Neuropharmacology

Circadian clock genes in the striatum modulate alcohol drinking behavior in female mice, but the underlying synaptic mechanisms remain unknown. Medium spiny neurons (MSNs) of the striatum express D1- or D2-type dopamine receptors, which are differentially modulated by alcohol. Here we used whole-cell patch-clamp recordings to examine how deletion of the clock gene Bmal1 affects spontaneous excitatory postsynaptic currents (sEPSCs) in electrophysiologically identified D1 and D2 neurons of the female mouse dorsomedial striatum (DMS). Prior intermittent access to alcohol increased sEPSC amplitude in putative D1 neurons of wildtype mice without affecting sEPSC frequency, and this increase was accompanied by greater dendritic branching and complexity in biocytin-filled D1 MSNs. Bmal1 knockout had the opposite effect in D1 neurons, reducing sEPSC amplitude in both alcohol-exposed and alcohol-naive animals. In D2 neurons, alcohol produced only a non-significant trend toward increased sEPSC amplitude in wildtype mice, but a significant increase in Bmal1 knockout mice. Thus, local disruption of Bmal1 appears to block the alcohol-induced enhancement of sEPSCs in D1 neurons while unmasking a similar enhancement in D2 neurons. These findings may help explain how Bmal1 deletion in the DMS reduces alcohol consumption in female mice.

N-methyl-D-aspartate receptor (NMDAR) neurotransmission plays a central role in the neurophysiological and behavioral adaptations produced by chronic alcohol (EtOH) exposure and withdrawal. Although alcohol-induced changes in canonical NMDAR subtypes have been extensively studied, the mechanisms by which prolonged alcohol exposure remodels NMDAR function remain incompletely understood. To investigate the impact of chronic voluntary alcohol consumption on glutamatergic signaling, adult C57BL/6J mice were exposed to 20% EtOH using the intermittent two-bottle choice (I2BC) approach for 8 weeks and NMDAR function was examined in ventral tegmental area (VTA) dopamine (DA) neurons using whole-cell electrophysiology. Chronic EtOH exposure reduced NMDA-evoked currents, an effect that became more pronounced during withdrawal. Withdrawal also reduced NMDAR outward current rectification, indicating that chronic alcohol exposure altered the biophysical properties of NMDAR-mediated signaling. These unexpected observations led us to investigate the contribution of GluN3A-containing NMDARs. Pharmacological analyses revealed a significant increase in the proportion of VTA DA neurons expressing functional GluN3A-containing receptors following chronic EtOH exposure, with all tested neurons expressing these receptors during withdrawal. Glycine activation directly depolarized VTA DA neurons and increased action potential firing, demonstrating that the recruited receptors function as excitatory NMDARs. Moreover, the alcohol-induced changes in NMDAR currents, receptor pharmacology, and glycine responsiveness were absent in GluN3A null mice. Together, these findings identify functional recruitment of GluN3A-containing NMDARs as a previously unrecognized neuroadaptation through which chronic alcohol remodels glutamatergic signaling in the adult VTA.

The lateral habenula (LHb) encodes aversive states and negative affect, positioning it as a candidate region for the negative reinforcement that drives alcohol withdrawal. However, little is known about how chronic ethanol exposure affects LHb neuronal function and glial biology during withdrawal. Here, we used chronic intermittent ethanol (CIE) vapor exposure, a well-established model of alcohol dependence that reliably produces somatic and affective signs of withdrawal, to examine LHb physiology and astrocytic markers during acute withdrawal in male and female rats. Whole-cell and cell-attached recordings revealed that withdrawal reduced evoked and spontaneous firing in LHb neurons, with rebound firing following a crossover pattern between males and females. Despite these excitability changes, the overall distribution of firing phenotypes was unchanged, suggesting a shift in gain rather than a reorganization of cell types. Immunofluorescence revealed increased Sox9+ and GFAP labeling in the LHb during withdrawal at the same time point when electrophysiology experiments uncovered impaired astrocytic regulation of glutamate clearance. Together, these findings reveal that withdrawal from chronic ethanol exposure produces neuronal and glial adaptations in the LHb, pointing to impaired glutamate regulation as a candidate mechanism relevant to the negative affective state of alcohol withdrawal. These findings position the LHb as a potential node linking astrocyte-neuron dynamics to withdrawal symptoms and relapse vulnerability in alcohol use disorder.

Alcohol use disorder is associated with persistent neuroadaptations and cognitive impairments, particularly during withdrawal, yet the temporal dynamics and interactions between these processes remain poorly understood. In the present study, we investigated time-dependent alterations in the oxytocin receptor (OTR), -opioid receptor (MOPr), metabotropic glutamate receptor 5 (mGlu5R), and translocator protein (TSPO) following chronic ethanol exposure and withdrawal, and their relationship to behavioral impairments. Male C57BL/6J mice were exposed to a 10-day escalating-dose ethanol liquid diet paradigm followed by 1, 4, or 7 days of withdrawal. Motor coordination and recognition memory were assessed using rotarod and novel object recognition tests, and receptor binding was quantified using autoradiography across multiple brain regions. Chronic ethanol exposure induced motor deficits that persisted during early withdrawal, whereas recognition memory impairments emerged after 7 days of withdrawal. Ethanol exposure and withdrawal produced region- and time-dependent alterations across all systems examined, with increased OTR binding in septal regions and transient reductions in basomedial amygdala OTR binding during intermediate withdrawal that normalized following prolonged withdrawal, transient increases in MOPr binding in striatal regions at 4 days of withdrawal, increased mGlu5R binding in the nucleus accumbens core, basolateral amygdala, and hippocampus, and early transient increases in TSPO binding indicative of neuroimmune activation. Notably, mGlu5R binding in the caudate-putamen and amygdala was associated with recognition memory deficits in a withdrawal stage-dependent manner. Bioinformatic analysis of human postmortem hippocampal transcriptomic datasets from individuals with alcohol use disorder revealed no consistent changes in GRM5 expression, suggesting that mGlu5R dysregulation may be driven by post-transcriptional mechanisms. These findings demonstrate that ethanol withdrawal is characterized by temporally distinct neuroadaptations across multiple systems and identify mGlu5R as a key correlate of withdrawal-associated memory impairment, highlighting its potential as a translational therapeutic target.

A growing body of evidence demonstrates that signaling pathways of endogenous lipids (endolipids) modulate the reinforcing properties of opioids including reward and withdrawal. Many of these studies are focused on the endocannabinoid (eCB) system, and its primary eCB ligands, Anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The central hypothesis is that modulation of the eCB system through eCB receptors and enzymes may improve therapeutic outcomes for opioid use disorder (OUD). However, outcomes in preclinical and clinical studies using the low efficacy, CB1/CB2 orthosteric agonist, THC, found limited to no effectiveness, suggesting that targeting this aspect of CB1/CB2 is not a useful therapeutic tool for OUD. Previous studies finding that genetic deletion or pharmacological inhibition of endolipid-regulating enzymes (including eCBs) can alter behavioral sensitivity to opioids provide insight into an alternative approach. If opioid use dysregulates a wide range of endolipid biosynthesis and metabolism, then a clearer understanding of these changes, especially in signaling ligands, will provide a novel avenue to both understand the underlying physiological changes with opioid use as well as providing novel targets for therapeutic interventions. In this study, we tested the hypothesis that if morphine dysregulates multiple endolipid signaling systems in the brain and body, and this dysregulation evolves over time, then these differential effects will be measurable by changes in endolipid levels. Using an Acute (30 minutes post injection 20mg/kg) and a Chronic paradigm (5 days, twice daily, 20mg to 100mg/kg escalating dose) we measured 100 targeted endolipids in 8 brain regions, plasma, liver, and feces in male mice. In the Acute condition, we found that the most screened endolipids were changed in the striatum (42%), while the fewest were changed in the thalamus (19%) and 38% in the plasma. In the Chronic condition, 79% of plasma endolipids were changed. The highest level of change in the CNS was in the cortex (39%). Levels of AEA and 2-AG were largely unchanged; however, levels of the N-acyl GABAs, N-acyl valines, N-acyl taurines, and specific bile acids (e.g. DCA, TCA) showed the most dynamic changes by treatment group. These results provide information on novel endolipid signaling pathways that may contribute to the unwanted side effects of opioids, such as dependence and withdrawal, and provide novel avenues for the development of therapeutic strategies.

Chronic ethanol consumption produces long-lasting effects on the brain that contribute to persistent behavioral changes. This has been modeled in animals using the chronic intermittent ethanol (CIE) paradigm, which induces escalation of consumption and alterations in affective and cognitive behavior. While this model has been well characterized in male subjects, the effects of CIE in females and long-term effects that persist beyond the acute withdrawal remain relatively underexplored. Here, female C57BL/6J mice were exposed to four weeks of chronic intermittent ethanol (CIE) vapor to induce dependence. Following CIE, mice were evaluated for physiological tolerance to ethanol and alterations in anxiety-like behaviors and memory processing. Using the ethanol responsivity battery (ERB) assay to assess physiological tolerance, we found blunted responsiveness to the intoxicating effects of ethanol in the CIE-exposed females, indicating heightened ethanol tolerance that persists beyond the acute withdrawal phase. A cohort underwent behavioral testing following CIE to characterize changes in anxiety-like behavior, exploration, and memory across abstinence. In early abstinence (<5 days post-CIE), CIE-exposed females showed increased exploratory behavior compared with Air controls. After one week of abstinence, CIE-exposed females displayed impaired object recognition memory. CIE-exposed females also showed increased anxiety-like behavior at protracted abstinence time points, exhibiting hyponeophagia in the novelty-suppressed feeding test at 2 weeks post-CIE. In addition, following cued fear conditioning, we found reduced extinction recall at 4 weeks post-CIE. Together, this work indicates that chronic alcohol exposure leads to increased alcohol tolerance and distinct, persistent alterations in memory and anxiety-like behavior in female mice.

Recent shifts in the illicit fentanyl supply have led to widespread exposure to xylazine, yet the biological consequences of chronic fentanyl-xylazine co-exposure remain poorly understood. Here, we compared the behavioral and molecular effects of chronic exposures to fentanyl and xylazine both alone and in combination in male and female mice. Combined fentanyl-xylazine produced distinct, sex-dependent alterations in sleep architecture, including changes in NREMS recovery, REMS dynamics, and reduced sleep bout duration compared with fentanyl alone. The 2-adrenergic receptor antagonist yohimbine partially reversed these sleep alterations in a sex-dependent manner. Fentanyl-xylazine also exacerbated respiratory depression relative to fentanyl alone, particularly in males. In contrast, despite its prevalence in humans no skin lesions were observed. Molecular analyses revealed that fentanyl-xylazine co-administration leads to increased peripheral Fibroblast growth factor 21, a hepatokine linked to reduced opioid preference and alcohol consumption. Together, these findings demonstrate that xylazine substantially alters the physiological and neurobiological consequences of chronic fentanyl exposure in a sex-dependent manner, providing a preclinical framework for understanding the growing public health impact of fentanyl-xylazine co-use.

RationaleThe opioid epidemic continues to be driven by synthetic opioids, particularly fentanyl, yet the long-term behavioral manifestations of withdrawal remain poorly characterized. Women exhibit unique vulnerabilities to opioid use disorder, including greater susceptibility to withdrawal-related symptoms and relapse, suggesting that defining the behavioral adaptations that emerge during prolonged fentanyl abstinence may identify mechanisms underlying female-specific relapse vulnerability. Thus, we used an oral fentanyl drinking model to longitudinally examine nociceptive, affective, and exploratory behaviors across chronic exposure and abstinence in female mice. MethodsAdult female C57BL/6J mice underwent a modified 5-week Drinking-in-the-Dark paradigm with 4-hour daily access to fentanyl (30 {micro}g/mL) or water. Somatic withdrawal was assessed following naloxone-precipitated withdrawal. Thermal and mechanical nociception, sucrose preference, exploratory behavior, acoustic startle, nest building, and locomotor activity were evaluated during fentanyl exposure and throughout one month of abstinence. ResultsFemale mice exhibited robust naloxone-precipitated somatic withdrawal, confirming physiological effects of opioid drinking. Chronic fentanyl exposure produced transient thermal hyperalgesia during weeks 2-4 of drinking that resolved by the final week, but re-emerged after 30 days of abstinence. After 30 days of abstinence, we also found increased mechanical sensitivity. During early abstinence, fentanyl-exposed mice exhibited increased sucrose consumption and greater exploration of anxiogenic environments without alterations in general locomotion. Increased exploratory behavior persisted into prolonged abstinence and was partially rescued in mice who previously received naloxone. Additionally, fDID mice exhibited impaired nesting behavior, while prior naloxone-precipitated withdrawal improved nest-building performance. ConclusionsWe demonstrate that chronic opioid exposure, precipitated withdrawal trials, and duration of abstinence interact to shape protracted withdrawal phenotypes in female mice that extend well beyond drug exposure. These results provide insight into persistent withdrawal symptoms that may shape relapse vulnerability using a novel translationally relevant framework.

Alcohol misuse is a leading cause of preventable death worldwide. Chronic alcohol is associated with disrupted circadian rhythms, yet molecular mechanisms linking circadian rhythm dysregulation and alcohol consumption are poorly understood. Current FDA-approved treatments for alcohol use disorder (AUD) do not target molecular rhythms or sleep-wake cycles. Mammalian circadian rhythms are regulated by transcription-translation feedback loops that regulate expression of 'clock genes' (e.g., Arntl encoding for BMAL1). Both human and rodent studies demonstrate associations between clock gene variants and changes in reward-seeking behavior. Evidence suggests astrocytes, non-neuronal brain cells with cell-autonomous rhythms, may regulate both circadian rhythms and reward. In the nucleus accumbens (NAc), a region responsible for modulating alcohol- and reward-related behavior, over 43% of the astrocyte transcriptome is expressed rhythmically. However, no studies to date have investigated roles of NAc astrocyte rhythmicity in regulating alcohol drinking. We used AAV8-Gfap-Cre to functionally ablate molecular rhythms in NAc astrocytes of BMAL1 floxed mice. Two-bottle choice (2BC), drinking-in-the-light (DIL), and drinking-in-the-dark (DID) assessed alcohol drinking. Behavioral assays included locomotor response to novelty, sucrose preference, and social interaction. Disrupting molecular clock function in NAc astrocytes increased binge-like drinking during both DIL and DID paradigms (d = 1.44), but not with any other drinking paradigm or behavior. This study carves out a unique role for astrocytes in controlling the temporal organization of reward circuitry and susceptibility to binge-drinking. Future studies will investigate clock-controlled astrocyte mechanisms, such as glutamate uptake and ATP release, that may underlie binge-like drinking behavior.

BackgroundCircadian disruption is strongly associated with alcohol use disorder (AUD), but insight into the underlying brain-region and sex-specific mechanisms is limited. The function of the circadian clock gene Bmal1 within the striatum has been linked to alcohol drinking, yet its role within functionally distinct striatal subregions has not been systematically examined. MethodsWe deleted Bmal1 in medium spiny neurons of the dorsomedial striatum (DMS) or dorsolateral striatum (DLS). Male and female mice were tested for anxiety-like behavior, depressive-like behavior, and motor coordination. Voluntary alcohol intake was measured with an intermittent two-bottle choice paradigm, followed by sucrose preference and quinine-adulterated alcohol tests. To assess hormonal contributions, a subset of female mice underwent ovariectomy before behavioral testing. ResultsDeletion of Bmal1 in the DLS did not alter alcohol intake, alcohol preference, or quinine-adulterated alcohol intake in either sex. In contrast, DMS Bmal1 deletion significantly reduced alcohol consumption and alcohol preference in female mice, with no effect in males. These effects were not accompanied by changes in depressive-like behavior or motor coordination and were not explained by generalized reward changes, as sucrose preference was unaffected. Ovariectomy eliminated the effect of DMS Bmal1 deletion on alcohol intake, indicating dependence on ovarian hormones. ConclusionsThe DMS is a critical site at which Bmal1 regulates alcohol consumption in a sex-specific manner. These findings support an interaction between local circadian mechanisms and ovarian hormones in controlling alcohol drinking and highlight a potential target for sex-specific therapeutics in AUD.

Binge alcohol drinking is a public health concern that can dramatically increase the risk for development of alcohol use disorder (AUD). Continued alcohol drinking in the face of negative consequences is another key feature of AUD. A better understanding of the neural circuitry that regulates these behaviors could provide insight as to novel treatments for AUD. Serotonin is a neurotransmitter that has been implicated in alcohol consumption in both human studies and animal models. The orbitofrontal cortex (OFC) is a brain region that both receives serotonergic input from the dorsal raphe and has been implicated in AUD. However, how volitional alcohol consumption impacts serotonin signaling within the OFC and how this contributes to alcohol related behaviors is unknown. Here, we show that a history of alcohol consumption alters the ability of 5-HT to hyperpolarize OFC pyramidal neurons in mice and monkeys. Consistent with this, a history of binge alcohol consumption decreases the expression of the 5-HT1A but not 5-HT2A receptor in the OFC from mice. Next, we show that deletion of the 5-HT1A receptor from the OFC increased alcohol intake and preference in male, but not female mice. Finally, we found that 5-HT1A receptor deletion led to increased quinine-adulterated alcohol intake, a measure of aversion-resistant drinking, in both male and female mice. Altogether, we identified serotonin signaling in the OFC as key target for modulation of binge and compulsive alcohol consumption.

Alcohol use disorders are heritable, and large-scale genome-wide association studies have recently identified novel genetic loci that are correlated with alcohol consumption and problematic alcohol use. One such gene is Slc39a8, which encodes the ion transporter ZIP8, responsible for cellular import of zinc and other metals. Slc39a8 is highly pleiotropic, and a common missense variant has been associated with decreased problematic alcohol use. Here, we used Slc39a8 heterozygous (HET) knockout mice and their wild type (WT) littermates to characterize the effects of reduced gene function on two models of voluntary alcohol consumption (drinking in the dark and intermittent access two-bottle choice), and anxiety- and depression-like behavior. We found that HET mice consumed less alcohol than WT in the intermittent access two-bottle choice procedure. No evidence was found for genotypic differences in baseline locomotor activity, anxiety-like, or depression-like behavior in the open field and forced swim tests. Slc39a8 mRNA expression and alcohol metabolism experiments confirmed decreased brain expression of Slc39a8 in HET mice and unchanged alcohol metabolism. Basal and post-alcohol measurement of zinc levels showed that HET mice after ethanol exposure had lower zinc concentrations in the liver and higher concentrations in blood than WT animals. Analysis of the spatial distribution of multiple trace metals in the brains of naive Slc39a8 HET and WT mice using Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) showed region-specific genotypic differences in levels of zinc and iron. These results provide evidence for a direct role of Slc39a8 in ethanol consummatory behavior that does not appear to be due to metabolism, affective state, or locomotor activity. Moreover, we confirm that this animal model shows tissue- and brain region-specific alterations in zinc levels, highlighting potential mechanisms for future study.

Caffeine is the most widely consumed psychoactive substance worldwide, yet the long-term consequences of exposure during critical periods of brain development remain incompletely understood. Synaptogenesis represents a vulnerable window during which environmental factors can shape the maturation of neuronal circuits and influence lifelong brain function. Here, we investigated the impact of caffeine exposure during hippocampal synaptogenesis on synaptic development, neuronal function, behavior, and seizure susceptibility, with a particular focus on sex-dependent effects. Developmental caffeine exposure induced distinct, sex-specific trajectories of hippocampal synaptic remodeling. In the CA1 region, caffeine produced opposite patterns of glutamatergic synapse regulation, characterized by a delayed reduction in excitatory synapse density in males and an increase in females. In contrast, inhibitory synapse organization was selectively altered in males, with a transient increase in CA3 inhibitory synaptic density during development associated with enhanced inhibitory transmission, whereas females exhibited no significant changes. These findings reveal sex-specific and temporally divergent effects of developmental caffeine exposure on hippocampal synaptic maturation and function. At the behavioral level, developmental caffeine exposure produced distinct sex-dependent phenotypes : males exhibited increased anxiety-like behavior, whereas females developed a delayed impairment in recognition memory that became apparent only in adulthood. Furthermore, caffeine exposure selectively increased PTZ-induced seizure susceptibility in juvenile females, an effect that was no longer detected in adulthood. Together, these findings demonstrate that caffeine exposure during hippocampal synaptogenesis induces sex-specific and temporally dynamic alterations in circuit maturation, resulting in distinct behavioral and neuronal excitability outcomes. These results highlight the importance of considering both sex and developmental timing when assessing the neurodevelopmental consequences of caffeine exposure.

Despite growing evidence that neuroimmune dysfunction contributes to Alcohol Use Disorder (AUD) pathology, the underlying neuroinflammatory mechanisms that may promote alcohol consumption are not as clear. We recently report that specific knockdown of interferon regulatory factor 7 (IRF7) in the anterior insula (aIC) mitigates escalation in ethanol self-administration in rats. In addition, we find pro-inflammatory activation of microglia contributes to other AUD-related behavioral impairments. Here, we sought to determine if pro-inflammatory activation of microglia from ethanol contributes to elevations in IRF7 and ethanol self-administration in rats. Male Wistar rats were trained under our ethanol self-administration paradigm (15% v/v; FR2 vs inactive lever) followed by 1-4 cycles of chronic intermittent ethanol exposure (CIE). To inhibit microglia, rats were treated with minocycline (30mg/kg, i.p.) before and after each ethanol vapor session. Escalation in self-administration and biochemical markers were assessed 72 hours into abstinence. We found CIE increased ethanol self-administration, which was positively correlated with aIC IRF7 levels. Minocycline treatment blunted IRF7 expression and alleviated ethanol self-administration following CIE. These data suggest a role for microglia in driving both IRF7 levels and escalation in ethanol self-administration in early abstinence.

Understanding the neurobiological systems that regulate alcohol cue-induced craving is of utmost importance for the development of novel intervention strategies for alcohol use disorders (AUDs). However, although a human experimenter is required to conduct alcohol self-administration studies in the lab, the cues associated with the experimenter are seldom if ever factored into the experimental design. Thus, although we have learned much to date about alcohol cue-induced behavior and neurobiology, and in particular about discrete cues presented many times throughout a single daily alcohol self-administration session, we know relatively little about how responses to alcohol availability cues might predict subsequent alcohol consumption. For the current experiment, mice were exposed daily to auditory cues that preceded 2 hours of alcohol or water access using drinking-in-the-dark (DID) methods. An additional control group experienced cues but were not otherwise manipulated. Importantly, cues were initiated remotely from outside the animal facility, avoiding the experimenter being the first cue predicting ethanol availability. Head direction, location in the home cage, and movement velocity were the primary variables on interest. Surprisingly, during the cue period, there were no significant differences between groups in any of these measures, despite meaningful alterations over days. However, we observed many significant correlations between behaviors and drinking variables. First, we observed significant positive associations between ambulatory velocity during cues and subsequent total alcohol (R2=0.14; p<0.0001) and total water (R2=0.12; p=0.0002) consumption, but only in females. We also observed a significant positive relationship (R2=0.25; p<0.0001) between the amount of time oriented toward the sipper port during the auditory cues and the average rate of subsequent alcohol consumption (i.e. front-loading), but only in females. In males, head direction was found to be positively associated with subsequent total water consumption (R2=-0.21; p<0.0001), but not alcohol (R2=-0.01; p=0.2267). We also observed a significant negative relationship (R2=-0.15; p<0.0001) between proximity to the sipper during the cue period and subsequent total 2-hour alcohol intake in males. Although these associations were modest in strength, they suggest potential sex-specific behavioral predictors of alcohol consumption that are regulated by different neural dynamics.

Alcohol-induced toxicity is driven largely by the accumulation of acetaldehyde and disruption of hepatic redox homeostasis during ethanol metabolism. Oxidation of ethanol by alcohol dehydrogenase (ADH) consumes nicotinamide adenine dinucleotide (NAD) while generating NADH, shifting the intracellular redox state toward a highly reduced environment that impairs mitochondrial function, limits endogenous aldehyde dehydrogenase (ALDH)-mediated acetaldehyde clearance, and promotes oxidative stress and tissue injury. We investigated whether UT-018, a novel metabolic intervention, could support endogenous metabolic resilience during acute ethanol exposure using complementary in vitro and in vivo models. Mechanistic in vitro studies evaluated ADH-dependent NADH generation and NAD add-back experiments, while in vivo investigations assessed serum ALDH-associated activity, circulating acetaldehyde concentrations, and gross gastrointestinal and hepatic morphology following acute ethanol challenge. UT-018 reduced ethanol-associated NADH accumulation in a concentration-dependent manner without evidence of irreversible ADH inhibition. Restoration of NADH generation following supplementation with exogenous NAD demonstrated reversible modulation of ethanol-associated redox biology rather than direct enzymatic inhibition. In vivo, UT-018 enhanced serum ALDH-associated activity, reduced circulating acetaldehyde concentrations by approximately 27 to 33% compared with ethanol-treated controls. Metabolic biomarkers were accompanied by preservation of gross colon and liver morphology following acute ethanol exposure. Collectively, these findings support coordinated biological activity across multiple interconnected stages of alcohol metabolism and support a systems-level mechanism in which restoration of redox homeostasis enhances endogenous aldehyde detoxification, reduces acetaldehyde burden, and preserves tissue integrity. These results identify alcohol metabolism restoration as a promising strategy for enhancing physiological resilience to acute alcohol exposure and provide a rationale for further preclinical and clinical evaluation of UT-018. HighlightsO_LIUT-018 restored ethanol-associated redox homeostasis by reducing excessive NADH accumulation without irreversible inhibition of alcohol dehydrogenase in vitro. C_LIO_LIRestoration of redox balance was associated with enhanced endogenous aldehyde dehydrogenase (ALDH)-associated activity following acute ethanol exposure in vivo. C_LIO_LIUT-018 reduced circulating acetaldehyde concentrations by approximately 30%. C_LIO_LIThe metabolic homeostasis was accompanied by preservation of gross gastrointestinal and hepatic morphology in an acute ethanol challenge model. C_LIO_LIThe collective findings support a systems-level mechanism in which modulation of endogenous alcohol related metabolic pathways enhances physiological resilience to acute alcohol exposure. C_LI

GLP-1 agonist drug repurposing efforts may establish new clinical niches in managing psychiatric and neurological disorders. However, a comprehensive understanding of GLP-1 neurobiology and an appreciation of specific neural mechanisms by which GLP-1 agonists might provide therapeutic effects is limited and stands as a barrier to these efforts. When considering current preclinical research evaluating GLP-1 agonist central mechanisms, a considerable knowledge gap remains regarding which specific cellular populations and systems define potential therapeutic effects in the brain. In this research, we used cFos immunohistochemistry to identify specific neuronal populations that exhibited altered cellular activity following acute administration of semaglutide to rats. We found that the orexin/hypocretin and basal forebrain cholinergic systems are activated following acute semaglutide administration in male and female young adult rats (3-5 months). Informed by the results of our histological analysis, we next employed in vivo microdialysis to test our hypothesis that semaglutide would acutely increase acetylcholine release in the rodent hippocampus. Here, we report that semaglutide acutely increases acetylcholine efflux in the ventral hippocampus of conscious and freely moving rats regardless of biological sex in both young adult and aged rats (23-26 months). Given the relevance of hippocampal cholinergic neurotransmission in learning and memory, our research mechanistically connects GLP-1 agonists with established targets in cognitive decline and dementia.

Opioid use disorder (OUD) remains a major public health concern, with potent synthetic opioids driving overdose deaths. Understanding how synthetic opioids like fentanyl alter brain circuits to promote persistent use is critical for advancing OUD treatment. The ventral tegmental area (VTA), which projects to several other brain regions including prefrontal cortex (PFC), nucleus accumbens (NAc) and amygdala (Amy), contributes to both the euphoric and dysphoric phases of OUD. Projection target influences the baseline properties and opioid-induced neuroadaptations of VTA neurons, yet less is known about the molecular adaptations that occur across distinct VTA projections during spontaneous opioid withdrawal. Here, we investigated transcriptional responses to acute spontaneous fentanyl withdrawal in projection-defined VTA neurons. Following chronic fentanyl exposure and 24h of spontaneous withdrawal in male and female mice, we sequenced ribosome-associated RNA from VTA neurons projecting to PFC, NAc, or Amy. We found that fentanyl withdrawal produced divergent transcriptional adaptations in VTA neurons projecting to PFC compared with VTA-NAc and VTA-Amy neurons, whereas adaptations in VTA-NAc and VTA-Amy neurons were more concordant. Weighted gene co-expression network analysis (WGCNA) identified fentanyl-responsive modules across VTA projections, with gene ontology analysis linking these modules to distinct biological processes. We further used regulatory enrichment analysis to identify candidate transcriptional regulators associated with fentanyl-responsive co-expression modules. Together, our data demonstrate that VTA neurons undergo distinct molecular adaptations during fentanyl withdrawal based on their projection target, providing new insight into molecular mechanisms relevant to OUD.

Fentanyl-related overdose deaths now commonly involve non-injection routes, yet preclinical opioid self-administration is modeled predominantly intravenously. Here we establish an oral fentanyl self-administration procedure in male and female inbred C57BL/6 and outbred CD1 mice that measures volitional intake, cue-driven seeking, extinction, and relapse. Mice self-administered oral fentanyl (70 {micro}g/mL) on a fixed-ratio 1 schedule across fifteen 3-hour sessions, followed by ten extinction sessions and a cued reinstatement test. A separate cohort underwent between-session dose thresholding across a quarter-log series from 222 to 22 {micro}g/mL. Seventy-five percent of mice acquired self-administration, with similar rates across genetic background and sex. Responding increased as fentanyl concentration fell, indicating dose-sensitivity toward a preferred drug level. C57BL/6 mice escalated intake and lever pressing across sessions, responded persistently early in extinction before declining, and reinstated pressing to a conditioned cue. CD1 mice consumed high levels from the outset with limited escalation and showed neither extinction nor cued reinstatement of pressing, but shortened their reward-port approach latency when cues returned. This shows that lever presses alone would have misclassified them as weakly conditioned. A composite severity score summing seven components of fentanyl-use risk varied continuously rather than splitting into high and low groups, even among inbred mice. Sex differences were largely confined to C57BL/6 mice, in which females showed stronger cue association and higher severity scores than males. These results reveal separable escalation-prone and relapse-prone phenotypes that track genetic background. Protocols, hardware specifications, and analysis code are openly available, lowering the barrier to adopting oral fentanyl self-administration.

Opioid Use Disorder (OUD) continues to be a pressing public health crisis, marked by high relapse rates and limited treatment options. Ketamine, a non-competitive N-Methyl-D-Aspartate receptor (NMDAR) antagonist, has emerged as a promising therapeutic for numerous psychiatric illnesses due to its robust antidepressant properties and glutamate-mediated synaptic plasticity. However, ketamine's therapeutic mechanisms remain elusive, with emerging evidence implicating the endogenous opioid system. Furthermore, few studies have investigated ketamine's potential as an intervention for OUD. Accordingly, this study assessed ketamine's ability to rewire endogenous opioid and glutamatergic circuitry and reduce heroin-seeking activity in male Long-Evans rats. Using a multi-cohort design, we investigated ketamine's effects on opioid-related transcriptional, protein, and signaling properties in key brain regions associated with reward and addiction, and subsequently on heroin-seeking using a self-administration paradigm. To investigate mechanisms of plasticity, we also quantified transcriptional changes to NMDAR subunits. Molecular analyses demonstrated increased expression of endogenous opioid system-related genes and endogenous opioid peptides at the protein level, elevated mu-opioid receptor protein levels, and enhanced G protein activity, suggesting increased receptor function. Notably, transcriptional changes were particularly prominent in the shell subregion of the nucleus accumbens (NAc). We also revealed that ketamine altered NMDAR subunit expression in the medial prefrontal cortex and NAc, indicating circuit-level synaptic remodeling. Behavioral analyses revealed that ketamine significantly reduced heroin-primed, but not cue-induced, seeking activity. Together, these findings suggest ketamine reduces acute relapse vulnerability to opioids through coordinated modulation of opioid and glutamatergic systems, highlighting its potential as a novel intervention for OUD.