Neuropharmacology

AbstractPrenatal exposure to alcohol (PAE) increases the risk for misusing alcohol and/or developing an alcohol use disorder (AUD) by adulthood. The corticotropin releasing factor (CRF) system is a major target of pre- and post-natal ethanol (EtOH) exposure. CRF and its receptor (CRFR1), in part, mediate EtOH potentiated GABA release in the medial nucleus of the central amygdala (CeM) of adult male rodents. Interestingly, our lab has shown a disruption in the function and expression of CeM CRFR1 and acute EtOHs effects on GABA transmission in PAE adolescent animals, but it is unknown whether these alterations to the CRF system persist into adulthood or alter the actions of acute EtOH on GABAergic transmission in the CeM. Using CRF1-Cre-tdTomato rats, this study examined how moderate PAE alters acute EtOH modulation of GABAergic neurotransmission onto CRFR1+ and CRFR1-CeM neurons in adult offspring (P80-105). Pregnant dams were exposed to vaporized ethanol or room air (control) on gestational day 12 (G12) for 6 hours and whole-cell electrophysiology was performed in the CeM to assess the actions of acute EtOH (44, 66, & 88 mM) on GABAergic transmission onto CRFR1+ and CRFR1-neurons. We found unique effects of PAE that were cell type- and concentration-dependent in males and females, suggesting PAE dysregulates acute EtOHs modulation of GABA transmission within the CeM in a sex-specific manner. This study contributes to the expanding body of research exploring the effects of PAE and how a single exposure can impact neurophysiological mechanisms in brain regions associated with AUD. HighlightsO_LIModerate PAE alters the actions of alcohol on synaptic transmission in adult rats C_LIO_LIPAE differentially impacts GABAergic transmission onto CeM CRFR1- and CRFR1+ C_LIO_LILong-term PAE effects in the CeM are sex-specific C_LI

Alcohol Use Disorders (AUD) is characterized by compulsion-like alcohol drinking (CLAD), and this intake despite negative consequences can be a major clinical obstacle. With the quite limited treatment options available for AUD, there is a significant and critical unmet need for novel therapies. The noradrenergic system is an important hub for the stress response as well as maladaptive drives for alcohol, and pre-clinical (including our own) and clinical studies have shown that drugs targeting the 1 adrenenergic receptors (ARs) may represent a pharmacological treatment for pathological drinking. However, the involvement of {beta} ARs for treating human drinking AUD has received somewhat scant investigation, and we sought to provide pre-clinical validation for possible AR utility for CLAD. Thus, we first examined whether {beta} AR antagonist propranolol, betaxolol ({beta}1), and ICI, 118 551 ({beta}2) impacted compulsion-like intake and alcohol-only drinking (AOD) in male Wistar rats through systemic injections. The systemic highest dose of propranolol (10mg/kg) reduced both AOD and CLAD. 5mg/kg propranolol affected CLAD more than AOD, with no effects of 2.5mg/kg. Similar to propranolol, betaxolol also only decreased CLAD at the lower dose (2.5mg/kg). ICI 118.551 had no effects, suggesting propranolol regulates alcohol intake through {beta}1. Also, while AR compounds might have utility for AUD, these compounds can also lead to undesirable cardiovascular system side effects; thus, any strategy incorporating lower doses of these compounds to reduce drinking could have broad utility. Importantly, here we found that a combination of ineffective doses of propranolol and prazosin administrated together did reduce both CLAD and AOD. Finally, we investigated the effect of propranolol and betaxolol into two brain areas related to pathological drinking, the anterior insula (aINS) and medial prefrontal cortex (mPFC). Surprisingly, propranolol (1-10g) in aINS or mPFC did not affect CLAD or AOD (although with a trend for aINS betaxolol to impact CLAD), suggesting propranolol regulation of alcohol drinking through a target other than aINS and mPFC. Together, our findings provide new pharmacological insights into noradrenergic regulation of alcohol consumption, which may inform AUD therapy.

BackgroundChronic alcohol exposure is a major driver of alcohol use disorders (AUD), in part through its ability to induce maladaptive plasticity within neural circuits that regulate reward, motivation, and affect. Excitatory projections from the hippocampus (Hipp) to the nucleus accumbens (NAc) play a pivotal role in regulating reward-related behaviors, and this pathway serves as a key locus for establishing associations between rewarding stimuli and related contextual information. Regulation of the strength of Hipp-NAc synapses is critical for supporting these behaviors, and aberrant Hipp-NAc plasticity is associated with anhedonia and disrupted reward learning. MethodsTo examine acute ethanol effects, we used whole-cell electrophysiology to record Hipp-NAc synaptic plasticity in acute brain slices in the presence or absence of 50mM ethanol. To examine the effects of chronic ethanol administration, mice were exposed to ethanol vapor in a 3-week chronic intermittent ethanol (CIE) paradigm. Slices from ethanol and air exposed mice were used for whole-cell electrophysiology to examine Hipp-NAc synaptic plasticity. ResultsHere, we demonstrate that acute ethanol application to ex vivo brain slices prevents long-term potentiation (LTP) at Hipp-NAc synapses, without altering presynaptic release probability. Furthermore, chronic intermittent exposure to ethanol abolishes LTP at these synapses, even during abstinence, indicating persistent synaptic dysfunction. ConclusionsTogether, our findings demonstrate that ethanol has immediate and long-lasting effects on Hipp-NAc plasticity. Given the behavioral relevance of these synapses, this work has important implications for the mechanisms underlying ethanol-dependent effects on reward processing and negative affective states associated with AUD.

Individuals with prenatal alcohol exposure (PAE) are at a higher risk for developing alcohol use disorder (AUD). Using a rat model of PAE on gestational day 12 (G12; ~2nd trimesters in humans), a critical period for amygdala development, we have shown disruptions in medial central amygdala (CeM) function, an important brain region associated with the development of AUD. In addition to this, acute ethanol (EtOH) increases GABA transmission in the CeM of rodents in a sex-dependent manner, a mechanism that potentially contributes to alcohol misuse. How PAE alters acute alcohols effects within the CeM is unknown. Given these findings, we investigated how PAE may interact with acute alcohol to alter neuronal and synaptic mechanisms in the CeM of adolescent rats in order to understand PAE-induced alcohol-related behaviors. Under basal conditions, PAE males showed reduced rheobase, indicative of reduced excitability, and females showed a reduction in GABA transmission, indicated by lower spontaneous inhibitory postsynaptic currents (sIPSCs). We found that acute EtOH increased sIPSCs in control males at a moderate concentration (66 mM), while PAE males showed increased sIPSCs only at a high concentration (88 mM). Adolescent females, regardless of PAE status, were largely insensitive to EtOHs effects at all tested concentrations. However, PAE females showed a significant increase in sIPSCs at the highest concentration (88 mM). Overall, these findings support the hypothesis that PAE leads to sex-specific changes in synaptic activity and neuronal function. Future research is needed to better understand the specific mechanisms by which acute EtOHs affects neurotransmission in the adolescent brain of individuals with a history of PAE.

Although FDA-approved medications for alcohol use disorder (AUD) are available, their efficacy varies across patients, highlighting the need for novel therapeutics that address inter-individual differences in disease etiology and treatment response. Genetic models, particularly heterogeneous stock (HS) rats, recapitulate human-like genetic diversity and behavioral heterogeneity, enabling the dissection of individual differences in vulnerability to AUD and pharmacotherapeutic sensitivity. P2X4 receptors, which are encoded by the gene P2rx4, are ATP-gated ion channels inhibited by ethanol and abundantly expressed in neurons found in reward and stress circuits. P2X4 receptors have emerged as key modulators of ethanol sensitivity and consumption in preclinical models. Here, we genetically predicted P2rx4 expression in whole brain in a cohort of 130 HS rats exposed to chronic intermittent ethanol (CIE) vapor and phenotyped for self-administration during acute abstinence. Rats were dichotomized into high- and low-predicted expression groups. Higher predicted P2rx4 expression was associated with increased post-vapor intake and escalation. In 32 CIE-escalated rats, ivermectin, a positive allosteric modulator of P2X4 receptors, dose-dependently reduced drinking. We stratified rats into three groups: non-responders, mild responders, and high responders. Electrophysiological recordings from CeA slices revealed that ivermectin differentially enhanced GABAergic IPSCs: high-responders exhibited sustained increases in IPSC frequency and selective amplitude reductions, while the two other groups showed transient frequency increases. All groups displayed prolonged rise times, however non-responders showed extended decay times. These findings suggest that P2rx4 upregulation serves as a vulnerability marker for dependence-like behaviors, with ivermectin attenuating withdrawal-driven alcohol consumption by enhancing CeA GABAergic inhibition.

The constantly evolving trends in substance abuse are a major concern for health authorities worldwide, and these trends include an increasing prevalence of poly-substance misuse. For instance, opioid overdoses are frequently accompanied by alcohol co-use, yet the combined effects of these substances remain poorly understood. Using zebrafish as a highly relevant vertebrate model of neuropharmacology, we investigated the interactions between fentanyl and alcohol, uncovering a unique and robust hyperlocomotor response characterized by initial locomotor suppression followed by persistent, erratic hyperlocomotion. Alcohol was found to be critical to this phenomenon, as substitution with other GABAA modulators failed to replicate the effect. However, the response was replicable with heroin and remifentanil, suggesting an opioid-class wide effect. In vivo whole brain imaging further demonstrated dysregulated neuronal activity, with co-administration with alcohol causing potentiated neuronal activity compared to individual drug exposures and controls. Collectively, these findings suggested an integral role of ethanol and fentanyl co-administration in dysregulated neuronal responses and reveals a complex neurobehavioral mechanism. These observations suggest further investigation is warranted into the use of the larval zebrafish model for studying the neuropharmacological interactions of multiple substances of abuse.

Rationale: The positive reinforcing effects of alcohol (ethanol) drive its repetitive use and contribute to alcohol use disorder (AUD). Ethanol alters the expression of glutamate AMPA receptor (AMPAR) subunits in reward-related brain regions, but the extent to which this effect regulates ethanols reinforcing properties is unclear. Objective: This study investigates whether ethanol self-administration changes AMPAR subunit expression and synaptic activity in the nucleus accumbens core (AcbC) to regulate ethanols reinforcing effects in male C57BL/6J mice. Results: Sucrose-sweetened ethanol self-administration (0.81 g/kg/day) increased AMPAR GluA2 protein expression in the AcbC, without effect on GluA1, compared to sucrose-only controls. Infusion of myristoylated Pep2m in the AcbC, which blocks GluA2 binding to N-ethylmaleimide-sensitive fusion protein (NSF) and reduces GluA2-containing AMPAR activity, reduced ethanol-reinforced responding without affecting sucrose-only self-administration or motor activity. Antagonizing GluA2-lacking AMPARs, through AcbC infusion of NASPM, had no effect on ethanol self-administration. AcbC neurons receiving projections from the basolateral amygdala (BLA) showed increased sEPSC area under the curve (a measurement of charge transfer) and slower decay kinetics in ethanol self-administering mice as compared to sucrose. Optogenetic activation of these neurons revealed an ethanol-enhanced AMPA/NMDA ratio and significantly reduced paired-pulse ratio, suggesting elevated GluA2 contributions specifically within the BLA[->]AcbC pathway. Conclusions: Ethanol use upregulates GluA2 protein expression in the AcbC and AMPAR synaptic activity in AcbC neurons receiving BLA projections and enhances synaptic plasticity directly within the BLA[->]AcbC circuit. GluA2-containing AMPAR activity in the AcbC regulates the positive reinforcing effects of ethanol through an NSF-dependent mechanism, highlighting a potential therapeutic target in AUD.

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.

Individuals with Fetal Alcohol Spectrum Disorders (FASDs) show reduced subicular volume, and preclinical studies compliment this by demonstrating that third-trimester-equivalent ethanol exposure induced apoptosis in corticolimbic regions, including the subiculum. The subiculum mediates hippocampal-cortical communication critical for long-term memory consolidation. Within the distal dorsal subiculum, a population of bursting neurons uniquely express VGLUT2 and they play a key role in memory processing. We hypothesized that third-trimester-equivalent ethanol exposure would reduce neuronal and VGLUT2+ cell density in the dorsal subiculum and reduce the excitability of bursting neurons, providing a mechanism for long-term memory impairments observed in FASD. To test this, postnatal day (P)7 mice received a subcutaneous injection of ethanol and long-term effects were assessed in adolescence (P35-62). Using transgenic mice with fluorescently labeled VGLUT2+ neurons, and immunohistochemistry we observed a significant reduction in neuronal density in males and an increase in VGLUT2+ cell density in females. Using whole-cell patch clamp electrophysiology, we observed a reduction in action potentials per burst in both sexes. Additionally, females showed reduced overall excitability, and a subset of neurons exhibited a shift to regular spiking. These findings suggest that development ethanol exposure disrupts subicular output by impairing burst firing, potentially weaking hippocampal-cortical communication and contributing to the cognitive deficits associated with FASD. HighlightsO_LIThird-trimester ethanol targets VGLUT2+ neurons in the dorsal subiculum C_LIO_LIEthanol reduced neuronal density in male dorsal subiculum C_LIO_LIEthanol increases VGLUT2+ cell density in females C_LIO_LIEthanol reduces action potential per burst in both sexes C_LIO_LIFemales show reduced excitability and loss of bursting in some cells C_LI

Alcohol induced peripheral neuropathy (AIPN) is a neurodegenerative disease caused by chronic alcohol intake and is associated with peripheral nerve damage and somatosensory symptoms, such as allodynia. Current treatments lack efficacy and do not target underlying pathology emphasizing the need for preclinical models of AIPN to elucidate mechanisms and novel targets. Thus, we performed a detailed characterization of a mouse model of AIPN and candidate mechanistic associations including the role of neuroinflammation and acetaldehyde. Our studies showed chronic alcohol induced mechanical and cold hypersensitivity and deficits in spontaneous behaviors in EtOH concentration-, time- and sex-dependent manners. Female mice drank more alcohol and developed more rapid and severe hypersensitivity but less robust deficits of spontaneous behaviors. The grimace test demonstrated chronic alcohol promoted spontaneous pain independent of sex. Duration of intake impacted alcohol-induced deficits in peripheral nerve electrophysiology amplitude and intra-epidermal nerve fiber density. We characterized an extensive time-course of chronic alcohol-induced neuroinflammation in the DRG and spinal cord and found significant time, sex and tissue effects. Polymorphisms of ALDH2 have been associated with alcohol-induced neuroinflammation, alcohol-related pain, and alcohol-induced peripheral neuropathy. We investigated the role of acetaldehyde, via inhibition of ALDH2, in the development of AIPN and showed that ALDH2 inhibition accelerated and exacerbated development of chronic alcohol-induced hypersensitivity in male and female mice. Overall, our studies in a well-controlled model of AIPN strongly point to neuroinflammation and inflammatory modulators such as acetaldehyde as important mechanistic targets for possible intervention in AIPN.

Alcohol use disorder (AUDs) commonly co-occurs in patients with chronic pain, and a major barrier to achieving abstinence and preventing relapse is the emergence of hyperalgesia during alcohol withdrawal. Elucidating novel therapeutic approaches to target hyperalgesia associated with alcohol withdrawal could have important implications for the treatment of AUD. Here we examined the role of 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid (eCB) signaling in the regulation of hyperalgesia associated with alcohol withdrawal in mice and tested the hypothesis that pharmacological augmentation of 2-AG signaling could reduce hyperalgesia during withdrawal. After 72 hours of withdrawal from a continuous access two-bottle choice drinking paradigm, male and female mice exhibited increased mechanical but not thermal hypersensitivity, which normalized by 7 days. This effect was reversed by pretreatment with the monoacylglycerol lipase (MAGL) inhibitor JZL184, which elevates levels of 2-AG. The effects of JZL184 were prevented by coadministration of either a CB1 or CB2 antagonist. Inhibition of the 2-AG synthetic enzyme diacylglycerol lipase (DAGL) with DO34 exacerbated mechanical hypersensitivity during alcohol withdrawal, causing an earlier onset and persistent hypersensitivity even one week into withdrawal. Our findings demonstrate the critical role of 2-AG signaling in the bidirectional regulation of mechanical sensitivity during alcohol withdrawal, with enhancement of 2-AG levels reducing sensitivity, and inhibition of 2-AG synthesis exacerbating sensitivity. These data suggest 2-AG augmentation could represent a novel approach to the treatment of alcohol withdrawal-associated hyperalgesia and AUD in patients with comorbid pain disorders.

IntroductionThe opioid epidemic has seen an increase in drug use among women of reproductive age. It is well established that Opioid Use Disorder (OUD) can have many negative consequences for the health of mothers and their babies, both during pregnancy and after delivery, but our understanding of the impact of fetal opioid exposure on behavior during adolescence and adulthood is less understood. Preclinical studies have unveiled some of the long-term effects of in utero morphine exposure primarily using injections as the route of drug delivery. Our study utilized a model for oral, voluntary morphine self-administration to investigate neonate, adolescent, and adult offsprings behavioral phenotypes and subsequent ethanol misuse liability. MethodsWe first validated a paradigm for maternal oral intake of morphine, where female mice became morphine dependent pre-pregnancy, and continued to voluntarily consume morphine in the continuous two-bottle choice (C2BC) paradigm during pregnancy and up to offspring postnatal day 7 (PND 7). Offspring were cross-fostered to a drug-naive dam at PND 7, to model first and second trimester in utero exposure in humans and to mimic the stress associated with NOWS. Bodyweight and ultrasonic vocalizations were assessed to determine alterations in the neonates. Offspring from control and morphine-exposed dams were then tested during adolescence and adulthood in a battery of behavioral tests to assess baseline behavioral phenotypes. We also computed a global behavioral score (GBS) to integrate offsprings multiple behavioral outcomes into a composite score that could be used to identify potential vulnerable and resilient populations in offspring exposed prenatally to morphine. Offspring that were tested during adolescence were also evaluated during adulthood in the ethanol intermittent 2BC to assess ethanol misuse risk. ResultsUsing an oral maternal morphine C2BC protocol, we demonstrated that morphine dams display signs of dependence, measured by somatic signs during withdrawal, and voluntarily drink morphine throughout gestation. Neonate cross-fostered offspring display changes in spontaneous activity, body weight, and ultrasonic vocalization parameters. During adolescence, offspring display both increased baseline anxiety-like/compulsive-like behavior, while in adulthood they display increased anxiety-like behavior. No changes were found for baseline physical signs, locomotion, and depressive-like behavior during adolescence or adulthood. In addition, a greater percentage of adult male offspring exposed to maternal morphine fell into moderate and high GBS classifications, signaling a more severe behavioral phenotype, compared to male control offspring. These effects were not observed in adult female offspring exposed to morphine in utero. Additionally, male adult offspring exposed to maternal morphine reduced their 2-hour ethanol intake in the intermittent two-bottle choice (I2BC) paradigm, although no changes in 24-hour ethanol intake and preference were found. No changes were observed in female offspring of morphine-exposed dams. ConclusionOverall, maternal morphine exposure leads to sex-specific changes in neonate, adolescent, and adult behavior, including ethanol intake.

Recent studies have implicated the ethanol metabolite, acetic acid, as neuroactive, perhaps even more so than ethanol itself. In this study, we investigated sex-specific metabolism of ethanol (1, 2, and 4g/kg) to acetic acid in vivo to guide electrophysiology experiments in the accumbens shell (NAcSh), a key node in the mammalian reward circuit. There was a sex-dependent difference in serum acetate production, quantified via ion chromatography only at the lowest dose of ethanol (males>females). Ex vivo electrophysiology recordings of NAcSh neurons in brain slices demonstrated that physiological concentrations of acetic acid (2 mM and 4 mM) increased NAcSh neuronal excitability in both sexes. N-methyl-D-aspartate receptor (NMDAR) antagonists, AP5, and memantine robustly attenuated the acetic acid-induced increase in excitability. Acetic acid-induced NMDAR-dependent inward currents were greater in females compared to males. These findings suggest a novel NMDAR-dependent mechanism by which the ethanol metabolite, acetic acid, may influence neurophysiological effects in a key reward circuit in the brain.

RationaleThe development and progression of alcohol use disorder (AUD) is widely viewed as maladaptive neuroplasticity. The transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory protein {gamma}8 (TARP {gamma}-8) is a molecular mechanism of neuroplasticity that has not been evaluated in AUD or other addictions. ObjectiveTo address this gap in knowledge, we evaluated the mechanistic role of TARP {gamma}-8 bound AMPAR activity in the basolateral amygdala (BLA) and ventral CA3 hippocampus (vHPC) in the positive reinforcing effects of alcohol, which drive repetitive alcohol use throughout the course of AUD, in C57BL/6J mice. These brain regions were selected because they exhibit high levels of TARP {gamma}-8 expression and send glutamate projections to the nucleus accumbens (NAc), which is a key nucleus in the brain reward pathway. Methods and ResultsSite-specific pharmacological inhibition of AMPARs bound to TARP {gamma}-8 in the BLA via bilateral infusion of the selective negative modulator JNJ-55511118 significantly decreased operant alcohol self-administration with no effect on sucrose self-administration in behavior-matched controls. Temporal analysis showed that reduction of alcohol-reinforced responding occurred >25 min after the onset of responding, consistent with a blunting of the positive reinforcing effects of alcohol in the absence of nonspecific behavioral effects. In contrast, inhibition of TARP {gamma}-8 bound AMPARs in the vHPC selectively decreased sucrose self-administration with no effect on alcohol. ConclusionsThis study reveals a novel brain region-specific role of TARP {gamma}-8 bound AMPARs as a molecular mechanism of the positive reinforcing effects of alcohol and non-drug rewards.

IntroductionConsidering sex as a biological variable (SABV) in preclinical research can enhance understanding of the neurobiology of alcohol use disorder (AUD). However, the behavioral and neural mechanisms underlying sex-specific differences remain unclear. This study aims to elucidate SABV in ethanol (EtOH) consumption by evaluating its reinforcing effects and regulation by glutamate AMPA receptor activity in male and female mice. MethodsC57BL/6J mice (male and female) were assessed for EtOH intake under continuous and limited access conditions in the home cage. Acute sensitivity to EtOH sedation and blood clearance were evaluated as potential modifying factors. Motivation to consume EtOH was measured using operant self-administration procedures. Sex-specific differences in neural regulation of EtOH reinforcement were examined by testing the effects of a glutamate AMPA receptor antagonist on operant EtOH self-administration. ResultsFemale C57BL/6J mice exhibited a time-dependent escalation in EtOH intake under both continuous and limited access conditions. They were less sensitive to EtOH sedation and had lower blood levels post-EtOH administration (4 g/kg) despite similar clearance rates. Females also showed increased operant EtOH self-administration and progressive ratio performance over a 30-day baseline period compared to males. The AMPAR antagonist GYKI 52466 (0-10 mg/kg, IP) dose-dependently reduced EtOH-reinforced lever pressing in both sexes, with no differences in potency or efficacy. DiscussionThese findings confirm that female C57BL/6J mice consume more EtOH than males in home-cage conditions and exhibit reduced acute sedation, potentially contributing to higher EtOH intake. Females demonstrated increased operant EtOH self-administration and motivation, indicating higher reinforcing efficacy. The lack of sex differences in the relative effects of GYKI 52466 suggests that AMPAR activity is equally required for EtOH reinforcement in both sexes.

The prefrontal cortex (PFC) develops until early adulthood in rodents and humans, but how synaptic plasticity evolves throughout postnatal development is not known. Here, we used a cross-sectional approach to establish the postnatal maturational trajectories of intrinsic properties and synaptic plasticity in the PFC of rats of both sexes. We found that while layer 5 PFC pyramidal neurons from rats of both sexes displayed similar current-voltage relationships, rheobases and resting potentials across all age groups, excitability was lower in female adults compared to the other developmental stages. NMDAR-dependent long-term potentiation and mGluR2/3-mediated long-term depression (LTD) were equally expressed at the juvenile, pubescent and adult developmental stages in animals of both sexes. However, the developmental course of endocannabinoid (eCB)-mediated LTD was sexually dimorphic. First, eCB-LTD emerged during the juvenile period in females. However, although CB1Rs were functional in both sexes at all developmental stages, eCB-LTDs first emerged during pubescence in male. Second, eCB-LTD engaged distinct receptors in males and females depending on their developmental stages. Female rats employ both CB1R and TRPV1R to produce eCB-LTD at the juvenile stage but solely CB1R at pubescence followed by only TRPV1R at adulthood. In contrast, in pubescent and adult males eCB-LTD always and exclusively depended on CB1R. Pharmacological blockade of 2AGs principal degrading enzyme allowed incompetent male juvenile synapses to express eCB-LTD. The data reveal different maturational trajectories in the PFC of male and female rats and provide new cellular substrates to the sex-specific behavioral and synaptic abnormalities caused by adolescent exposure to cannabinoids.

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.

Although most adults in the United States will drink alcohol in their life, only about 6% will go on to develop an alcohol use disorder (AUD). While a great deal of work has furthered our understanding of the cycle of addiction, it remains unclear why certain people transition to disordered drinking. Altered activity in regions implicated in AUDs, like the basolateral amygdala (BLA), has been suggested to play a role in the pathophysiology of AUDs, but how these networks contribute to alcohol misuse remains unclear. Our recent work demonstrated that alcohol can modulate BLA network states and that GABAergic parvalbumin (PV) interneurons are crucial modulators of network activity in the BLA. Further, our lab has demonstrated that {delta} subunit-containing GABAA receptors, which are modulated by alcohol, are highly expressed on PV interneurons in the BLA. These receptors on PV interneurons have also been shown to influence alcohol intake in a voluntary binge drinking paradigm and anxiety-like behavior in withdrawal. Therefore, we hypothesized that alcohol may impact BLA network states via {delta} subunit-containing GABAA receptors on PV interneurons to impact the extent of alcohol use. To test this hypothesis, we measured the impact of acute alcohol exposure on oscillatory states in the basolateral amygdala and then assessed the relationship to the extent of voluntary ethanol consumption in the Intermittent Access, Drinking-in-the-Dark-Multiple Scheduled Access, and Chronic Intermittent Ethanol exposure paradigms. Remarkably, we demonstrate that the average alcohol intake negatively correlates with {delta} subunit-containing GABAA receptor expression on PV interneurons and gamma power in the BLA after the first exposure to alcohol. These data implicate {delta} subunit-containing GABAA receptor expression on PV interneurons in the BLA in voluntary alcohol intake and suggest that BLA network states may serve as a useful biomarker for those at risk for alcohol misuse. Significance StatementOscillatory states in the BLA have been demonstrated to drive behavioral states involved in emotional processing, including negative valence processing. Given that negative emotional states/hyperkatifeia contribute to the cycle of AUDs, our previous work demonstrating the ability of alcohol to modulate BLA network states and thereby behavioral states suggests that this mechanism may influence alcohol intake. Here we demonstrate a relationship between the ability of alcohol to modulate oscillations in the BLA and future alcohol intake such that the extent to which alcohol influences BLA network states predict the extent of future voluntary alcohol intake. These findings suggest that individual variability in the sensitivity of the BLA network to alcohol influences voluntary alcohol consumption.

Compulsions, defined by debilitating repetitive actions, permeate many mental illnesses and are challenging to treat partly because of a limited understanding of their neurobiological underpinnings. Accumulating evidence suggests the rodent model of Schedule-Induced Polydipsia (SIP) as a promising pre-clinical assay to elucidate the neurobiological and behavioural manifestations of compulsivity. In the rodent SIP paradigm, susceptible rats develop adjunctive excessive drinking when they are chronically food restricted and presented with food pellets according to a fixed-time schedule. We found that normally, bi-directional plasticity of GABA synapses in the oval bed nucleus of the stria terminalis (ovBNST) tightly followed the rats satiety state where low-frequency stimulation-induced potentiation (LTPO_SCPCAPGABAC_SCPCAP) prevailed in sated rats whilst food restriction uncovered long-term depression (LTDO_SCPCAPGABAC_SCPCAP). In rats that developed excessive drinking during SIP, removing the caloric restriction failed at reverting LTDO_SCPCAPGABAC_SCPCAP to LTPO_SCPCAPGABAC_SCPCAP whereas bi-directional plasticity at ovBNST GABA synapses was unaltered in low-drinking SIP-trained rats. Excessive drinking ceased in polydipsic rats removed from their caloric restriction; however, these rats retained a form of compulsive schedule-induced checking (SIC) and impaired plasticity at ovBNST GABA synapses for several days following termination of the caloric restriction. We conclude that altered bi-directional plasticity at ovBNST GABA synapses is a neurophysiological trace of compulsivity in susceptible rats in the SIP model.

Male rats escalate intravenous self-administration of entactogen psychostimulants, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxymethamphetamine (MDMA) under extended access conditions, as with typical psychostimulants. Here, we investigated whether female rats escalate self-administration of methylone, 3,4-methylenedioxypentedrone (pentylone), and MDMA and then studied consequences of MDMA and pentylone self-administration on GABAA receptor and kappa opioid receptor (KOR) signaling in the central nucleus of the amygdala (CeA), a brain area critically dysregulated by extended access self-administration of alcohol or cocaine. Adult female Wistar rats were trained to self-administer methylone, pentylone, MDMA (0.5 mg/kg/infusion), or saline-vehicle using a fixed-ratio 1 response contingency in 6-hour sessions (long-access: LgA) followed by progressive ratio (PR) dose-response testing. The effects of pentylone-LgA, MDMA-LgA and saline on basal GABAergic transmission (miniature postsynaptic inhibitory currents, mIPSCs) and the modulatory role of KOR at CeA GABAergic synapses were determined in acute brain slices using whole-cell patch-clamp. Methylone-LgA and pentylone-LgA rats similarly escalated their drug intake (both obtained more infusions compared to MDMA-LgA rats) however, pentylone-LgA rats reached higher breakpoints in PR tests. At the cellular level, baseline CeA GABA transmission was markedly elevated in pentylone-LgA and MDMA-LgA rats compared to saline-vehicle. Specifically, pentylone-LgA was associated with increased CeA mIPSC frequency (GABA release) and amplitude (postsynaptic GABAA receptor function), while mIPSC amplitudes (but not frequency) was larger in MDMA-LgA rats compared to saline rats. In addition, pentylone-LgA and MDMA-LgA profoundly disrupted CeA KOR signaling such as both KOR agonism (1mM U50488) and KOR antagonism (200nM nor-binaltorphimine) decreased mIPSC frequency suggesting recruitment of non-canonical KOR signaling pathways. This study confirms escalated self-administration of entactogen psychostimulants under LgA conditions in female rats which is accompanied by increased CeA GABAergic inhibition and altered KOR signaling. Collectively, our study suggests that CeA GABA and KOR mechanisms play a critical role in entactogen self-administration like those observed with escalation of alcohol or cocaine self-administration.