Neuropharmacology

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.

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.

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.

BackgroundConcurrent cocaine and alcohol use is one of the most prevalent forms of polysubstance consumption and is associated with poorer clinical outcomes than cocaine use alone. However, the regional molecular adaptations induced by combined exposure remain poorly understood. Here, we used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to characterize peptide/protein alterations in addiction-related brain regions following cocaine and cocaine-alcohol self-administration. MethodsYoung adult male and female Wistar rats underwent intravenous self-administration of saline, cocaine (1 mg/kg/infusion) or cocaine plus ethanol (1 mg/kg cocaine and 133 mg/kg ethanol per infusion), followed by extinction of drug-seeking behaviour. Coronal brain sections containing the anterior cingulate cortex (ACC) and ventral hippocampus (vHPC) were analysed by MALDI-IMS. Differential molecular features were identified using an exploratory statistical approach (FDR q < 0.20) and subsequently subjected to MS/MS analysis. ResultsThe ACC exhibited a substantially greater number of treatment-associated molecular alterations than the vHPC, suggesting a higher regional susceptibility to cocaine-induced molecular remodelling. Several molecular features were shared between the cocaine and cocaine-alcohol groups, indicating persistent cocaine-driven neuroadaptations. In contrast, additional signals were selectively associated with combined cocaine-alcohol exposure, while others present after cocaine alone were absent following alcohol co-exposure, supporting a modulatory effect of alcohol on specific cocaine-induced molecular responses. Overall, combined exposure generated a distinct regional molecular profile rather than simply reproducing the effects of cocaine alone. ConclusionsThis exploratory study demonstrates that MALDI-IMS enables the identification of region-specific peptide/protein alterations associated with cocaine and cocaine-alcohol exposure while preserving their spatial distribution within the brain. These findings highlight the ACC as a particularly responsive region and provide a framework for future studies aimed at validating molecular pathways involved in cocaine-alcohol polysubstance use.

Background: The nicotine analog 6-methyl nicotine (6-MN) has appeared in commercial e-cigarette liquids, and other products, spurring interest in determining the extent to which it conveys similar effects to those of nicotine. Objective: To determine if 6-MN acts like nicotine to decrease body temperature, decrease nociception, suppress wheel activity and reinforce operant behavior when delivered by vapor inhalation using an Electronic Nicotine Delivery System (ENDS; "e-cigarette") approach in a rat model. Methods: Male and female (N=8 per sex) young adult Sprague-Dawley rats were evaluated for rectal temperature and nociceptive responses (warm water tail-withdrawal) to the inhalation of vapor from (-)-6-MN or (-)-nicotine in concentrations ranging from 5-30 mg/mL in the propylene glycol vehicle. Rats were then assessed for the reinforcing effects of nicotine and 6-MN using a vapor self-administration procedure and the rate suppressing effects of nicotine and 6-MN on wheel activity following injection. Results: Inhalation of nicotine or 6-MN for 30 minutes decreased the rectal temperature and increased tail-withdrawal latency of female and male rats in a concentration-dependent manner. The magnitude of the effects of 6-MN and nicotine were similar at similar vapor concentrations. Operant responding for 6-MN vapor was increased by pre-treatment with the antagonist mecamylamine. 6-MN was more potent than nicotine at suppressing wheel activity after injection. Conclusions: 6-MN induces effects very similar to those of nicotine, at a similar potency when inhaled and at a slightly increased potency when injected.

Tobacco use is the leading preventable cause of death worldwide. Anxiety increases the risk for smoking, and smoking in turn increases the risk for anxiety disorders. There is therefore a need to identify interventions that reduce anxiety, in general and in the context of smoking, without producing sedation. Kava (Piper methysticum), a natural product with a long history of indigenous use, has been shown to have anxiolytic and calming effects and reduce nicotine withdrawal. The current study examined whether kava without the hepatotoxic flavokavains A and B (AB-free) could reduce anxiety-like behavior in mice repeatedly treated with nicotine. Male and female C57BL/6NCrl mice received either a control diet or an AB-free kava-supplemented diet and underwent two blocks of nicotine treatments. Mice underwent a first block of five every-other-day injections of nicotine (0.5 mg/kg) or saline, with open field testing after each injection, followed one week later by a nicotine challenge. A second block of injections was given using the same injection schedule, followed by a second challenge one week later, and two weeks afterward mice received a final challenge in a novel open field. During the first treatment block, AB-free kava significantly increased center time overall, an effect most pronounced in saline-treated animals, and increased locomotor activity, while nicotine decreased both measures. During the second challenge, nicotine reduced center time but not locomotor activity, and AB-free kava increased center time in saline-treated animals only. During the final challenge, nicotine reduced both measures, whereas AB-free kava increased center time regardless of nicotine treatment, and kava-treated animals also showed a near-significant increase in center entries. These results suggest that AB-free kava reduces anxiety-like behavior without inducing sedation but does not prevent nicotine-induced suppression of exploratory behavior.

Astrocytes play an important role in neuronal health. A critical function of astrocytes is to clear excess extracellular glutamate and prevent excitotoxicity. STAT3 is a transcription factor that promotes astrocyte development and astrocyte reactivity in neurodegenerative diseases and following central nervous system injury. To determine the innate molecular and behavioral functions of adult astrocyte-expressed STAT3 in a non-pathological state, we created conditional Stat3 astrocyte knockout mice (Stat3 aKO) using Stat3flox and the tamoxifen-activated Cre line, Aldh1l1-Cre/ERT2. We measured transcript levels of Gfap, a known STAT3 target gene, and glutamate transporter genes in the medial prefrontal cortex (PFC) of Stat3 aKO. Gfap, Slc1a2 and Slc17a8 transcripts were decreased in the PFC of Stat3 aKO of both sexes. GLT-1 protein, encoded by Slc1a2, was also reduced in the PFC of male Stat3 aKO. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) in male Stat3 aKO and control prelimbic pyramidal neurons and found increased sEPSC amplitude, consistent with a hyper-glutamatergic state due to impaired glutamate clearance. To determine the behavioral consequences of STAT3 depletion in astrocytes, Stat3 aKO were tested for locomotor activity, anxiety-like behavior and binge ethanol consumption, behaviors linked to dysregulation of glutamate homeostasis. Stat3 aKO mice did not differ in locomotor activity or anxiety-like behavior; however, male Stat3 aKO mice consumed significantly less ethanol than controls. These results indicate that STAT3 in adult astrocytes is crucial for maintaining glutamate transporter levels in the adult brain and that astrocytic STAT3 promotes ethanol consumption in male mice. Main pointsO_LIGfap, Slc1a2 and Slc17a8 expression are lower in the cortex of Stat3 astrocyte knockout mice (Stat3 aKO) C_LIO_LIGLT-1 protein is decreased and glutamate neurotransmission is elevated in the cortex of male Stat3 aKO C_LIO_LIMale Stat3 aKO consume less ethanol C_LI

Opioid addiction treatment is often hampered by the severe dysphoria of opioid withdrawal, but withdrawal treatments are limited by incomplete understanding of brain mechanisms involved. One area frequently implicated in withdrawal symptoms is the central amygdala, whose capsular portion (CeC) is particularly strongly activated during withdrawal. Additionally, a ventral posterior striatal region that resides near CeC, the interstitial nucleus of the posterior limb of the anterior commissure (IPACc), is also activated as strikingly as CeC. However, it is still unknown how these regions are activated, nor whether their activation explains the high intensity of withdrawal dysphoria. Using RNAscope, we found that c-fos expression is induced in the parabrachial nucleus (PB), a key glutamatergic afferent of CeC, after precipitated morphine withdrawal. Chemogenetic inhibition of PB glutamatergic neurons (VG2PB) nearly eliminated withdrawal-induced CeC c-Fos, without affecting IPACc c-Fos, indicating these two nuclei are activated by distinct sources. Furthermore, VG2PB inhibition markedly reduced somatic (jumping) and modestly reduced affective (place avoidance) withdrawal behavior. On the other hand, inhibition of CeC-projecting PB neuronal subtypes expressing calcitonin gene-related peptide (CGRP) or mu opioid receptor (MOR) reduced place avoidance without affecting jumping, indicating their specific role in withdrawal aversion. Strikingly, simultaneous inhibition of VG2PB and posterior striatal region containing IPACc robustly reduced withdrawal-induced place avoidance much more than the modest effects of either inhibition alone, suggesting their cooperative action in driving aversion. Our data suggests that PB-CeC circuit and posterior striatal area constitute a cooperative system driving opioid withdrawal aversion.

Psilocybin is a psychedelic which has been shown to induce neural plasticity through activation of intracellular serotonergic 5-HT2A receptors. It also produces brain-wide changes in structural and functional connectivity and holds promise as a therapeutic compound for treating anxiety and depression. Despite links between psilocybin-induced plasticity, the psychedelic experience, and reduction in depressive symptoms, little is known about the effects of psilocybin on the function of the highly plastic hippocampus, a region crucial for memory whose dysfunction is linked to neural disorders such as depression and anxiety. In this study, we investigated the acute and lasting effects of psilocybin on rodent sharp-wave ripples (SWRs), transient high frequency oscillations observable in the hippocampal local field potential which are linked to memory consolidation. We found that a 10 mg/kg dose of psilocybin robustly decreased the peak SWR frequency and increased the duration of SWRs immediately following administration compared to control sessions the day before and after. Psilocybin also perturbed sleep architecture, resulting in a pronounced reduction in non-rapid eye movement (NREM) sleep which lasted for hours. Therefore, psilocybin could impact memory processing by modulating hippocampal SWRs.

Relapse following prolonged abstinence is a primary challenge in the treatment of opioid and cocaine use disorders, driven in part by enduring dysfunction of medial prefrontal cortex (mPFC) circuits that impair inhibitory control over drug-seeking. The molecular substrates underlying this dysfunction, and whether they differ across drug classes, remain unknown. Here, we performed label-free quantitative proteomics of mPFC synaptosomes isolated from rats after 30-day abstinence following intravenous heroin or cocaine self-administration to profile synaptic adaptations that may contribute to relapse vulnerability. Heroin abstinence induced extensive synaptic proteomic remodeling characterized by coordinated downregulation of mitochondrial proteins involved in oxidative phosphorylation, including pyruvate dehydrogenase complex subunits that regulate carbon entry into mitochondrial metabolism. Targeted metabolomic profiling of whole mPFC revealed accumulation of upstream glycolytic and pentose phosphate pathway intermediates, consistent with altered pyruvate utilization and mitochondrial oxidation. Several bioenergetic metabolites also correlated positively with the severity of escalation of heroin intake. Consistent with the bioenergetic remodeling observed during protracted heroin abstinence, whole-cell patch-clamp recordings from layer V mPFC pyramidal neurons revealed lasting suppression of intrinsic excitability and a decreased spontaneous excitatory synaptic activity. Cocaine abstinence, by contrast, produced limited changes in synaptic bioenergetics while inducing a distinct cytoskeletal remodeling signature. Overall, these findings identify synaptic bioenergetic remodeling as a previously underappreciated feature of prolonged heroin abstinence and reveal a marked divergence in the molecular adaptations induced by heroin versus cocaine within the mPFC. These results implicate mitochondrial bioenergetic pathways as therapeutic targets for reducing relapse vulnerability specifically associated with opioid use disorder.

BackgroundAlthough nicotine reinforcement is often attributed to mesolimbic dopamine neurons in the ventral tegmental area, accumulating evidence indicates that additional brain circuits contribute to its reinforcing effects. AimsThe hypothalamic supramammillary region (SuM) has been implicated as one such substrate, yet the cellular targets and circuit mechanisms through which nicotine engages this region remain poorly understood. MethodsWe combined RNAscope in situ hybridization to identify nicotinic acetylcholine receptor (nAChR) subunits, intravenous nicotine self-administration in mice to determine doses that reliably support reinforcement, and fiber photometry to monitor calcium activity in SuM VGluT2 neurons in vivo. ResultsMice exhibited reliable nicotine self-administration across a range of doses under fixed-ratio and progressive-ratio schedules. RNAscope analysis revealed prominent expression of the {beta}2 nAChR subunit in VGluT2-expressing neurons projecting from the SuM to the medial septum. Fiber photometry recordings showed that reinforcing doses of nicotine produced rapid, infusion-locked increases in GCaMP signals in SuM VGluT2 neurons. ConclusionsThese findings identify nAChR-expressing SuM neurons as a candidate circuit substrate engaged by reinforcing doses of nicotine and extend current models of nicotine reinforcement beyond canonical mesolimbic dopamine pathways.

Long-term opioid therapy is limited by analgesic tolerance and opioid-induced hyperalgesia, but the roles of genetic background, sex, and drug exposure remain unclear. We used 20 inbred strains from the Hybrid Rat Diversity Panel to examine thermal sensitivity, oxycodone analgesia, tolerance, and hyperalgesia-like changes following voluntary intravenous oxycodone or saline self-administration. Rats underwent tail-immersion testing before self-administration (Pre-SA) and after self-administration (Post-SA). Oxycodone analgesia was assessed using the percent maximum possible effect time course and the corresponding area under the curve. Pre-SA thermal sensitivity differed across strains and between sexes, and Pre-SA oxycodone analgesia also differed across strains. Oxycodone self-administration produced a sex-dependent increase in thermal sensitivity that was most evident in males. During Post-SA testing, oxycodone self-administering rats showed reduced analgesic responsiveness compared with saline controls, and the magnitude of this difference varied across strains. Within-strain Pre-SA-to-Post-SA comparisons identified tolerance-like reductions in several strains. Across strains and sexes, oxycodone self-administering rats showed a greater Pre-SA-to-Post-SA reduction in analgesic responsiveness than saline controls, consistent with analgesic tolerance. Total oxycodone intake was not associated with tolerance at either the strain-mean or individual-animal level. Heritability estimates were higher for thermal sensitivity and analgesia (H2 {approx} 0.28-0.40) than for changes in thermal sensitivity and tolerance (H2 {approx} 0.18-0.27). These findings demonstrate strain variation in thermal sensitivity and oxycodone analgesia, sex-dependent hyperalgesia-like effects, and reduced analgesic responsiveness following voluntary oxycodone intake.

Cannabis is the most common illicit drug abused worldwide, and its consumption has substantially increased among pregnant women. We previously demonstrated that male preadolescent offspring prenatally exposed to {Delta}9-tetrahydrocannabinol (THC), a model of prenatal cannabinoid exposure (PCE), exhibit a mesolimbic dopamine (DA) neuron dysfunction contributing to at-risk psychotic-like (endo)phenotypes that are unmasked by acute THC exposure at preadolescence. Dysregulation of mesocortical DA signaling along with prefrontal cortex (PFC) function is also a central feature of psychotic disorders. Furthermore, studies investigating the impact of PCE on PFC in the offspring at preadolescence, a window of heightened plasticity and vulnerability, are limited. To fill this gap, we applied a multiscale analysis of mesocortical DA transmission and PFC function in PCE preadolescent offspring by integrating behavioral, neurochemical, electrophysiological, and molecular approaches. PCE enhanced spontaneous repetitive behaviors in a male-specific manner. PCE also abolished sex differences in the intrinsic excitability of PFC pyramidal neurons and Netrin-1 expression. In addition, PCE altered the expression of genes associated with endocannabinoid signaling without changing basal and THC-induced extracellular levels of DA in the PFC. Collectively, these findings demonstrate that prenatal THC exposure disrupts both proper maturation and sexual differentiation of PFC circuitry, thus extending the impact of PCE from previously described mesolimbic abnormalities to mesocortical pathway. Finally, our data identify early cortical molecular and cellular alterations that may contribute to neuropsychiatric vulnerability later in life. Highlights* Preadolescent male rats exposed in utero to THC display repetitive behavior * Prenatal cannabinoid exposure (PCE) does not alter dopamine transmission in the PFC * PCE potentiates AMPA-mediated transmission in male pyramidal cells * PCE abolishes sex differences in Netrin-1 expression levels in the PFC

RationalePsychedelic compounds such as psilocybin have attracted growing interest for their potential therapeutic effects in psychiatric disorders, with improvements in cognitive flexibility proposed as a possible mechanism of action. However, the effects of psychedelics on cognitive flexibility remain poorly understood. ObjectiveThis study aimed to examine the acute and post-acute effects of psilocybin (0.1, 0.3, 1 mg/kg) and lysergic acid diethylamide (LSD, (0.02, 0.04, 0.08 mg/kg) on cognitive flexibility in male rats. MethodsThis was tested using two variants of the probabilistic reversal learning task (PRLT): a touchscreen-based operant task and a more ethological foraging-based task. ResultsIn the touchscreen PRLT, acute psilocybin disrupted task engagement, with animals completing fewer trials and showing increased trial initiation latency, although psilocybin also showed a trend toward faster initial rule acquisition. However, psilocybin did not significantly alter the number of rule changes achieved, a canonical measure of cognitive flexibility, or feedback sensitivity. LSD similarly produced limited acute effects, although the highest dose reduced lose-shift probability, suggesting decreased sensitivity to negative feedback under some conditions. Post-acute effects of psilocybin were minimal in both PRLT variants and, where LSD effects were observed these occurred across different doses and timepoints without a consistent pattern. ConclusionsOverall, these findings suggest that serotonergic psychedelics do not robustly enhance reversal learning in these paradigms and that apparent learning effects may reflect transient disruptions in task engagement rather than improvements in cognitive flexibility. These results also highlight potential limitations of these PRLT paradigms for detecting psychedelic-induced changes in cognitive flexibility in rodents.

Alcohol use disorder (AUD) disrupts the gut-liver-brain axis, yet mechanistically grounded and therapeutically actionable targets within this network remain poorly defined. To identify microbial modulators of alcohol-induced tissue pathology, longitudinal advanced diffusion MRI and fecal 16S rRNA profiling were integrated across Marchigian Sardinian alcohol-preferring rats evaluated at baseline, after four weeks of voluntary alcohol intake, and following six weeks of abstinence. Machine learning, specifically random forest models combining neuroimaging and microbiota data, improved phase classification and identified Akkermansia as the microbial feature most strongly associated with alcohol-related white matter microstructural abnormalities. Alcohol exposure induced widespread white matter alterations alongside gut dysbiosis characterized by reduced microbial diversity. To evaluate functional relevance, Akkermansia muciniphila was administered during the abstinence phase. Supplementation with A. muciniphila restored intestinal mucus, reduced liver injury markers, and elevated myelin basic protein levels within affected white matter regions. Collectively, these findings highlight Akkermansia as a critical modulator of alcohol-induced gut-liver-brain pathology and provide experimental support for a causal contribution of specific gut bacteria to persistent white matter damage in AUD. More broadly, this work establishes a robust multimodal framework for microbiome-based target discovery with clear translational relevance for disorders characterized by dysfunction along the gut-liver-brain axis. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAlcohol use disorder (AUD) is associated with gut dysbiosis, impaired intestinal barrier function, liver injury, and persistent white matter abnormalities. Previous studies in patients and animal models have linked alcohol exposure to reduced microbial diversity, altered gut permeability, and white matter microstructural damage, particularly during abstinence. Other work has shown that microbiota-derived interventions can ameliorate peripheral consequences of alcohol exposure, especially in the gut and liver. However, the specific microbial features linked to alcohol-induced brain pathology remain poorly defined, and no prior study has integrated longitudinal microbiota and neuroimaging data to identify candidate microbial modulators of alcohol-related white matter damage and then functionally test them in vivo across the gut-liver-brain axis. Added value of this studyWe developed a multimodal framework that integrates longitudinal advanced diffusion MRI with fecal microbiota profiling and machine learning in alcohol-preferring rats. This approach identified Akkermansia as the microbial feature most strongly associated with alcohol-induced white matter abnormalities. Guided by this result, we administered Akkermansia muciniphila during abstinence and observed coordinated beneficial effects across multiple organs, including restoration of intestinal mucus, reduction of liver injury markers, and recovery of myelin basic protein in affected white matter regions. To our knowledge, this is the first study to combine longitudinal microbiota-MRI integration with experimental validation of a microbiota-based intervention that mitigates alcohol-induced pathology across the gut-liver-brain axis while restoring central white matter integrity. Implications of all the available evidenceOur findings support a mechanistic contribution of specific gut bacteria to persistent alcohol-induced tissue damage and identify Akkermansia as a candidate modulator of gut-liver-brain axis dysfunction in AUD. More broadly, this study establishes a generalizable strategy for integrating microbiota and neuroimaging data to discover biologically meaningful and therapeutically actionable targets in complex disorders involving coordinated peripheral and central pathology.

Alcohol exposure perturbs intracellular calcium (Ca2+) homeostasis in digestive organs, yet whether common or organ-specific mechanisms coordinate this response remains unclear. Using an acute ethanol paradigm in zebrafish, single-cell transcriptomics revealed broad up-regulation of Ca2+-signaling genes in hepatocytes and pancreatic acinar cells. In vivo Ca2+ buffering with SpiCee, a genetically encoded chelator, demonstrated a shared requirement for Ca2+ flux: in hepatocytes, lineage-restricted buffering was associated with pronounced cytoplasmic vacuolation composed of lipid-negative vesicles, consistent with stalled lysosomes or autophagosomes; in pancreatic acinar cells, it was associated with accumulation of aggregated/misfolded protein. Mechanistic experiments using pharmacological inhibitors implicated distinct molecular contributors in each tissue. In hepatocytes, inhibition of Pikfyve or its downstream effector, the lysosomal Ca2+ channel TRPML1, phenocopied Ca2+ buffering. While, in acinar cells, Pick1 inhibition produced analogous associations. These data position Pikfyve and Pick1 as organ-specific components linked to the Ca2+-coupled alcohol response. Notably, pharmacologic activation of TRPML1 in hepatocytes recapitulated alcohol-like Ca2+ dynamics but increased macrophage recruitment and cell death, indicating that Ca2+ signaling is required for the alcohol response yet can be detrimental when amplified. Together, our results support a model in which alcohol elicits a shared Ca2+ dynamics across liver and pancreas, modulated by tissue-specific molecular nodes.

The GABAA positive allosteric modulator diazepam is taken systemically by millions of people daily. GABAA signaling is essential for hippocampal circuit function, but the effects of systemic diazepam on hippocampal information processing during behavior has not been studied. To answer this question, large neural ensembles were recorded from rats running a linear track under systemic diazepam administration. A cross-correlation of spiking activity revealed significantly increased inhibition from interneurons, aligned with the timescale of GABAA, suggesting a direct effect on local circuits. Local field potentials (LFP) showed an increase in theta and lo-gamma (30-50 Hz) power but a decrease in hi-gamma (80-120 Hz) power. We also found decreased amplitude and rate of sharp wave ripple (SWR) events and a reduction of firing rate and proportion of cells recruited to the SWRs. An autocorrelation of single-cell spike trains revealed a decrease and shift from shorter to longer timescales, aligning differently with theta frequencies. Phase coupling measurements showed decreased cellular coupling to theta and increased coupling to lo-gamma and hi-gamma. Finally, entropy of decoding along the track was increased, suggesting less precise spatial representations under diazepam. These changes suggest mechanisms that would likely disrupt hippocampal memory storage and consolidation processes under systemic diazepam.

Stress-related experiences alter glutamatergic signaling and neuronal excitability, but the mechanisms that couple experience to dentate granule cell function remain incompletely understood. Here, we examined how protein kinase N1a (PKN1a), a protein kinase C-like serine/threonine kinase, and repeated swim exposure regulate mouse hippocampal dentate granule cell excitability, with a focus on the neuronal glutamate transporter excitatory amino acid transporter 3 (EAAT3) and group I metabotropic glutamate receptors (mGluRs). Five days of repeated swim exposure increased spike firing in mature dentate granule cells from wild-type mice. PKN1a knockout produced a similar increase, and repeated swim did not further enhance firing in knockout mice. The enhanced firing observed after repeated swim exposure and in PKN1a knockout mice was reduced by co-application of an mGluR1 antagonist (LY367385) and an mGluR5 antagonist (MPEP). Inhibition of glutamate transporters with DL-TBOA increased granule cell firing in control wild-type mice but did not further increase firing in repeated-swim wild-type or PKN1a knockout mice, suggesting occlusion of transporter-dependent regulation of excitability. Repeated swim exposure and PKN1a knockout also reduced total and surface expression of EAAT3 in the hippocampus, whereas expression of the glial glutamate transporter EAAT2 was not significantly altered. Finally, PKN1a knockout and repeated swim exposure reduced anxiety-related behavior in the elevated plus maze test. Thus, PKN1a-dependent regulation of EAAT3 may restrain group I mGluR-dependent excitability in dentate granule cells, whereas repeated swim exposure and PKN1a knockout shift this system toward a lower-EAAT3, higher-excitability state accompanied by reduced anxiety-related behavior.

BackgroundThe pre-supplementary motor area (preSMA) is increasingly being explored as a neuromodulation target for impulsive behaviour in several clinical populations. Treatment effects are generally interpreted as improvements in inhibitory control. However, healthy studies report improved/impaired/unchanged inhibitory control following identical preSMA stimulation protocols, and few studies examine accompanying neurophysiological changes. We therefore investigated whether preSMA stimulation influences downstream corticomotor excitability to modify a general stopping mechanism, other components of action control, or wider cue-dependent attentional processes relevant to impulsive behaviour. MethodsIn a preregistered, double-blind crossover study, 18 healthy adults received active and sham continuous theta burst stimulation (cTBS) over right preSMA. Motor-evoked potentials (MEPs), anticipatory response inhibition task measures, and alcohol dot-probe reaction times were collected before and after stimulation and analysed with linear mixed models. ResultsMEPs increased during sham (p = .028) but not after active cTBS (p = .741). Active cTBS did not affect complete or partial stopping on the response inhibition task. Instead, active cTBS slowed the continuing response after partial stopping (p < .001) whereas response execution sped up across the sham session (p < .001). No alcohol attentional bias or stimulation effect was detected. ConclusionsPreSMA cTBS did not impair general inhibitory or attentional control. Instead, it attenuated session-related corticomotor facilitation and selectively slowed reinitiation of a partially inhibited action. These findings suggest that clinical effects to impulsive behaviour from preSMA neuromodulation are primarily rooted in changes to motor preparation and action updating rather than a unitary stopping mechanism.