Alcohol

Sweetened alcoholic beverages are thought to contribute to developing Alcohol Use Disorder by increasing palatability. One monosaccharide, glucose, readily enters the brain more than fructose and directly impacts the activity of central neurons. The objective of this study is to determine the impact of glucose versus fructose on alcohol drinking patterns in female and male rats. Rats drank alcohol cocktails (1.25%-10%) containing either glucose or fructose (10%) in 4-hour sessions. We sought to parse orosensory effects from post-ingestive central effects by analyzing drinking microstructure. We compared measures of palatability and post-ingestive feedback between early and later in the session when brain levels of alcohol and glucose are different. We found that rats of both sexes drank more low alcohol glucose cocktails than cocktails containing fructose by volume and by overall calories. When considering the dose of alcohol, glucose potentiated alcohol intake by shifting the dose-response curve leftward compared to similar fructose cocktails. We found that drinking patterns associated with palatability remained stable for both types of cocktails over the entire drinking session. In contrast, post-ingestive behavior related to brain mediated satiety or positive feedback showed a greater influence of the session time, as well as a greater interaction with sex. Overall, our results suggest that glucose and alcohol interact to impact central regulation of cocktail drinking. This highlights that the type of sugar within cocktails interacts and ultimately have different effects on brain regulated alcohol drinking.

RationalePrenatal alcohol exposure (PAE) can result in Fetal Alcohol Spectrum Disorder (FASD), which consists of a group of diagnosable medical conditions that can include an increased risk for anxiety disorders and/or alcohol misuse, and sensory issues, such as increased mechanical sensitivity. ObjectiveThis study investigated how a single moderate PAE on gestational day 12 (G12) alters anxiety-like behavior, ethanol (EtOH) intake, and mechanical sensitivity across the lifespan of Sprague Dawley rats. MethodsPregnant dams were exposed to vaporized EtOH or room air (control) for 6 hours (BECs [~]108 mg/dL). Testing in male and female offspring began at three different ages: juveniles ([~]postnatal day (P) 25), adolescents ([~]P45) and adults ([~]P80). ResultsThe greatest PAE effects were observed in adolescent animals, with alterations in anxiety-like behaviors demonstrated in the light-dark box and elevated plus maze. Additionally, adolescent female animals consumed more sweetened EtOH compared to males. However, PAE adolescent animals consuming less sweetened EtOH compared to their counterparts, which was also observed in adult PAE females. Interestingly, this effect is reversed in juvenile and adolescent males when tested with unsweetened EtOH, with juvenile females consuming more EtOH also. Finally, PAE and air animals exhibited increased mechanical sensitivity following post-natal EtOH consumption across all ages. ConclusionThese data demonstrate that there are age- and sex-specific effects of PAE on anxiety-like behaviors, EtOH intake, and mechanical sensitivity that are more distinct in adolescent animals.

BackgroundAlcohol use disorder (AUD) is linked to increased neuroinflammation. Alcohol (ethanol) may activate toll-like receptors, which leads to the release of inflammatory molecules that could influence AUD-related behaviors, such as increased alcohol intake. Activation of toll-like receptor 3 (TLR3) by Polyinosinic:polycytidylic acid (Poly(I:C) or PIC) is associated with escalation of alcohol consumption in male, but not female F1 hybrid mice from reciprocal crosses between FVB/NJ (FVB) and C57BL/6J (B6) strains. Little is known about the underlying mechanisms of these sex-specific behavioral effects. In this study, we investigated the effects of TLR3 activation by PIC on temporal profiles of several pro- and anti-inflammatory molecules in the blood and brain of FVB/B6 F1 hybrid male and female mice at multiple time points. We hypothesized that TLR3 - dependent immune profiles would differ between males and females, which may, at least in part, explain the observed differences in drinking behavior. MethodsMale and female FVB/B6 F1 hybrid alcohol-naive mice were injected intraperitoneally with PIC (10 mg/kg) or saline. Blood and perfused brain tissues from the prefrontal cortex (PFC) and striatum were collected at 6-, 24-, and 48-hours post-injection. The expressions of Ccl2, Ccl5, Tnf, Il-6, Il-1{beta}, Ifng, Ifnb1, and Mmp9 genes were analyzed using qPCR. Protein levels of a subset of these molecules and IL-17r/a, IL-4, and IL-10 were measured in striatal samples from the same animals using ELISA. ResultsActivation of TLR3 by PIC triggered time-dependent, sex- and tissue-specific responses in immune genes and their proteins. PIC induced a time-dependent increase in expression of majority of the genes peaking at the 6 hr time point. Temporal immune profiles for pro-inflammatory chemokines, Ccl2 and Ccl5 differed between males and females in the PFC and striatum, suggesting possible sex-specific effects of these molecules on behavior. Protein levels of CCL2, CCL5, and IL-6 increased in the striatum of both sexes and correlated strongly with gene expression, with females showing somewhat higher protein fold changes. MMP-9, a key regulator of blood-brain barrier (BBB) permeability and synaptic plasticity, showed an increase in protein levels, but not mRNA levels in striatum. This pattern suggests altered blood-brain barrier (BBB) permeability, although this would require further investigation. ConclusionOur results revealed distinct TLR3-dependent immune gene and protein expression profiles in blood and brain between males and females and suggested different roles for these molecules in regulating alcohol consumption. We identified CCL2, CCL5 and MMP-9 as target molecules for investigating sex-specific behavior in the immune modulation of alcohol consumption.

BackgroundAlcohol use disorder (AUD) is a chronic condition characterized by compulsive drinking and high relapse risk. Craving in early abstinence is a strong predictor of relapse, yet its underlying neurobiological mechanisms remain unclear. Guided by Menons Triple Network Model (TNM) of psychopathology, this study investigates whether altered connectivity between the salience (SN), default mode (DMN), and central executive (CEN) networks --previously implicated in alcohol-related behaviours -- underlies craving during early abstinence. MethodsA final cohort of 27 individuals with AUD recruited from an inpatient alcohol withdrawal program completed resting-state fMRI scans on day 1 of withdrawal and 18 days later. Additionally, 17 healthy controls underwent fMRI at two sessions spaced two weeks apart. Craving was assessed in the AUD group at both timepoints using the obsessive thoughts subscale of the Obsessive Compulsive Drinking Scale (OCDS). Functional connectivity between brain networks was computed by referencing each individuals between-network connectivity to normative models derived from large-scale reference data to generate scores reflecting their deviations from normative values. Proposed analysesPlanned analyses will leverage large-scale lifespan normative models to test associations between patient deviation scores in SN-DMN connectivity and craving during acute withdrawal, along with longitudinal associations during abstinence. Exploratory analyses will assess correlations between craving and connectivity of other network pairs of the TNM. ConclusionsThis report aims to identify functional neurobiological markers of craving during early abstinence in AUD employing normative models. Findings may advance understanding of relapse vulnerability and inform personalized interventions targeting large-scale brain network dysfunctions in AUD. This submission corresponds to Level 3 of the Peer Community In (PCI) Registered Report bias-control taxonomy: data were collected and pre-processed prior to hypothesis formulation, but key variables (subject-level values) have not been observed and no statistical analyses have been performed.

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

IntroductionNeural cue reactivity is increasingly being investigated as a biomarker of treatment response and relapse prediction in addiction disorders. Whilst aberrant brain responses to salient cues (e.g. drugs) have been widely reported in addiction, it is unclear whether these brain responses persist during longer-term abstinence, how they compare between substance use disorder and obesity, and relate to potential differences in eating behaviours. As part of the Gut Hormones in ADDiction (GHADD) neuroimaging study, we investigated how salient cue reactivity to drugs or food, craving and eating behaviours compare in three clinical populations where alterations have been previously observed: abstinent nicotine use disorder (NUD) and alcohol use disorder (AUD), and obesity. MethodsThis study compared group differences in salient cue reactivity and eating behaviours between ex-smokers (n=25, ExS), adults with alcohol dependence who are abstinent (n=26, AAD), adults with obesity who were actively dieting (n=26, OB). Participants completed a high-energy food, preferred alcohol and cigarette functional magnetic resonance imaging (fMRI) cue reactivity task, along with eating behaviour questionnaires, appetite visual analogues scales and an ad libitum test meal. ResultsExS exhibited greater blood oxygen level dependent (BOLD) signal to high-energy food pictures in several reward processing regions in both whole brain and region of interest (ROI) analyses, compared with the OB and AAD groups, with no difference in their appeal rating. Compared with the OB group, ExS exhibited greater BOLD signal to cigarette pictures in the frontal gyrus, orbitofrontal cortex, frontal pole and insula, with no difference in their appeal rating. There were no group differences in preferred alcohol cue reactivity. The AAD group rated sweet taste as more pleasant, and consumed more calories from sweet dishes in the ad libitum meal than the OB and ExS groups. ConclusionsThe presence of heightened cue reactivity to high-energy foods in ex-smokers could contribute to post-quitting weight gain after smoking cessation. Neuroimaging findings were consistent with persistence of some salient drug cue reactivity, despite absence of craving, after medium term abstinence in ExS, but not in AAD. This study also adds to the body of evidence supporting a sweet taste preference endophenotype predisposing individuals to AUD. These changes in eating behaviour in NUD and AUD may provide targets for treatments to reduce substance misuse and facilitate abstinence.

BackgroundLoss of control over drinking is a hallmark feature of alcohol use disorder (AUD) that is modeled preclinically through escalation of ethanol consumption and aversion-resistant drinking. Prior work with other reinforcers suggests that within-session unpredictable, intermittent access (uIntA) promotes loss of control over intake. However, the effect of uIntA on voluntary ethanol consumption is unknown. MethodsMale and female Long-Evans rats (n=9-10/group) underwent seven weeks of daily voluntary ethanol (20% v/v) drinking sessions under either a continuous access (ContA) or uIntA schedule. Following four weeks of baseline, rats were rendered dependent using a two-week chronic intermittent ethanol vapor exposure procedure. Daily testing was maintained through one week into withdrawal from vapor exposure. On the final day of testing, ethanol was adulterated with quinine (30 mg/L) to assess aversion-resistant drinking. ResultsRats drinking under ContA and uIntA exhibited similar levels of average daily ethanol consumption at baseline. However, uIntA elicited a more robust dependence-induced escalation of ethanol consumption compared to ContA, with uIntA sustaining escalation through early protracted withdrawal. Additionally, while rats with ContA to ethanol remained sensitive to quinine even after chronic ethanol vapor exposure, uIntA promoted aversion-resistant drinking in ethanol dependent rats. ConclusionsThese results demonstrate that, compared to ContA, uIntA maintains ethanol drinking and exacerbates AUD-related symptomatology while also providing researchers with the ability to capture additional measures of motivation and drinking patterns without increasing experimental burden. This work positions uIntA as a powerful tool to assess psychological and neurobiological factors underlying loss of control over drinking.

There is a dearth of information on how different cocktails sweetened with different sugars impact brain activity. Glucose enters the brain faster and in greater concentration than fructose and directly affects neuronal activity of melanin-concentrating hormone (MCH) neurons. MCH signaling promotes both glucose drinking and alcohol intake by integrating central and sensory inputs, but it is currently unknown how MCH neuronal activity relates to sweetened cocktail drinking. This study sought to investigate the relationship between MCH activity and sugar-sweetened alcoholic cocktail drinking. We also sought to compare MCH neuronal responses to the sugar solutions without alcohol as well as their response to sensory stimuli. In female and male rats, we used fiber photometry to monitor MCH neurons in response to sensory stimuli and during drinking of 10% glucose, 10% fructose, and glucose or fructose cocktails with 1.25% or 10% alcohol. We found that MCH activity rises in response to a variety of sensory stimuli and peaks before the start of drinking for all cocktails, before returning to baseline near the start of drinking. The cocktail type impacted the dynamics of MCH activity, where increased alcohol concentration resulted in earlier MCH activity for fructose but not glucose cocktails. Finally, we found that peak MCH activity during drinking is correlated with approach behavior for all sugar and cocktail types. These findings suggest that glucose and alcohol may interact to directly influence MCH activity. Further, MCH neurons may regulate cocktail drinking in response to sugar type and alcohol concentration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/719280v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@54685org.highwire.dtl.DTLVardef@59003eorg.highwire.dtl.DTLVardef@11f0358org.highwire.dtl.DTLVardef@114b524_HPS_FORMAT_FIGEXP M_FIG C_FIG New and noteworthyFiber photometry was used to monitor lateral hypothalamic melanin-concentrating hormone (MCH) neurons in male and female rats during sensory stimuli and drinking of glucose, fructose, or glucose- or fructose-sweetened alcoholic cocktails. Subsecond-scale changes in MCH activity occurred after stimuli. Peak MCH activity during drinking was correlated with approach behavior. Alcohol concentration only impacted MCH activity with fructose cocktails. We discuss the implications of MCH dynamics towards brain function, associative learning, and alcohol use disorder.

Introduction: The Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) is a widely utilized screening tool in large-scale electronic health record (EHR) biobanks. However, its categorical, range-based survey responses present a significant challenge for epidemiological research, especially where continuous quantitative variables may be preferred. Standard workarounds, such as assigning categorical midpoints or utilizing aggregate ordinal scores for regression mapping often introduce false mathematical precision or obscure critical behavioral nuances between drinking frequency and quantity. This report presents a novel framework for presenting and bounding categorical alcohol survey data. Materials and Methods: I developed two complementary descriptive techniques: (1) a two-dimensional cross-tabulation matrix that preserves the interaction between drinking frequency and typical quantity, and (2) a systematic bounding algorithm that applies time-interval correction factors to calculate strict lower and upper estimates of average daily alcohol consumption. To demonstrate the real-world utility of this framework, I applied these methods to three analytical descriptive scenarios within a European ancestry (EUR) cohort of the All of Us Research Program: Generalized Anxiety Disorder (GAD) prevalence (n=104,893), minor allele frequency (MAF) for the rs1229984 genetic variant (n=104,890), and self-reported active duty military service history (n=104,893). Results: Application of the cross-tabulation matrix revealed patterns across all three descriptive scenarios. For example, participants reporting the highest frequency ("4 or more times a week") combined with the highest quantity ("10 or More" drinks) demonstrated a GAD prevalence of 13.5%, compared to 5.8% among those reporting the same frequency but a low quantity ("1 or 2" drinks). A general trend of increased anxiety in higher quantity drinkers contrasts with a general trend of decreased anxiety in higher frequency drinkers. Bounding estimates for average daily consumption ranged from 0.299 to 0.730 drinks for individuals with GAD, and 0.303 to 0.787 for those without. Those who reported having been active duty in the US Armed Forces demonstrated a general trend toward more frequent drinking and higher average daily consumption estimates (0.339 to 0.875) than those who had not (0.297 to 0.770). The minor allele of the genetic variant rs1229984 exhibited a clear effect reducing both frequency and quantity, resulting in lower average daily consumption estimates. Conclusions: This bounding and mapping framework provides researchers with an additional method to traditional midpoint and aggregate scoring methods. By explicitly defining the uncertainty inherent in categorical survey instruments and visualizing cohort distributions across intersecting behavioral axes, this methodology improves the resolution, reproducibility, and interpretability of lifestyle exposure data.

BackgroundAlcohol use disorder is a chronic relapsing condition characterized by excessive drinking and withdrawal symptoms. Alcohol dependence disrupts function across multiple brain regions, and recent evidence implicates the cortical amygdala (CoA) as a critical node in alcohol-related circuits. However, how CoA activity influences alcohol intake and brain-wide network function during withdrawal remains unclear. MethodsAlcohol dependence was induced using chronic intermittent ethanol vapor (CIE). In one cohort, electrophysiological activity of CoA neurons was assessed during withdrawal. In a second cohort, mice underwent CIE paired with two-bottle choice drinking, and inhibitory DREADDs (hM4Di) were used to suppress CoA activity during drinking and withdrawal while behavioral outcomes were measured. Brains were then collected for Fos immunolabeling and iDISCO+ based whole-brain activity mapping to determine how CoA inhibition during withdrawal altered network organization. ResultsRepeated CIE increased alcohol sensitivity in CoA neurons during withdrawal. Chemogenetic inhibition of the CoA reduced alcohol intake in dependent mice without affecting withdrawal-related behaviors. Whole-brain Fos mapping showed that CoA inhibition reduced activity within the CoA while enhancing functional connectivity across multiple brain regions, particularly in the isocortex, thalamus, and anterior hypothalamic nucleus. During withdrawal without CoA inhibition, thalamic regions exhibited negative connectivity, consistent with disrupted network function; CoA inhibition reversed this pattern, producing strongly positive thalamic and medial prefrontal cortex connectivity. ConclusionsThese findings demonstrate that alcohol dependence alters CoA sensitivity, alcohol dependence-induced drinking and brain-wide network organization during withdrawal. The CoA appears to selectively regulate withdrawal-associated alcohol drinking, and its inhibition may reduce intake by restoring thalamic and cortical connectivity. HighlightsO_LIThis study identifies the cortical amygdala as a previously underexplored brain region involved in alcohol-related behaviors. C_LIO_LIBy integrating chemogenetic inhibition with brain-wide network analysis, the study reveals candidate circuit connections through which the CoA may regulate alcohol dependence-related brain activity. C_LIO_LIThis study establishes the CoA as a potential driver of excessive alcohol drinking and alcohol-related network dysfunction. C_LI

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

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

BackgroundFetal alcohol spectrum disorder (FASD) encompasses a variety of disorders that occur after a fetus has been exposed to alcohol. Hippocampal related issues are a common neurological deficit found in FASD. The dentate gyrus within the hippocampus is a unique area of the brain that continues to generate new neurons into adulthood. This neurogenesis can be enhanced by an enriched environment (EE); however in prenatal alcohol exposed (PAE) mice this EE-mediated neurogenesis is impaired. In addition to EE, selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, also promote neurogenesis. Here we examine if fluoxetine restores the impaired EE-mediated neurogenesis of PAE mice. MethodPAE mice were generated using a voluntary limited access model where mice received a 10% ethanol (w/v) solution during gestation. To evaluate neurogenesis, we use a NestinCreT2:tdTomato transgenic mouse line in which newborn dentate granule cells (nDGCs) can be evaluated by tdTomato flourescence. PAE and saccharine control (SAC) mice were placed in either standard housing (SH) or enriched environment (EE). Subsequently, we administered fluoxetine (FLX) or vehicle (VEH) after which neurogenesis was evaluated. ResultsPAE resulted in impaired EE-mediated neurogenesis. This neurogenic impairment was not restored by FLX. Interestingly, FLX did increase neurogenesis in PAE mice while housed in SH. ConclusionThese results suggest that there is a neurogenic ceiling in PAE mice that cannot be increased by fluoxetine in EE. However, fluoxetine can increase neurogenesis while the environment is less complex.

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.

Craving - the powerful urge to seek and consume alcohol in response to alcohol-associated cues does not diminish after drinking cessation but rather is magnified throughout abstinence. This phenomenon, termed "incubation of craving", contributes to the relapsing nature of alcohol use disorder. Despite its occurrence in human populations and being well-studied in rodent models of psychostimulant drug relapse, the underlying neural mechanisms and potential treatments remain largely unexplored for alcohol-related incubation of craving. Our research seeks to meet this gap, and this particular study investigated the neural correlates of the incubation of craving for alcohol-associated cues and assessed whether exercise could prevent increased relapse propensity in rats. Male Long Evans rats were trained to lever press for an alcohol reward delivered with simultaneous presentation of a discrete cue. This response was then extinguished and reinstated by presenting the discrete cue alone when rats pressed the lever. Cue-induced reinstatement occurred either on day 1 following extinction (No Abstinence) or on day 29 (Abstinence). A third group was tested on day 29 and had 4-hour daily voluntary running wheel access throughout this abstinence period (Exercise). All rats were perfused 90 minutes following test, and relative activation across the brain was estimated by quantifying c-Fos protein immunoreactivity. The brain-wide coordination of neural activity was also mapped. We found a robust incubation of craving effect for alcohol-associated cues, which was mitigated by exercise. Immunohistochemistry revealed that the Abstinence group demonstrated higher c-Fos immunoreactivity compared to the No Abstinence group in multiple reinstatement-related brain regions. This effect was reversed in the Exercise group. Brain-wide neural mapping demonstrated that the Abstinence group had decreased modularity (groups of coordinating brain regions) compared to the no-abstinence group. Although network connectivity profile in the exercise group was different from no abstinence, we found that overall neural activation returned to a similar modularity profile of clustered regions as this condition, indicating that exercise does not attenuate the incubation of craving effect by reversing all the neural effects of abstinence. Rather, exercise may be acting upon select brain regions or pathways to exert relapse protective effects by restoring widespread interconnectivity. This is the first study to investigate neural activation in incubated alcohol-seeking, and provides supporting evidence for promoting voluntary exercise as an adjunctive treatment for relapse prevention in alcohol-use disorder.

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

BackgroundMaladaptive drinking is often sustained by automatic approach tendencies toward alcohol cues that override conscious self-control. While cognitive and behavioral modification techniques show some promise, their effects remain limited, highlighting the need for alternative neuromodulatory strategies. The current study examined the feasibility of a single session of 10 Hz repetitive transcranial magnetic stimulation (rTMS) to the right dorsolateral prefrontal cortex (dLPFC) as a targeted approach to reduce automatic alcohol approach tendencies. MethodForty-five healthy alcohol-using participants completed an alcohol approach- avoidance task (A-AAT) with concurrent electroencephalographic recording before and after active or sham stimulation. Primary analyses focused on participants with baseline alcohol approach tendencies (n = 35). ResultsAt baseline, individuals with approach tendencies exhibited attenuated N2 and P3b amplitudes to alcohol relative to non-alcohol cues, indicating reduced cognitive control and attentional mechanisms irrespective of group. Following stimulation, active rTMS selectively facilitated alcohol avoidance responses and enhanced prefrontal N2 amplitudes, suggesting strengthened top-down control and protection against repetition-induced automaticity, which was evident in the sham group. ConclusionThese findings suggest that high-frequency rTMS over the right dLPFC can modulate automatic alcohol-related action tendencies by strengthening neural control mechanisms, supporting its further evaluation as a neuromodulatory adjunct for maladaptive drinking. Baseline motivational profiles may additionally influence rTMS response and warrant consideration when tailoring such approaches.

ObjectiveMinimum unit pricing (MUP) aims to reduce use of cheap, high strength alcoholic beverages that drive harm, yet concerns remain about inequitable effects for structurally vulnerable groups. As part of the Costs, Harms, Expenditures and Alcohol Prices (CHEAP) study, we linked individual-level, product-specific alcohol consumption data from a customized survey with provincial retail price data to estimate prices per standard drink (PPSD) and examine their association with alcohol-related outcomes across sociodemographic groups. MethodA cross-sectional survey of past-week drinkers in British Columbia, Canada, was linked to provincial product-level alcohol sales data. The population weighted sample included 1,217 adults aged [≥] 19 years (716 men; mean age 49.34, SD 16.98). Participants reported product-specific consumption, which was matched to retail prices to calculate individual-level PPSD. Survey weighted quasibinomial models then examined associations between PPSD and three outcomes: (1) causing harm to self or others in the past year, (2) scoring [≥] 8 on the Alcohol Use Disorder Identification Test, and (3) consuming [≥] 15 standard drinks per week. Analyses were stratified by income, education, subjective social status, and race/ethnicity. ResultsLower price per standard drink was associated with higher odds of harm (OR 3.05, 95% CI 1.25-7.40) and scoring [≥] 8 on the AUDIT (OR 2.34, 95% CI 1.37-3.99). Associations were stronger among structurally disadvantaged groups, including low-income respondents and Indigenous participants. ConclusionsLower alcohol affordability is linked to risky alcohol use, with the strongest effects among structurally disadvantaged groups. MUP would reduce this risk and promote health equity.

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

Previous studies have linked opioid use to altered metabolic profiles, but findings have been inconsistent and mechanisms remain unclear. One potential mechanism involves increased adiposity, leading to chronic low-grade inflammation that elevates metabolic risk. Here, we examined metabolic profiles in individuals with opioid use disorder (OUD) and matched non-OUD controls, focusing on the sequential mediating roles of BMI and inflammation. Data from individuals with OUD (n=281) and non-OUD (n=246) were drawn from a natural history screening protocol from the National Institute on Alcohol Abuse and Alcoholism intramural program. Groups were matched on age, sex, race, ethnicity, socioeconomic status, and education via propensity score matching. Metabolic measures included BMI, blood glucose, hemoglobin A1c (HbA1c), and lipid profiles, with lipid imbalance indexed by the atherogenic index of plasma (AIP). Inflammatory markers included C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Individuals with OUD had significantly higher BMI (F1,481=12.9, p<0.001), HbA1c (F1,481=10.5, p=0.001), lower high-density lipoprotein cholesterol (HDL-C; F1,481= 46.2, p< 0.001), higher low-density lipoprotein cholesterol (LDL-C; F1, 481=11.9, p< 0.001), and higher AIP (F1,481=20.7, p< 0.001) compared to non-OUD. Inflammatory markers were also elevated in individuals with OUD, including CRP (F1,481=9.4, p=0.002) and ESR (F1,481=7.4, p= 0.007), and statistically mediated group differences in AIP and HbA1c, respectively. Our results are consistent with prior evidence of metabolic dysfunctions in individuals with OUD and suggest inflammation as a contributing mechanism. Targeting metabolic health and inflammation may offer new avenues for improving long-term health outcomes in OUD.