsolateral and dorsomedial striatum. Together, the study shows that the bottle exchange during DID promotes fast, voluntary ethanol drinking and that this intermittent pattern of ethanol drinking causes a depression of GABAergic transmission in the dorsal striatum.

Exposure to trauma is a risk factor for the development of a number of mood disorders, and may enhance vulnerability to future adverse life events. Recent data demonstrate that ventral tegmental area (VTA) neurons expressing the vesicular glutamate transporter 2 (VGluT2) signal and causally contribute to behaviors that involve aversive or threatening stimuli. However, it is unknown whether VTA VGluT2 neurons regulate transsituational outcomes of stress and whether these neurons are sensitive to stressor controllability. This work adapted an operant mouse paradigm to examine the impact of stressor controllability on VTA VGluT2 neuron function as well as the role of VTA VGluT2 neurons in mediating transsituational stressor outcomes. Uncontrollable (inescapable) stress, but not physically identical controllable (escapable) stress, produced social avoidance and exaggerated fear in male mice. Uncontrollable stress in females led to exploratory avoidance of a novel brightly lit environment. Both controllable and uncontrollable stressors increased VTA VGluT2 neuronal activity, and chemogenetic silencing of VTA VGluT2 neurons prevented the behavioral sequelae of uncontrollable stress in male and female mice. Further, we show that stress activates multiple genetically-distinct subtypes of VTA VGluT2 neurons, especially those that are VGluT2+VGaT+, as well as lateral habenula neurons receiving synaptic input from VTA VGluT2 neurons. Our results provide causal evidence that mice can be used for identifying stressor controllability circuitry and that VTA VGluT2 neurons contribute to transsituational stressor outcomes, such as social avoidance, exaggerated fear, or anxiety-like behavior that are observed within trauma-related disorders.

The current opioid epidemic necessitates a better understanding of human addiction neurobiology to develop efficacious treatment approaches. Here, we perform genome-wide assessment of chromatin accessibility of the human striatum in heroin users and matched controls. Our study reveals distinct neuronal and non-neuronal epigenetic signatures, and identifies a locus in the proximity of the gene encoding tyrosine kinase FYN as the most affected region in neurons. FYN expression, kinase activity and the phosphorylation of its target Tau are increased by heroin use in the post-mortem human striatum, as well as in rats trained to self-administer heroin and primary striatal neurons treated with chronic morphine in vitro. Pharmacological or genetic manipulation of FYN activity significantly attenuates heroin self-administration and responding for drug-paired cues in rodents. Our findings suggest that striatal FYN is an important driver of heroin-related neurodegenerative-like pathology and drug-taking behavior, making FYN a promising therapeutic target for heroin use disorder.

Foraging is a universal behavior that has co-evolved with predation pressure. We investigated the role of bed nucleus of the stria terminalis (BNST) GABA neurons in robotic and live predator threat processing and their consequences in post-threat encounter foraging. Mice were trained to procure food in a laboratory-based foraging apparatus in which food pellets were placed at discrete and incrementally greater distances from a nest zone. After mice learned to forage, they were exposed to either a robotic or live predator threat, while BNST GABA neurons were chemogenetically inhibited. Post-robotic threat encounter, mice spent more time in the nest zone, but other foraging parameters were unchanged compared to pre-encounter behavior. Inhibition of BNST GABA neurons had no effect on foraging behavior post-robotic threat encounter. Following live predator exposure, control mice spent significantly more time in the nest zone, increased their latency to successfully forage, and their overall foraging performance was significantly a ltered. I nhibition o f BNST GABA neurons during live predator exposure prevented changes in foraging behavior from developing after live predator threat. BNST GABA neuron inhibition did not alter foraging behavior during robotic or live predator threat. We conclude that while both robotic and live predator encounter effectively intrude on foraging behavior, the perceived risk and behavioral consequence of the threats are distinguishable. Additionally, BNST GABA neurons may play a role in the integration of prior innate predator threat experience that results in hypervigilance during post-encounter foraging behavior.

NMDA-type ionotropic glutamate receptors are critical for normal brain function and are implicated in central nervous system disorders. Structure and function of NMDA receptors composed of GluN1 and GluN3 subunits are less understood compared to those composed of GluN1 and GluN2 subunits. GluN1/3 receptors display unusual activation properties in which binding of glycine to GluN1 elicits strong desensitization, while glycine binding to GluN3 alone is sufficient for activation. Here, we explore mechanisms by which GluN1-selective competitive antagonists, CGP-78608 and L-689,560, potentiate GluN1/3A and GluN1/3B receptors by preventing glycine binding to GluN1. We show that both CGP-78608 and L-689,560 prevent desensitization of GluN1/3 receptors, but CGP-78608-bound receptors display higher glycine potency and efficacy at GluN3 subunits compared to L-689,560-bound receptors. Furthermore, we demonstrate that L-689,560 is a potent antagonist of GluN1FA+TL/3A receptors, which are mutated to abolish glycine binding to GluN1, and that this inhibition is mediated by a non-competitive mechanism involving binding to the mutated GluN1 agonist binding domain (ABD) to negatively modulate glycine potency at GluN3A. Molecular dynamics simulations reveal that CGP-78608 and L-689,560 binding or mutations in the GluN1 glycine binding site promote distinct conformations of the GluN1 ABD, suggesting that the GluN1 ABD conformation influences agonist potency and efficacy at GluN3 subunits. These results uncover the mechanism that enables activation of native GluN1/3A receptors by application of glycine in the presence of CGP-78608, but not L-689,560, and demonstrate strong intra-subunit allosteric interactions in GluN1/3 receptors that may be relevant to neuronal signaling in brain function and disease.

The dorsal striatum (DS) is implicated in behavioral and neural processes including action control and reinforcement. Alcohol alters these processes in rodents, and it is believed that the development of alcohol use disorder involves changes in DS dopamine signaling. In nonhuman primates, the DS can be divided into caudate and putamen subregions. As part of a collaborative effort examining the effects of long-term alcohol self-administration in rhesus macaques, we examined DS dopamine signaling using fast-scan cyclic voltammetry. We found that chronic alcohol self-administration resulted in several dopamine system adaptations. Most notably, dopamine release was altered in a sex- and region-dependent manner. Following long-term alcohol consumption, male macaques, regardless of abstinence status, had reduced dopamine release in putamen, while only male macaques in abstinence had reduced dopamine release in caudate. In contrast, female macaques had enhanced dopamine release in the caudate, but not putamen. Dopamine uptake was also enhanced in females, but not males (regardless of abstinence state). We also found that dopamine D2/3 autoreceptor function was reduced in male, but not female, alcohol drinkers relative to control groups. Finally, we found that blockade of nicotinic acetylcholine receptors inhibited evoked dopamine release in nonhuman primates. Altogether, our findings demonstrate that long-term alcohol consumption can sex-dependently alter dopamine release, as well as its feedback control mechanisms in both DS subregions.

A unique population of ventral tegmental area (VTA) neurons co-transmits glutamate and GABA as well as functionally signals rewarding and aversive outcomes. However, the circuit inputs to VTA VGluT2+VGaT+ neurons are unknown, limiting our understanding of the functional capabilities of these neurons. To identify the inputs to VTA VGluT2+VGaT+ neurons, we coupled monosynaptic rabies tracing with intersectional genetic targeting of VTA VGluT2+VGaT+ neurons in mice. We found that VTA VGluT2+VGaT+ neurons received diverse brain-wide inputs. The largest numbers of monosynaptic inputs to VTA VGluT2+VGaT+ neurons were from superior colliculus, lateral hypothalamus, midbrain reticular nucleus, and periaqueductal gray, whereas the densest inputs relative to brain region volume were from dorsal raphe nucleus, lateral habenula, and ventral tegmental area. Based on these and prior data, we hypothesized that lateral hypothalamus and superior colliculus inputs were glutamatergic neurons. Optical activation of glutamatergic lateral hypothalamus neurons robustly activated VTA VGluT2+VGaT+ neurons regardless of stimulation frequency and resulted in flee-like ambulatory behavior. In contrast, optical activation of glutamatergic superior colliculus neurons activated VTA VGluT2+VGaT+ neurons for a brief period of time at high stimulation frequency and resulted in head rotation and arrested ambulatory behavior (freezing). For both pathways, behaviors induced by stimulation were uncorrelated with VTA VGluT2+VGaT+ neuron activity, suggesting that VGluT2+VGaT+ neurons are integrators of signals related to aversive outcomes but not of aversion-induced behavioral kinematics. We interpret these results such that VTA VGluT2+VGaT+ neurons may integrate diverse inputs related to the detection and processing of motivationally-salient outcomes.

Hypothalamic agouti-related peptide (AgRP) neurons potently stimulate food intake, whereas proopiomelanocortin (POMC) neurons inhibit feeding. Whether AgRP neurons exert their orexigenic actions, at least in part, by inhibiting anorexigenic POMC neurons remains unclear. Here, the connectivity between GABA-releasing AgRP neurons and POMC neurons was examined in brain slices from male and female mice. GABA-mediated spontaneous IPSCs (sIPSCs) in POMC neurons were unaffected by disturbing GABA release from AgRP neurons either by cell type-specific deletion of the vesicular GABA transporter or by expression of botulinum toxin in AgRP neurons to prevent vesicle-associated membrane protein 2-dependent vesicle fusion. Additionally, there was no difference in the ability of μ-opioid receptor (MOR) agonists to inhibit sIPSCs in POMC neurons when MORs were deleted from AgRP neurons, and activation of the inhibitory designer receptor hM4Di on AgRP neurons did not affect sIPSCs recorded from POMC neurons. These approaches collectively indicate that AgRP neurons do not significantly contribute to the strong spontaneous GABA input to POMC neurons. Despite these observations, optogenetic stimulation of AgRP neurons reliably produced evoked IPSCs in POMC neurons, leading to the inhibition of POMC neuron firing. Thus, AgRP neurons can potently affect POMC neuron function without contributing a significant source of spontaneous GABA input to POMC neurons. Together, these results indicate that the relevance of GABAergic inputs from AgRP to POMC neurons is state dependent and highlight the need to consider different types of transmitter release in circuit mapping and physiologic regulation.SIGNIFICANCE STATEMENT Agouti-related peptide (AgRP) neurons play an important role in driving food intake, while proopiomelanocortin (POMC) neurons inhibit feeding. Despite the importance of these two well characterized neuron types in maintaining metabolic homeostasis, communication between these cells remains poorly understood. To provide clarity to this circuit, we made electrophysiological recordings from mouse brain slices and found that AgRP neurons do not contribute spontaneously released GABA onto POMC neurons, although when activated with channelrhodopsin AgRP neurons inhibit POMC neurons through GABA-mediated transmission. These findings indicate that the relevance of AgRP to POMC neuron GABA connectivity depends on the state of AgRP neuron activity and suggest that different types of transmitter release should be considered when circuit mapping.

A two-neuron model of ventral tegmental area (VTA) opioid function classically involves VTA GABA neuron regulation of VTA dopamine neurons via a mu-opioid receptor dependent inhibitory circuit. However, this model predates the discovery of a third major type of neuron in the VTA: glutamatergic neurons. We found that about one-quarter of VTA neurons expressing the mu-opioid receptor are glutamate neurons without molecular markers of GABA co-release. Glutamate-Mu opioid receptor neurons are largely distributed in the anterior VTA. The majority of remaining VTA mu-opioid receptor neurons are GABAergic neurons that are mostly within the posterior VTA and do not express molecular markers of glutamate co-release. Optogenetic stimulation of VTA glutamate neurons resulted in excitatory currents recorded from VTA dopamine neurons that were reduced by presynaptic activation of the mu-opioid receptor ex vivo, establishing a local mu-opioid receptor dependent excitatory circuit from VTA glutamate neurons to VTA dopamine neurons. This VTA glutamate to VTA dopamine pathway regulated dopamine release to the nucleus accumbens through mu-opioid receptor activity in vivo. Behaviorally, VTA glutamate calcium-related neuronal activity increased following oral oxycodone consumption during self-administration and response-contingent oxycodone-associated cues during abstinent reinstatement of drug-seeking behavior. Further, chemogenetic inhibition of VTA glutamate neurons reduced abstinent oral oxycodone-seeking behavior in male but not female mice. These results establish 1) a three-neuron model of VTA opioid function involving a mu-opioid receptor gated VTA glutamate neuron pathway to VTA dopamine neurons that controls dopamine release within the nucleus accumbens, and 2) that VTA glutamate neurons participate in opioid-seeking behavior.

The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons.

Extensive evidence suggests that the hypocretins/orexins influence cocaine reinforcement and dopamine signaling via actions at hypocretin receptor 1. By comparison, the involvement of hypocretin receptor 2 in reward and reinforcement processes has received relatively little attention. Thus, although there is some evidence that hypocretin receptor 2 regulates intake of some drugs of abuse, it is currently unclear to what extent hypocretin receptor 2 participates in the regulation of dopamine signaling or cocaine self-administration, particularly under high effort conditions. To address this, we examined the effects of hypocretin receptor 1, and/or hypocretin receptor 2 blockade on dopamine signaling and cocaine reinforcement. We used in vivo fast scan cyclic voltammetry to test the effects of hypocretin antagonists on dopamine signaling in the nucleus accumbens core and a progressive ratio schedule to examine the effects of these antagonists on cocaine self-administration. Results demonstrate that blockade of either hypocretin receptor 1 or both hypocretin receptor 1 and 2 significantly reduces the effects of cocaine on dopamine signaling and decreases the motivation to take cocaine. In contrast, blockade of hypocretin receptor 2 alone had no significant effects on dopamine signaling or self-administration. These findings suggest a differential involvement of the two hypocretin receptors, with hypocretin receptor 1 appearing to be more involved than hypocretin receptor 2 in the regulation of dopamine signaling and cocaine self-administration. When considered with the existing literature, these data support the hypothesis that hypocretins exert a permissive influence on dopamine signaling and motivated behavior via preferential actions on hypocretin receptor 1.

Fetal alcohol exposure (FAE) is the leading preventable developmental cause of cognitive dysfunction. Even in the absence of binge drinking, alcohol consumption during pregnancy can leave offspring deficient. However, the mechanisms underlying these deficiencies are unknown. Using a mouse model of gestational ethanol exposure (GEE), we show increased instrumental lever-pressing and disruption of efficient habitual actions in adults, indicative of disrupted cognitive function. In vivo electrophysiology reveals disrupted action encoding in dorsolateral striatum (DLS) associated with altered habit learning. GEE mice exhibit decreased GABAergic transmission onto DLS projection neurons, including inputs from parvalbumin interneurons, and increased endocannabinoid tone. Chemogenetic activation of DLS parvalbumin interneurons reduces the elevated lever pressing of GEE mice. Pharmacologically increasing endocannabinoid tone mimics GEE effects on cognition and synaptic transmission. These findings show GEE induces long-lasting deficits in cognitive function that may contribute to human FAE, and identify potential mechanisms for future therapeutic targeting.

Tools for refined cell-specific targeting have significantly contributed to understanding the characteristics and dynamics of distinct cellular populations by brain region. While advanced cell-labeling methods have accelerated the field of neuroscience, specifically in brain mapping, there remains a need to quantify and analyze the data. Here, by modifying a toolkit that localizes electrodes to brain regions (SHARP-Track; Slice Histology Alignment, Registration, and Probe-Track analysis), we introduce a post-imaging analysis tool to map histological images to established mouse brain atlases called SHARCQ (Slice Histology Alignment, Registration, and Cell Quantification). The program requires MATLAB, histological images, and either a manual or automatic cell count of the unprocessed images. SHARCQ simplifies the post-imaging analysis pipeline with a step-by-step GUI. We demonstrate that SHARCQ can be applied for a variety of mouse brain images, regardless of histology technique. In addition, SHARCQ rectifies discrepancies in mouse brain region borders between atlases by allowing the user to select between the Allen Brain Atlas or the digitized and modified Franklin-Paxinos Atlas for quantifying cell counts by region. SHARCQ produces quantitative and qualitative data, including counts of brain-wide region populations and a 3D model of registered cells within the atlas space. In summary, SHARCQ was designed as a neuroscience post-imaging analysis tool for cell-to-brain registration and quantification with a simple, accessible interface. All code is open-source and available for download (https://github.com/wildrootlab/SHARCQ).