Neuropharmacology

The use of neuromodulation techniques for the treatment of alcohol use disorder is receiving increasing attention, especially non-invasive approaches, such as repetitive transcranial magnetic stimulation or transcranial direct current stimulation, while the hypothetical use of electroconvulsive therapy remains unexplored. Given our experience inducing electroconvulsive seizures (ECS) for therapeutic purposes in psychopathology rodent models, we evaluated the role of ECS on reducing the increased voluntary ethanol consumption caused by adolescent ethanol exposure in our validated preclinical model. Rats were treated in adolescence with a binge paradigm of ethanol (2 g/kg, i.p.; 3 rounds of 2 days at 48-h intervals; post-natal day, PND 29-30, PND 33-34 and PND 37-38) or saline. Following persistent withdrawal until adulthood, rats were allowed to: voluntarily drink ethanol (20%) by a two-bottle choice test, for 3 days (PND 80-82); treated with ECS (95 mA for 0.6 s, 100 Hz, pulse width 0.6 ms; ear-clip electrodes) or SHAM for 5 days (PND 86-90); re-exposed to voluntarily ethanol exposure (PND 94-96). Brains were collected on PND 97 to evaluate hippocampal markers of ethanol toxicity and/or treatment response (e.g., NeuroD, NF-L, BDNF and NF-L/BDNF ratio). Our results reproduced the increased voluntary ethanol consumption in adult rats induced by adolescent ethanol exposure and demonstrated that ECS could improve this abuse-prone response. Moreover, we suggested a possible role for BDNF in the beneficial effects induced by ECS, especially reducing the neurotoxic ratio NF-L/BDNF. Overall, we provide preclinical evidence for the potential use of ECS as an efficacious treatment for alcohol use disorder.

BackgroundFatty foods and alcohol (i.e., ethanol) produce strong reinforcing effects, in part by altering cholinergic interneuron (CIN) activity and tonic dopamine (DA) release within the nucleus accumbens (NAc). Ethanol and fatty foods also both stimulate hepatic and possibly local brain bile acid (BA) synthesis, which raises the possibility that BAs may act as a common upstream regulator of these substances shared mesolimbic effects. MethodsThe current study investigated whether BAs can directly alter mesolimbic activity. Electrophysiological data from acute mouse brain slices was collected to assess BA effects on NAc CIN firing, as well as on excitatory and inhibitory postsynaptic CIN inputs. Bile effects on NAc DA release and clearance rates were measured through voltammetry. ResultsWe found that low concentrations of a 1:1 mixture of BAs cholic acid (CA) and deoxycholic acid (DCA; 1-10 M) increased CIN firing rate, whereas high BA concentrations (1-10 mM) decreased CIN firing. We further demonstrated that BA-induced excitatory effects on CIN firing are independently mediated by at least two mechanisms: Takeda G-protein-coupled receptor 5 (TGR5) activation and suppression of inhibitory CIN currents. Additionally, our results indicate that BAs modulate inhibitory input in a complex manner, reducing frequency at low concentrations, but increasing at high concentrations, and increasing amplitude at low concentrations current amplitude, and the distribution of postsynaptic current amplitude sizes across concentrations. Finally, our voltammetry data indicate that while low BA concentrations enhance NAc DA release without affecting DA uptake, high BA concentrations robustly inhibit accumbal DA release. ConclusionOur findings provide evidence that BAs exert direct modulatory effects on neural activity in the striatum.

Alcohol use disorder is a major public health concern worldwide and there is a high comorbidity with psychiatric disorders. The basolateral amygdala (BLA) has been implicated in both mood and alcohol use disorders; however, the mechanisms contributing to the shared pathophysiology remain unknown. Extensive evidence indicates that ethanol modulates GABAergic signaling in the BLA, including actions on neurosteroid-sensitive, extrasynaptic {delta} subunit-containing GABAA receptors (GABAARs), which has been suggested to mediate many of the behavioral effects. In fact, several studies have suggested that 5-reduced neurosteroids, such as allopregnanolone, may mediate some of the behavioral effects of alcohol. Here we demonstrate that chronic intermittent ethanol (CIE) exposure impairs endogenous neurosteroidogenesis via downregulation of key neurosteroidogenic enzymes, 5-reductase type 1 and type 2. To examine the impact of impaired endogenous neurosteroidogenesis of the behavioral consequences of chronic alcohol exposure, including withdrawal-induced anxiety and increased alcohol consumption, we used CRISPR/Cas9 mediated knockdown of 5-reductase in the BLA. Reduced expression of 5-reductase in the BLA did not impact post-CIE alcohol intake or anxiety-like behaviors during withdrawal, perhaps because endogenous neurosteroidogenesis is already impaired following CIE. Therefore, we examined the impact of enhancing neurosteroid levels, treating mice post-CIE with SGE-516, a synthetic GABAAR positive allosteric modulator, which increased voluntary alcohol intake. These findings implicate endogenous neurosteroidogenesis in behavioral outcomes associated with withdrawal from chronic alcohol exposure. Further, this study suggests that targeting endogenous neurosteroidogenesis may be a novel and useful therapeutic target.

Alcohol use disorder (AUD) affects over 28 million people in the U.S and is associated with neurobiological alterations, including in the basal ganglia. Within the basal ganglia, the caudate nucleus (caudate) and putamen are implicated in AUD due to their roles in ethanol reinforcement, with the caudate receiving inputs from cortico-associative areas and the putamen receiving inputs from somatosensory areas, supporting goal-directed and habitual behaviors respectively. These distinct behavioral roles are supported by dopamine signaling, including phasic dopamine, involved in assessing action-outcome associations, and tonic dopamine, which reflects ongoing dopaminergic tone that biases action initiation. Intrastriatal dopamine release is modulated by cholinergic interneurons via nicotinic acetylcholine receptors. Dysregulation of these mechanisms can contribute to the transition from occasional to habitual ethanol drinking. Here, we used in-vitro fast-scan cyclic voltammetry to measure dopamine signaling in male (n=6) and female (n=6) rhesus macaques following six months of ethanol self-administration. In putamen, ethanol increased tonic dopamine in both sexes, with females exhibiting greater release and faster dopamine uptake rates than males. In the caudate, ethanol self-administration enhanced dopamine uptake rates only in males. Phasic dopamine release was enhanced in caudate of both sexes but only putamen in males. nAChR blockade revealed that phasic dopamine release in males, but not females, was dependent on cholinergic modulation. These results demonstrate basal and sex-specific dopamine release and uptake are uniquely altered in rhesus macaque caudate and putamen in conjunction with chronic ethanol drinking.

Cannabis contains many bioactive compounds, including {Delta}9-Tetrahydrocannabinol (THC) and cannabidiol (CBD), which influence behavior through complex pharmacological interactions with endogenous targets. This study examines whether CBD influences THC-induced changes in motor activity, hypothermia, and antinociception traits across different THC:CBD ratios, sexes, and genetic backgrounds. Traits were measured in C57BL/6J (B6) and DBA/2J (D2) mice of both sexes following baseline intraperitoneal (i.p.) injection of vehicle (VEH) and two consecutive daily doses of VEH or THC (10 mg/kg) alone or in combination with 0.56, 5, or 10 mg/kg CBD (THC:0.56CBD, THC:5CBD, or THC:10CBD, respectively). Motor activity and hypothermia were quantified daily from 0 to 120 min following injection and antinociception was measured daily at 60 min. We found that CBD alters THC-induced changes in motor activity and hypothermia as a function of day, dose, time, sex, and strain. In D2 females, CBD dose-dependently attenuated the hypolocomotor effects of THC immediately following acute injection and enhanced these effects later at 75 min. Following repeated exposure, CBD dose-dependently enhanced THC-induced hypolocomotion in B6 females at 75 min and in D2 males at 30 min while attenuating THC-induced hypolocomotion in D2 females immediately following injection. In D2 females, CBD dose-dependently attenuated THC-induced hypothermia at 15 min and enhanced hypothermia relative to THC at 30 min in D2 males following acute injection. After repeated exposure, CBD dose-dependently enhanced THC-induced hypothermia in B6 females at 15 min and in D2 males from 30 to 120 mins, while attenuating hypothermia in D2 females at 30 min. No significant effects of CBD on antinociception were observed. Our results indicate that CBD can modulate some THC-induced traits acutely and after repeated exposure. Regulation of THC-induced behavioral responses is dependent on CBD dose, genetic background, and sex. A candidate gene search using brain gene expression in recombinant inbred mice revealed greater genetic variation in ion channel genes relative to key metabolic genes, suggesting an underlying pharmacodynamic mechanism. Future research and validation of molecular mechanisms underlying these differences is expected to enhance our understanding of potential health risks and clinical relevance of cannabis and cannabinoid compounds containing THC and CBD.

Alcohol use disorder (AUD) is a chronic relapsing condition with limited pharmacological treatments. Ethanol modulates neuronal excitability in part through activation of G-protein-gated inwardly rectifying potassium (GIRK/Kir3) channels, which dampen neuronal activity in reward- and stress-related circuits implicated in AUD pathophysiology. In this study, we investigated the therapeutic potential of targeting activation of GIRK channels in mouse models of ethanol intoxication. GiGA1 (G protein-independent GIRK activator type 1) is a selective activator of GIRK1/GIRK2 channels and has good brain bioavailability. Systemic GiGA1 administration prevented acquisition of ethanol-induced conditioned place preference (CPP) in both male and female mice. GiGA1 also significantly reduced voluntary ethanol intake and decreased blood alcohol concentrations, when administered to mice after they developed high preference and consumption of ethanol. Similarly, Baclofen, a GABAB receptor agonist that leads to activation of GIRK channels also decreased ethanol consumption. However, systemic Baclofen did not prevent acquisition of ethanol-dependent CPP, suggesting a broader efficacy of direct GIRK1/GIRK2 activation by GiGA1. Whole-brain c-Fos mapping as a proxy for neuronal activity revealed that GiGA1 blunted ethanol-induced neuronal activation in several AUD-relevant brain regions, including the central amygdala, paraventricular thalamus, paraventricular hypothalamus, and Edinger-Westphal nucleus. These findings demonstrate that pharmacological activation of GIRK channels modulates key neural circuits involved in ethanol reward and intake, supporting GiGA1 as a promising lead compound for targeted AUD therapy.

Astrocytes are the most abundant glial cells in the brain and an integrative component of the neural network. Studies have shown that ethanol altered expression of an astrocyte marker, i.e., glial fibrillary acidic protein (GFAP), in two key corticolimbic regions, the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). These regions comprise anatomically and functionally different subregions, i.e., the prelimbic (PL) and infralimbic (IL) cortex of the mPFC, the shell and core subregions of the NAc. However, ethanol effects on GFAP expression within these subregions remain largely unknown. In addition, effects of pharmacological manipulation of astrocytes on alcohol drinking have been understudied. Western blot was conducted to determine GFAP expression in subregions of the mPFC and NAc after chronic ethanol drinking. Fluorocitrate, an astrocyte-specific metabolic inhibitor, was administered to inhibit astrocytes and was tested on ethanol drinking. Ethanol drinking enhanced GFAP protein expression in the PL cortex and NAc core, but not in the IL cortex or NAc shell. Intra-ventricular administration of fluorocitrate reduced ethanol intake and preference, but increased water consumption during choice ethanol drinking. In addition, fluorocitrate did not affect total fluid consumption or basal locomotor activity. These results indicate that chronic ethanol drinking induced GFAP elevation in a subregion-specific manner within the mPFC and NAc, and that metabolic inhibition of astrocytes selectively attenuated ethanol drinking without non-specific effects on water drinking or general activity. Together, these results suggest that astrocytes may play an important role in ethanol drinking. HighlightsO_LIEthanol drinking enhanced GFAP levels in the PL cortex and NAc core. C_LIO_LIFluorocitrate inhibition of astrocytes reduced intermittent ethanol drinking. C_LIO_LIFluorocitrate did not alter total fluid consumption or basal locomotor activity. C_LI

Excessive alcohol drinking is a leading cause of preventable death in the United States. High alcohol consumption and persistent drinking despite adverse events, also known as compulsive drinking, are key criteria that contribute to the development and progression of alcohol use disorder (AUD). There is a clear need to better understand the mechanisms that support these related but distinct behaviors. The serotonin (5-HT) system has been associated with alcohol consumption and risk of alcohol dependence, however given the complexity of this system, there remains much to discover regarding specific alcohol related phenotypes. The current study uses a combination of volitional home-cage drinking and operant conditioning to phenotype mice based on ethanol intake and persistence of alcohol drinking following quinine adulteration, a model to study compulsive drinking. Brain tissue of 10 regions known to be implicated in regulating executive function, reward, and stress was collected, and gene expression of serotonergic receptors, transporters, and enzymes was quantified. Three opioid receptors were included given their well-established roles in alcohol-related behaviors and interactions with the 5HT system. Region-specific gene expression patterns emerged, with serotonergic and opioid receptor expression differentially associated with alcohol drinking phenotype. 5-HT and opioid receptors displayed opposing directionality across regions, consistent with functional heterogeneity within the system. These findings identify region-specific molecular alterations following chronic alcohol that may contribute to individual differences in alcohol drinking phenotypes, highlighting candidate targets for biomarkers of increased alcohol use disorder susceptibility or as interventions aimed at preventing the progression of AUD.

Tobacco use disorder is a chronic condition characterized by compulsive nicotine use, withdrawal, and relapse following abstinence. Impulsivity contributes to persistent nicotine use and poor cessation outcomes. This study examined whether nicotinic acetylcholine receptor (nAChR) modulators alter impulsive action in a nicotine self-administration Go/No-Go task in male and female rats. Rats acquired intravenous nicotine self-administration and were then trained in a Go/No-Go procedure in which active lever presses were reinforced during Go periods but not during No-Go periods. We then assessed the effects of varenicline (0.1-3 mg/kg), nicotine (0.1-0.6 mg/kg), and the nAChR antagonist mecamylamine (0.5-2 mg/kg) in the Go/No-Go procedure. Varenicline and nicotine pretreatment reduced active responding during both Go and No-Go periods, whereas mecamylamine selectively reduced responding during No-Go periods. Mecamylamine decreased the percentage of active responses during No-Go trials, indicating reduced bias toward the nicotine-associated lever. In contrast, nicotine and varenicline did not alter response allocation, suggesting that their effects reflected nonspecific reductions in responding rather than changes in impulsive action. No sex differences were observed. Substituting saline for nicotine during self-administration did not alter active responding during Go periods, but rats in the saline group had fewer active responses during No-Go periods than rats in the nicotine group. These results show that chronic nicotine self-administration increases impulsive action and that nAChR antagonism, but not agonism or partial agonism, reduces nicotine-related impulsive action. This work supports the utility of the Go/No-Go self-administration task for investigating nAChR-dependent mechanisms underlying nicotine-induced impulsivity.

Dopamine (DA) neurons of the midbrain project throughout the striatum, including the nucleus accumbens core (NAc) and are thought to co-release ATP with DA from vesicles. The mechanisms of evoked NAc ATP release and clearance and their relationship to exocytotic DA transmission are largely unexplored and the focus of the present work. Using fast scan cyclic voltammetry (FSCV), we measured simultaneous ATP and DA transmission in response to pharmacological manipulations of release and reuptake cellular machinery. ATP transmission is tightly coupled to that of DA, though ATP release concentrations are typically smaller. Manipulations that increase DA transmission (increased release via 4-aminopyridine Kv channel blockade or decreased uptake via cocaine) also increase ATP transmission, though to a smaller extent. Blocking DA vesicular packaging (reserpine) or action potentials (lidocaine), results in attenuated DA and ATP release. Interestingly, reserpine or lidocaine can result in completely abolished DA release, but not a complete prevention in ATP release, suggesting a secondary source for ATP transmission thats not dependent on DA terminals. Both transmitters were reduced to a similar extent following nAChR blockade, demonstrating that nAChR activation regulates ATP in addition to DA. Surprisingly, cocaine inhibition of DATs reduced clearance for both ATP and DA, which correlated with one another when cocaine concentration was highest. There was also a strong relationship between the effect of cocaine on release of ATP and DA. As the first FSCV study to examine evoked NAc ATP release, this paper bridges prior work to confirm the strong association between ATP and DA in the mesolimbic circuit and identifies unexpected overlap in mechanisms regulating their transmission. Our results contribute novel evidence of both vesicular and non-vesicular ATP release in the NAc and demonstrate that extracellular ATP is a modulator of DA terminal function.

Alternative polyadenylation (APA) is a common posttranscriptional mechanism to regulate gene expression. APA generates mRNAs with varying lengths of 3' UTRs or transcripts that encode distinct protein carboxy-terminal ends. APA is especially important in neurons, where different mRNA variants are often asymmetrically localized to dendrites and axons, and can be locally translated into proteins. Local protein synthesis is crucial for axon guidance, synaptic plasticity, and learning and memory, key processes associated with the development of alcohol use disorder (AUD). We investigated the role of APA in AUD using a mouse model of alcohol dependence characterized by increased voluntary drinking after chronic intermittent ethanol (CIE) exposure. We examined APA during protracted withdrawal from alcohol in three brain regions of male and female mice. Our analyses revealed hundreds of genes undergoing APA in males, but substantially fewer in females, suggesting sex-specific effects of CIE on APA. Notably, male and female mice displayed distinct APA signatures. APA genes were different from differentially expressed genes (DEGs), suggesting that these molecular processes are regulated independently. We also determined that the expression of APA genes was associated with neurons, while DEGs were associated with non-neuronal cells. Many of the APA genes were involved in synaptic integrity, neuroplasticity, and neuronal maintenance, which was consistent with their enrichment in neurons. Our study suggests that APA is a crucial sex- and cell type-specific mechanism in AUD with the potential to influence localized neuronal protein expression during protracted withdrawal and to modify alcohol consumption behavior. HIGHLIGHTSO_LIChronic ethanol exposure in mice results in profound changes of APA genes in brain. C_LIO_LICommonly regulated cleavage and polyadenylation sites and genes were identified in male but not in female mice. C_LIO_LIThere was a minimal overlap between APA and differentially expressed genes (DEGs). C_LIO_LIAPA genes were primarily associated with neurons, whereas DEGs were associated with non-neuronal cells. C_LI

Neuroimmune signaling is increased in postmortem brain tissue from individuals with alcohol use disorder (AUD), and growing evidence suggests that it contributes to persistent alcohol-related neuroadaptations. Interferon regulatory factor 7 (IRF7), a transcription factor downstream of endosomal Toll-like receptor signaling, is induced in alcohol-relevant brain regions and may contribute to escalated drinking. Here, we tested whether chronic intermittent ethanol (CIE) vapor exposure engages IRF7 signaling during subsequent alcohol self-administration and whether this is associated with altered molecular E/I balance in the aIC and altered functional E/I balance in aICnucleus accumbens projection neurons. Female Wistar rats (n=30) were trained to self-administer alcohol (15% v/v; FR2 vs inactive lever) during 30-minute sessions. After establishing baseline drinking, rats underwent 1-3 cycles of CIE, which increased alcohol self-administration at the 72 h post vapor test. This increase positively correlated with IRF7 levels in the anterior insular cortex (aIC) and nucleus accumbens, while molecular, and immunofluorescence showed that CIE shifted aIC excitatory/inhibitory (E/I) balance toward reduced excitation. Electrophysiological recordings further showed reduced functional E/I balance in aIC neurons projecting to the nucleus accumbens. Knockdown of IRF7 in the aIC attenuated CIE induced escalation of alcohol self-administration, supporting a role for insular IRF7 signaling in alcohol related neuroadaptations that promote escalated drinking.

Opioid use disorder (OUD) is a chronic, relapsing disease driven by the reinforcing properties of opioids and perpetuated by avoidance of the negative affective states associated with the absence of the drug. Most available OUD treatments directly engage the {micro}-opioid receptor and may induce side effects that can compromise their therapeutic efficacy, thus underscoring the need for novel therapeutic alternatives. Calcitonin gene-related peptide (CGRP) is produced by a small population of neurons in the parabrachial nucleus (PBN) that has been shown to modulate itch, pain, as well as appetitive behaviors. Using a cell-specific nuclear labeling approach coupled with RNA-sequencing, we generated a baseline transcriptome of CGRPPBN neurons and confirmed expression of multiple genes associated with behavioral responses to appetitive stimuli, as well as enrichment of the {micro}-opioid receptor, suggesting that CGRPPBN neuron function may be sensitive to the presence of opioids. Indeed, cFos immunostaining showed that CGRPPBN neuron activity increases during early morphine abstinence and reduces gradually over 48 hours. Given the inhibitory effects of opioids on CGRPPBN neuron activity, we next tested whether these neurons could regulate opioid reinforcement. Using a mouse model of morphine intravenous self-administration, we found that chemogenetic inhibition of CGRPPBN neurons significantly reduced the number of morphine rewards earned in both single-dose and dose-response tests but did not affect context-induced morphine seeking after 21 days of abstinence. These results suggest that CGRPPBN neurons are sensitive to opioid administration and can regulate appetitive behaviors such as morphine-taking. Considering that CGRP signaling is regulated by opioid administration, molecular targets that regulate CGRP neurotransmission without direct -opioid receptor engagement may therefore serve as novel therapeutic avenues for the treatment of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/712659v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1fb9c9borg.highwire.dtl.DTLVardef@1e6ba79org.highwire.dtl.DTLVardef@dc60f5org.highwire.dtl.DTLVardef@61adaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Stress is a major trigger of relapse in alcohol use disorder (AUD), and dysfunction of prefrontal cortex (PFC) circuits has been implicated in this process. Epigenetic regulators may contribute to relapse by shaping transcriptional programs within these circuits. Here, we investigated the role of the histone methyltransferase PRDM2 in stress-induced alcohol seeking. Analysis of postmortem human tissue showed that PRDM2 expression in the PFC was reduced in both men and women with AUD compared with control individuals. To examine the functional significance of this reduction, we used viral-mediated knockdown of Prdm2 in the dorsomedial prefrontal cortex (dmPFC) of male and female rats. Prdm2 knockdown enhanced stress-induced reinstatement of alcohol seeking in both sexes, particularly at intermediate shock intensity, without altering pain sensitivity or being influenced by estrous cycle stage. To determine whether this effect was mediated through specific prefrontal output pathways, we selectively reduced Prdm2 expression in dmPFC neurons projecting to the nucleus accumbens (NAc). Projection-specific knockdown also increased stress-induced reinstatement of alcohol seeking in male and female rats in an intensity-dependent manner. Together, these findings suggest that reduced PRDM2 expression in the PFC may contribute to stress-induced relapse-like behavior and identify the dmPFC-NAc projection as a circuit through which PRDM2 can influence alcohol seeking.

The misuse of opioid medications is a significant health issue in the United States. Very few studies have investigated the effect of opioids on perineuronal nets (PNNs), scaffold-like structures that surround neurons and are involved in the regulation of plasticity-dependent mechanisms such as development, learning and memory, and acquisition of addiction-like phenotypes. Regulation of PNNs in the orbitofrontal cortex (OFC) during periods of drug intoxication or withdrawal is widely unknown. In this study, male Wistar rats were injected with fentanyl (0.125 mg/kg, s.c.) or 0.9% saline twice daily for 7 days and once on day 8 (7continuous days following by 3 days of abstinence) or twice daily for 15 days (5 continuous days followed by 2 days of abstinence for more than 3 weeks) and twice on day 16. Antinociception was evaluated using the tail immersion test immediately before and 30 minutes after injections. Whole-brain coronal slices were collected, and immunohistochemistry was used to identify Wisteria Floribunda Agglutinin (WFA)-positive PNNs and parvalbumin (PV)-expressing cells. Results confirmed that repeated fentanyl injections induced tolerance to the antinociceptive effects, which normalized following acute abstinence periods. WFA intensity decreased following 8 days of injections. Analyses confirmed significant correlations between PV+ density and tail withdrawal latency following 8 days of fentanyl injections. These data confirm that repeated fentanyl injections modulate both WFA+ and PV+ expression in the rodent brain and antinociceptive tolerance in a duration-dependent manner. Overall, these data suggest that perineuronal nets may mediate opioid-induced behavioral effects, such as antinociceptive tolerance, following repeated administration and abstinence in rats.

Background: Alcohol use disorder is associated with dysregulated glutamatergic signaling within mesocorticolimbic circuits that govern reinforcement and excessive ethanol intake. Group II metabotropic glutamate receptors (mGlu2/3) act primarily as presynaptic autoreceptors that regulate glutamate release. However, how voluntary alcohol intake alters mGlu2/3 expression within reward circuitry remains unclear. Methods and Results: We examined the effects of operant alcohol self-administration on mGlu2/3 protein expression and assessed the functional impact of group II receptor modulation on binge-like ethanol intake. Male C57BL/6J mice self-administered sweetened ethanol or sucrose under behaviorally matched conditions for 35 days. Immediately after the final session, tissue punches from the nucleus accumbens (NAc), amygdala, and prefrontal cortex were collected for Western blot analysis. Operant ethanol self-administration selectively reduced mGlu2/3 protein expression in the NAc, with no changes detected in the amygdala or prefrontal cortex. Both monomeric and dimeric mGlu2/3 protein levels were reduced, and a composite index revealed coordinated downregulation of receptor expression. In separate cohorts, systemic administration of the mGlu2/3 agonist LY379268 dose-dependently reduced binge-like ethanol intake in a limited-access home-cage drinking model, whereas positive allosteric modulation of mGlu2 receptors with LY487379 was ineffective. Conclusions: These results show that low-dose operant ethanol self-administration produces an ethanol- and region-specific reduction of mGlu2/3 protein expression in the NAc and that pharmacological activation of group II receptors, potentially involving mGlu3-specific receptors, is sufficient to suppress binge-like ethanol consumption. These data identify presynaptic mGlu2/3 dysregulation as a mechanism contributing to ethanol-related behaviors and support group II metabotropic glutamate receptors as therapeutic targets for alcohol use disorder.

Endocannabinoid (eCB) signaling is a key regulator of reward-related dopaminergic signaling, particularly in response to drugs of abuse, such as cocaine. To date, our understanding of this mechanism has primarily been limited to male subjects. Prior work establishes that female cocaine users have more adverse outcomes, and female rats show greater sensitivity to cannabinoid type 1 receptor (CB1R) regulation of cocaine self-administration. Therefore, we hypothesize that female rats exhibit enhanced eCB regulation of cocaine-evoked dopamine (DA). We used in vivo fiber photometry recording of the dopamine biosensor, dLight 1.3b, in the nucleus accumbens medial shell (NAcms) in response to cocaine in male and female rats. Rats were pretreated with cannabinoid-targeting drugs to investigate the effects of CB1R inactivation or augmentation of the eCB 2-AG on cocaine-evoked DA. Our results revealed that CB1R inactivation attenuates cocaine-evoked DA in male and female rats, but females showed enhanced sensitivity for CB1R regulation of cocaine-evoked DA. Cocaine-evoked DA was enhanced by augmenting 2-AG levels, and females again showed increased sensitivity to this manipulation. Finally, females show greater cocaine-evoked DA when in a non-estrous cycle compared to estrous, reinforcing that estrous cycle is a determinant of cocaine-evoked DA. These data indicate that females show enhanced eCB regulation of cocaine-evoked DA signaling, underscoring the importance of sex as a biological variable in our understanding of endocannabinoid regulation of drug reward. HighlightsO_LICB1R inactivation attenuates cocaine-evoked DA in NAcms, preferentially in females C_LIO_LI2-AG augmentation via MAGL inhibition enhances cocaine-evoked DA, with female bias C_LIO_LIEstrous phase modulates the dopamine response to a high dose of cocaine in females C_LIO_LIMale and female rats show similar baseline DA and locomotor responses to cocaine C_LI

Astrocytes play essential roles in maintaining brain homeostasis and in contributing to synaptic functions, but, in response to injury, infection, or disease, astrocytes can downregulate their homeostatic and physiological functions while increasing neuroinflammatory responses. The central amygdala (CeA) is important for stress responsivity and the development of alcohol (ethanol) dependence. Using a multi-omics approach in Aldh1l1-EGFP/Rpl10a mice and the chronic intermittent ethanol two-bottle choice (CIE-2BC) model, we have characterized the translational response of CeA astrocytes, as well as the proteomic and phosphoproteomic changes in ethanol dependent, non-dependent, and naive mice. We identified astrocyte-specific alterations in neuroimmune functions and antioxidant/oxidative stress pathways in ethanol dependent mice as well as cytoskeletal plasticity related pathways in non-dependent mice. Proteomic analysis showed down-regulation of astrocyte physiological functions in dependent animals while phosphoproteomic analysis identified pathways associated with cytoskeleton remodeling in both dependent and non-dependent mice. Reconstructions of astrocyte morphologies demonstrated increased CeA astrocyte complexity in dependent and non-dependent groups compared to naive mice. The astrocyte-specific activation of neuroimmune and antioxidant pathways, down-regulation of homeostatic functions, alteration in protein phosphorylation-mediated cytoskeleton remodeling, and increased astrocyte morphological complexity demonstrate that ethanol dependence induces astrocyte reactivity in the CeA consistent with both adaptive and maladaptive changes. These findings highlight the role of CeA astrocytes in the progression from alcohol intake to dependence and represent a first step toward identifying astrocyte-specific therapeutic strategies to treat Alcohol Use Disorder (AUD) aimed at potentiating reactive astrocyte adaptive changes and inhibiting maladaptive responses.

Cognitive impairment is a major component of Alcohol Use Disorder. Optimal cognitive performance requires anterior cingulate input activation of the claustrum, a subcortical nucleus that orchestrates cortical activity. Yet the impact of chronic alcohol exposure on the ability for the anterior cingulate cortex to drive activity of claustrum projection neuron subtypes is unknown. In adult male and female mice, we found that the majority of non-burst firing Type 1 claustrum projection neurons did not express the vesicular glutamate transporter 2 (VGLUT2), while the majority of burst firing Type 2 projection neurons were VGLUT2-expressing. Following chronic intermittent vaporized ethanol exposure (CIE), we found that both Type 1 and VGLUT2-non-expressing neurons exhibited increased responsivity to anterior cingulate cortex input activation that was mediated by increased postsynaptic membrane excitability. In contrast, Type 2 and VGLUT2-expressing projection neurons exhibited increased responsivity to anterior cingulate cortex input due to strengthened pre- and post-synaptic transmission mechanisms. Altogether, we uncovered a hyper-excitatory drive of the claustrum by the anterior cingulate cortex following chronic alcohol exposure. The data provide a foundational resource for the complex effects of chronic alcohol exposure on the claustrum, a critical cognitive control nucleus.

The fear circuit orchestrates defensive responses to environmental threats and is essential for survival. Dysregulation of this system is thought to contribute to the pathophysiology of several psychiatric disorders. Within this fear circuit, the corticolimbic network, particularly the amygdala and the medial prefrontal cortex (mPFC), is strongly modulated by serotonin. Previous studies have shown that Htr3a knockout (Htr3a-KO) mice exhibit deficits in the extinction of cued fear memory; however, the circuit level mechanisms underlying these impairments remain unknown. Here, we investigated this question by recording local field potentials evoked by auditory conditioned stimuli (CS) in the prelimbic (PrL), infralimbic (IL), and basolateral amygdala (BLA) of head-fixed wild-type (WT) and Htr3a-KO mice prior to fear conditioning and during fear memory retrieval. Behaviorally, Htr3a-KO mice displayed a delayed attenuation of fear-induced freezing during cued fear memory retrieval, whereas WT mice showed a rapid attenuation in freezing. Electrophysiologically, Htr3a-KO mice exhibited reduced fear-evoked theta power in the PrL, IL, and BLA, along with diminished mPFC-BLA theta synchrony. Moreover, theta-phase modulation of gamma oscillations within the BLA, which has been shown to increase during fear states, was perturbed in the absence of Htr3a signaling. Together, these findings indicate that Htr3a is critical for maintaining proper oscillatory dynamics within the mPFC-BLA circuit and for supporting effective attenuation of learned fear. Highlights- Attenuation of fear responses during fear memory retrieval sessions is protracted in Htr3a knock-out mice - The fear-induced theta response in the medial prefrontal cortex and the basolateral amygdala is less powerful in the Htr3a knock-out mice than in wild-type - Htr3a knock-out mice show a deficit in fear-induced synchronization as well as in theta modulation of gamma power in the cortico-limbic network - These results suggest that malfunction of the Htr3a receptor cause alterations in fear network circuit mechanisms that might be linked to deficits in fear responses attenuation