Cocaine use disorder is marked by deficits in dopamine signaling; however, the molecular mechanisms driving these deficits remain unclear. The synaptic vesicle protein Synaptogyrin-3 (Syngr3) has recently garnered attention for its association with disorders involving dopamine dysfunction and impaired cognition. Here, we found low Syngr3 expression in the ventral tegmental area (VTA) of men who died of cocaine overdose and in male rats that had chronically self-administered cocaine. Syngr3 was confirmed to be in dopamine neurons, and its expression was correlated with dopamine markers in both humans and rats. Syngr3 levels showed a robust inverse correlation with motivation to self-administer cocaine in the rat model. Moreover, viral overexpression of Syngr3 in VTA dopamine neurons improved cognitive flexibility and substantially reduced cocaine reinforcement and drug-taking behavior. Finally, Syngr3 overexpression prevented cocaine-induced dopamine deficits. These findings establish Syngr3 as a key dopamine regulator and potential therapeutic target for cocaine use disorder.

Acute ethanol modulates an incredibly diverse number of neurotransmitter receptors and intracellular pathways. Presynaptic modulation by acute ethanol has been identified at some GABAergic synapses; but descriptions of acute ethanol regulation of presynaptic glutamatergic function are sparse. Recent reports suggest that acute ethanol can inhibit glutamatergic synaptic responses arising from recycling vesicles during tetanic stimulation of Stria terminalis inputs onto basolateral amygdala (BLA) principal neurons. This finding gives rise to the hypothesis that acute ethanol may modulate actively priming synaptic vesicles in glutamate synapses. To test this, we used whole cell patch clamp recordings in BLA principal neurons and measured the effects of acute ethanol on asynchronous synaptic transmission. We found Stria teminalis inputs express EGTA-sensitive asynchronous synaptic glutamatergic responses produced from both increased presynaptic activation and experimental elevation of presynaptic calcium using partial substitution of extracellular Na+ with Li+. Under both conditions, asynchronous events were acutely inhibited by ethanol in a concentration-dependent fashion. Ethanol inhibition of asynchronous glutamate release was present in DBA/2J Stria terminalis synapses but absent when measuring inputs in C57BL/6J mice. EGTA, which robustly suppresses expression of asynchronous release, also occluded ethanol modulation under both conditions. Surprisingly, EGTA did not alter the expression or occlude ethanol modulation of synaptic responses arising from recycling vesicles. Our findings demonstrate a unique acute effect of ethanol on asynchronous glutamate vesicle priming pathways.

Unique populations of basolateral amygdala (BLA) neurons regulate anxiety and reward through projections targeting downstream regions like the bed nucleus of the stria terminalis (BNST) and nucleus accumbens (NAC). We showed previously that withdrawal from chronic ethanol exposure (CIE/WD) produced population- and sex-specific alterations to distinct glutamatergic inputs. The current study examined GABAergic function in these distinct populations (BLANAC and BLABNST neurons). We found that CIE/WD diminished feed-forward GABA release from lateral paracapsular cells (LPCs) specifically onto male BLANAC neurons. Pharmacological manipulations showed this dysregulation was caused by the enhanced activity of μ-opioid receptors. CIE/WD did not alter evoked GABA release from local interneurons onto either population. However, females expressed greater GABA release from these local interneurons compared to males. Immunostaining and confocal microscopy revealed lower colocalization between the GABA vesicular transporter, vGAT and parvalbumin in females, indicating that greater GABA releases from local interneurons in this sex may be a compensatory response to lower levels of perisomatic innervation by PV+ interneurons. Consistent with this, there were no sex differences related to spontaneous GABAergic synaptic events although CIE/WD decreased their frequency specifically in BLABNST neurons from both sexes. Altogether, these findings demonstrate that CIE/WD dynamically alters GABAergic function in an input-, sex- and population-specific fashion. Moreover, there are basal sex differences in both the anatomy of BLA GABAergic synapses and their function.

Synaptogyrin-3, a functionally obscure synaptic vesicle protein, interacts with vesicular monoamine and dopamine transporters, bringing together dopamine release and reuptake sites. Synaptogyrin-3 was reduced by chronic cocaine exposure in both humans and rats, and synaptogyrin-3 levels inversely correlated with motivation to take cocaine in rats. Synaptogyrin-3 overexpression in dopamine neurons reduced cocaine self-administration, decreased anxiety-like behavior, and enhanced cognitive flexibility. Overexpression also enhanced nucleus accumbens dopamine signaling and prevented cocaine-induced deficits, suggesting a putative therapeutic role for synaptogyrin-3 in cocaine use disorder.

ID 25402 Poster Board 371 Background: The trimeric G-proteins, composed of a Gα and a dimeric Gβg subunit, function to relay extracellular stimuli from G-protein-coupled-receptors (GPCRs) and transduce intracellular signaling cascades by directly interacting with their cognate coupling partners in confined membrane microdomains. Chronic intermittent ethanol (CIE) exposure alters the function of many GPCRs, although the molecular mechanism is largely unknown. Our preliminary data indicate that CIE exposure increases membrane cholesterol content in the brain. Membrane cholesterol critically regulates the compartmentalization of membrane proteins and the assembly of signaling complexes. Thus, this project aimed to test the hypothesis that CIE exposure may result in spatial alterations of membrane localization of G-proteins and their coupling partners within lipid raft and non-raft microdomains, disrupting GPCR-stimulated G-protein and downstream signaling. Method:Ethanol Vapor Exposure: Male Sprague Dawley rats were exposed to either ethanol vapor (CIE) or room air (AIR) during the light cycle (12 hr/day) for seven consecutive days. Following 24 hr withdrawal from the last ethanol exposure, all animals were euthanized, and prefrontal cortex (PFC) tissue was dissected. Sucrose Density Gradient Ultracentrifugation: PFC tissue was homogenized and then fractionated by discontinuous sucrose density (5%/30%/40%) ultracentrifugation. Fifteen fractions were collected along the sucrose density gradient for each sample. Western blotting was performed to determine the localization of G-protein subunits and their interacting proteins within lipid raft and non-lipid raft microdomains. Results: We found that CIE induced differential changes in the localization of G-protein subunits within lipid rafts and non-raft regions. In CIE-exposed animals, Gαi and Gαo subunits, but not Gαs and Gαq subunits, translocated from lipid rafts toward non-rafts, while Gβγ subunits translocated from non-rafts toward lipid rafts when compared to AIR-exposed control animals. Interestingly, CIE exposure did not alter the compartmentalization of the protein kinases adenylyl cyclase type 1 and phospholipase Cβ1, which are direct G-protein interacting proteins. Further, CIE exposure induced an increase in lipid raft localization of mGluR2, a Gαi/o-interacting protein, and Kv1.2, a Gβγ-interacting protein, but had no effect on Gαq-coupled mGluR1 localization. Finally, CIE exposure significantly increased rat PFC membrane cholesterol content. Conclusions: Our data suggest that spatial localization of G-proteins and their interacting proteins is susceptible to regulation by CIE. Importantly, the present study highlights a potential role of lipid metabolism in GPCR dysregulation and may open a new avenue for targeting cholesterol metabolism as a treatment for alcohol use disorder. Support/Funding Information: T32-AA007565 R21DA056857 R01DA042862

Nucleus basalis magnocellularis (NBM) cholinergic projections to the basolateral amygdala (BLA) regulate the acquisition and consolidation of fear-like and anxiety-like behaviors. However, it is unclear whether the alterations in the NBM-BLA circuit promote negative affect during ethanol withdrawal (WD). Therefore, we performed ex vivo whole-cell patch-clamp electrophysiology in both the NBM and the BLA of male Sprague Dawley rats following 10 d of chronic intermittent ethanol (CIE) exposure and 24 h of WD. We found that CIE exposure and withdrawal enhanced the neuronal excitability of NBM putative “cholinergic” neurons. We subsequently used optogenetics to directly manipulate NBM terminal activity within the BLA and measure cholinergic modulation of glutamatergic afferents and BLA pyramidal neurons. Our findings indicate that CIE and withdrawal upregulate NBM cholinergic facilitation of glutamate release via activation of presynaptic nicotinic acetylcholine receptors (AChRs). Ethanol withdrawal-induced increases in NBM terminal activity also enhance BLA pyramidal neuron firing. Collectively, our results provide a novel characterization of the NBM-BLA circuit and suggest that CIE-dependent modifications to NBM afferents enhance BLA pyramidal neuron activity during ethanol withdrawal.

Chronic intermittent ethanol and withdrawal (CIE/WD) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections to the nucleus accumbens (NAC) and bed nucleus of the stria terminalis (BNST), respectively. Previous studies revealed that CIE/WD induces input- and sex-specific adaptations to glutamatergic function in the BLA. The BLA receives information from two distinct input pathways. Glutamatergic afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE/WD increases presynaptic glutamatergic function at stria terminalis synapses and postsynaptic function at external capsule synapses. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative "reward" and "aversion" BLA neurons, and showed that CIE/WD alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE/WD increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE/WD increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE/WD enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE/WD-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.

The basolateral amygdala (BLA) is intimately involved in the development of neuropsychiatric disorders such as anxiety and alcohol use disorder (AUD). These disorders have clear sex biases, with women more likely to develop an anxiety disorder and men more likely to develop AUD. Preclinical models have largely confirmed these sex-specific vulnerabilities and emphasize the effects of sex hormones on behaviors influenced by the BLA. This review will discuss sex differences in BLA-related behaviors and highlight potential mechanisms mediated by altered BLA structure and function, including the composition of GABAergic interneuron subpopulations, glutamatergic pyramidal neuron morphology, glutamate/GABA neurotransmission, and neuromodulators. Further, sex hormones differentially organize dimorphic circuits during sensitive developmental periods (organizational effects) and initiate more transient effects throughout adulthood (activational effects). Current literature indicates that estradiol and allopregnanolone, a neuroactive progestogen, generally reduce BLA-related behaviors through a variety of mechanisms, including activation of estrogen receptors or facilitation of GABAA-mediated inhibition, respectively. This enhanced GABAergic inhibition may protect BLA pyramidal neurons from the excitability associated with anxiety and alcohol withdrawal. Understanding sex differences and the effects of sex hormones on BLA structure and function may help explain sex-specific vulnerabilities in BLA-related behaviors and ultimately improve treatments for anxiety and AUD.

Chronic intermittent ethanol (CIE) exposure dysregulates glutamatergic and GABAergic neurotransmission, facilitating basolateral amygdala (BLA) pyramidal neuron hyperexcitability and the expression of anxiety during withdrawal. It is unknown whether ethanol-induced alterations in nucleus basalis magnocellularis (NBM) cholinergic projections to the BLA mediate anxiety-related behaviors through direct modulation of GABA and glutamate afferents. Following 10 days of CIE exposure and 24 h of withdrawal, we recorded GABAergic and glutamatergic synaptic responses in BLA pyramidal neurons with electrophysiology, assessed total protein expression of cholinergic markers, and quantified acetylcholine and choline concentrations using a colorimetric assay. We measured alpha(7) nicotinic acetylcholine receptor (nAChR) dependent modulation of presynaptic function at distinct inputs in AIRand CIE-exposed BLA coronal slices as a functional read-out of cholinergic neurotransmission. CIE/withdrawal upregulates the endogenous activity of alpha(7) nAChRs, facilitating release at both GABAergic' local' interneuron and glutamatergic synaptic responses to stria terminalis (ST) stimulation, with no effect at GABAergic lateral paracapsular cells (LPCs). CIE caused a three-fold increase in BLA acetylcholine concentration, with no changes in alpha(7) nAChR or cholinergic marker expression. These data illustrate that alpha(7) nAChRdependent changes in presynaptic function serve as a proxy for CIE-dependent alterations in synaptic acetylcholine levels. Thus, cholinergic projections appear to mediate CIE-induced alterations at GABA/glutamate inputs. (c) 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

This review highlights literature relating the anatomy, physiology, and behavioral contributions by projections between rodent prefrontal cortical areas and the basolateral amygdala. These projections are robustly modulated by both environmental experience and exposure to drugs of abuse including ethanol. Recent literature relating optogenetic and chemogenetic dissection of these circuits within behavior both compliments and occasionally challenges roles defined by more traditional pharmacological or lesion-based approaches. In particular, cortico-amygdala circuits help control both aversive and reward-seeking. Exposure to pathology-producing environments or abused drugs dysregulates the relative 'balance' of these outcomes. Modern circuit-based approaches have also shown that overlapping populations of neurons within a given brain region frequently govern both aversion and reward-seeking. In addition, these circuits often dramatically influence 'local' cortical or basolateral amygdala excitatory or inhibitory circuits. Our understanding of these neurobiological processes, particularly in relation to ethanol research, has just begun and represents a significant opportunity. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

Adolescent alcohol use in human populations dramatically increases the likelihood of adult alcohol use disorder. This adolescent vulnerability is recapitulated in preclinical models which provide important opportunities to understand basic neurobiological mechanisms. We provide here an overview of GABAergic and glutamatergic neurotransmission and our current understanding of the sensitivity of these systems to adolescent ethanol exposure. As a whole, the preclinical literature suggests that adolescent vulnerability may be directly related to region-specific neurobiological processes that continue to develop during adolescence. These processes include the activity of intrinsic circuits within diverse brain regions (primarily represented by GABAergic neurotransmission) and activity-dependent regulation of synaptic strength at glutamatergic synapses. Furthermore, GABAergic and glutamatergic neurotransmission within regions/circuits that regulate cognitive function, emotion, and their integration appears to be the most vulnerable to adolescent ethanol exposure. Finally, using documented behavioral differences between adolescents and adults with respect to acute ethanol, we highlight additional circuits and regions for future study.

A key feature of alcohol use disorder (AUD) is negative affect during withdrawal, which often contributes to relapse and is thought to be caused by altered brain function, especially in circuits that are important mediators of emotional behaviors. Both the agranular insular cortex (AIC) and the basolateral amygdala (BLA) regulate emotions and are sensitive to ethanol-induced changes in synaptic plasticity. The AIC and BLA are reciprocally connected; and the effects of chronic ethanol exposure on this circuit have yet to be explored. Here, we use a combination of optogenetics and electrophysiology to examine the pre- and postsynaptic changes that occur to AIC-BLA synapses following withdrawal from 7- or 10-days of chronic intermittent ethanol (CIE) exposure. While CIE/withdrawal did not alter presynaptic glutamate release probability from AIC inputs, withdrawal from 10, but not 7, days of CIE increased AMPA receptor-mediated postsynaptic function at these synapses. Additionally, NMDA receptor-mediated currents evoked by electrical stimulation of the external capsule, which contains AIC afferents, were also increased during withdrawal. Notably, a single subanesthetic dose of ketamine administered at the onset of withdrawal prevented the withdrawal-induced increases in both AMPAR and NMDAR postsynaptic function. Ketamine also prevented the withdrawal-induced increases in anxiety-like behavior measured using the elevated zero maze. Together, these findings suggest that chronic ethanol exposure increases postsynaptic function within the AIC-BLA circuit and that ketamine can prevent ethanol withdrawal-induced alterations in synaptic plasticity and negative affect.