Addiction Biology

Pavlovian conditioned stimuli can compromise goal-directed behavior, particularly when they conflict with the required instrumental response. Previous research linked an increased impact of such Pavlovian-to-instrumental transfer (PIT) interference to risky drinking and alcohol dependence. However, it remained unclear whether the observed behavioral and neural alterations generalize to the substantially larger population of non-treatment-seeking individuals with predominantly mild-to-moderate alcohol use disorder (AUD) and to other substances like nicotine. We therefore studied 200 participants from the general population who were stratified by AUD and smoking status before completing a single-lever PIT task during functional imaging. PIT interference was measured as the difference in error rates between incongruent PIT trials, characterized by conflicts of Pavlovian and instrumental stimuli, and congruent trials. Associations with AUD and smoking status, as well as dimensional associations of behavioral PIT interference and neural responses to PIT conflicts with substance-related measures, were examined. As expected, participants with AUD showed greater PIT interference and reduced conflict-related activation in the lateral prefrontal, dorsomedial prefrontal, and anterior cingulate cortex than participants without AUD. The magnitude of these alterations also correlated with higher AUD severity and alcohol consumption, while being independent of smoking status and other smoking-related measures. These findings suggest that enhanced PIT interference generalizes to the broader AUD population but may not extend to other substances like nicotine. They further implicate a reduced recruitment of prefrontal control networks during PIT conflicts as a potential mechanism underlying enhanced PIT interference in AUD and its association with clinically relevant outcome measures.

Genetics shape cocaine behavior and abuse liability, but the underlying neurophysiological effects remain understudied. In male C57BL/6J, B6129SF2/J, and DO/J mice, we report conserved and divergent cocaine induced neuroplasticity in the accumbens shell following 5 days of cocaine and 10 to 14 days of abstinence.

Background: Deficits in inhibitory control are considered a central feature of alcohol use disorder (AUD), yet behavioral and neuroimaging findings have been inconsistent. One potential explanation is the marked heterogeneity of AUD, which encompasses multiple symptom dimensions that may differ in their underlying neurocognitive mechanisms. In this study, we investigated whether behavioral and neural markers of inhibitory control are associated with specific DSM-5 AUD symptom domains. Methods: A total of 589 participants (432 with non-treatment seeking AUD, 157 controls) completed a Stop-Signal Task (SST) during fMRI. Associations between DSM-5 symptom domains (impaired control over alcohol use, risky use, social problems, physical dependence) and SST performance, inhibition-related brain activation, and alcohol consumption were examined. Real-life drinking behavior was additionally assessed over 12 months using ecological momentary assessment (EMA). Results: Greater impaired control symptom severity was associated with poorer performance on go trials and diminished post-error slowing during SST. At the neural level, higher impaired control symptom severity was associated with reduced activation across fronto-insular, parietal, cingulate, supplementary motor, and cerebellar regions. In contrast, other symptom domains showed little to no associations with inhibition-related behavioral or neural measures. Higher impaired control symptom severity was associated with greater alcohol consumption across questionnaire-based and EMA-based assessments. Conclusions: In this large cohort, altered behavioral performance and recruitment of inhibition-related brain regions in the SST was selectively linked to impaired control symptoms. These findings highlight the importance of symptom-based approaches for understanding heterogeneity in addiction and may inform the development of more targeted interventions.

In the KARE trial, 96 adults with severe alcohol use disorder, abstinent following detoxification, were randomised in a 2x2 factorial design to 3 weekly infusions of ketamine (0.8 mg/kg) or saline, each combined with mindfulness-based psychological support (PS) or alcohol psychoeducation (PE). We re-expressed the trial data on endpoints acceptable to regulators and compared the most and least intensive arms. Across pooled weeks 17-24, ketamine plus PS gave 78.5% versus 57.8% days abstinent (difference, 20.7 percentage points; P = .032); adjusted for baseline, the difference at weeks 21-24 was 26.9 percentage points (P = .011). At weeks 21-24, 50.0% versus 26.1% had no heavy drinking days (P = .127). Relative reductions in heavy drinking days were 55% in KARE, against 25%, -17%, 46% and 35% reported for naltrexone, exenatide, semaglutide and pemvidutide. These post hoc differences, measured 4 to 6 months after a 4 week course ended, are of magnitude similar to agents requiring continuous administration.

Genome-wide association studies (GWAS) have identified numerous loci associated with substance use disorders (SUDs). To connect common genetic variation to cell type- and region-specific gene expression and chromatin accessibility, we examined the enrichment of genetic liability for several SUDs (alcohol, cannabis, tobacco, and opioid use disorders) and substance consumption phenotypes (drinks per week, cannabis ever-use, and cigarettes per day) in available single-nucleus RNAseq data spanning 10 brain regions and snATACseq data spanning 42 brain regions. Significant cell-type enrichment after multiple testing corrections was observed only for drinks per week and tobacco use disorder in the snRNAseq data and for cannabis use disorder and problematic alcohol use in snATACseq data. Across both transcriptomic and chromatin accessibility annotations, enrichment patterns were concentrated in excitatory neuronal populations, particularly upper layer intratelencephalic neurons, amygdala excitatory neurons, and regionally resolved striatal medium spiny neuron subtypes. Drinks per week, problematic alcohol use, and cannabis use disorder showed enrichment values that were significantly correlated within cell-type across snRNAseq and snATACseq, suggesting convergent biological signals across independent single-nucleus modalities. These findings implicate chromatin-mediated expression differences in specific excitatory neuronal populations as potential mediators of SUD genetic liability and highlight the value of integrating transcriptomic and chromatin accessibility data for characterising the neurobiology of addiction.

A single nucleotide polymorphism (rs2304297) in the 3' untranslated region (UTR) of CHRNA6, encoding the alpha6 nicotinic acetylcholine receptor subunit, is associated with adolescent smoking behavior. Humanized rats carrying the 'C' or 'G' allele show sex- and genotype-dependent responses to adolescent nicotine, yet the variant does not alter the protein sequence and the mechanism has remained unknown. We examined the molecular consequences of rs2304297 in vivo in the adolescent ventral tegmental area (VTA). Chrna6 transcript abundance did not differ by genotype, nor did transcript stability or polyadenylation site selection. The variant was instead associated with a transcriptional signature spanning myelin maintenance (Enpp6), lipid metabolism (Retsat), and genomic repair (Polq), and with elevated basal lipid peroxidation in alpha6GG animals. Computational folding predicted greater conformational flexibility of the 'G' allele, and spatial colocalization of Chrna6 with miRNA-365-5p was reduced in alpha6GG animals of both sexes. Coordinated expression of the two transcripts, however, followed a sex-by-genotype pattern, absent in alpha6CC females and alpha6GG males. These same groups showed elevated tyrosine hydroxylase expression and dopaminergic neuron number, and expansion of the Chrna6 co-expression network following nicotine. We propose that rs2304297 acts through two distinct layers: genotype determines spatial engagement of miRNA-365-5p with the transcript, while sex determines whether that engagement produces functional regulation. These findings identify a post-transcriptional mechanism for a non-coding nicotine addiction risk variant and demonstrate that its molecular effect can be uniform while its consequences are not.

Following cocaine self-administration, male and female rats received daily semaglutide during a 30-day abstinence period before cue- and cocaine-induced reinstatement tests. Semaglutide attenuated cue-induced reinstatement and cocaine-induced stereotypy, although operant responding was elevated following cocaine priming. These findings support further investigation of semaglutide as a treatment for cocaine use disorder.

Background Cocaine use disorder (CUD) is estimated to be 50% heritable, yet few genetic variants have been robustly associated with risk. During January 2021 to June 2024, cocaine was involved in 30% of U.S. overdose deaths. Identifying genetic contributors to CUD could inform diagnostics and treatments. The missing heritability may stem from small sample sizes, models that inadequately account for small-effect common variants, and the exclusion of large-effect structural variants like copy number changes. Epistasis likely contributes but remains challenging to detect genome-wide. Results We focused on epistasis and haplotypes rather than individual SNPs by applying the custom correlation coefficient in BlocBuster to construct allele networks from eQTL SNPs. Testing these regulatory haplotype networks for association with cocaine use in 7,826 individuals with opioid use disorder identified 12 significant associations across two ancestries. For one haplotype, raw genotyping array intensity data revealed a copy number increase at the CYP2D6 locus in individuals of European ancestry. In a subcohort of individuals who co-use cocaine and heroin (i.e., speedball), two haplotypes were also significantly associated with HIV infection. One of these involved GPR75, whose ligand, CCL5 (RANTES), is shared with CCR5, a coreceptor for HIV entry. Conclusions Our findings, integrated with long-read human pan-genomes, support a model in which non-functional copies of CYP2D6 disrupt dopaminergic, glutamatergic, and kynurenine pathways in the brain, contributing to cocaine use severity. The association of additional haplotypes with HIV status in speedball users highlights the genetic contribution to comorbid risk in high-exposure populations.

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.

4-hydroxytamoxifen (4-OHT) is commonly used to facilitate activity-dependent recombination in neurons. Here we show that one dose of 4-OHT devalues alcohol, contexts, and tastants, indicating that experimental designs using 4-OHT should control for its aversiveness. Further, reactivation of 4-OHT-responsive neurons causes conditioned taste avoidance. An activity screen coupled with graph theoretical analysis identifies candidate brain areas mediating these effects. We suggest these areas could be therapeutic targets for treating addiction.

Background and Purpose High novelty-seeking is an endophenotype associated with greater vulnerability to drug addiction, marked by a hyperdopaminergic state and reduced dopamine D2 receptor (D2R) levels in the dorsal striatum (DS). G protein-coupled receptor kinases (GRKs) regulate desensitization, internalization, and recycling of these receptors, including the D2R. This study investigated the relevance of GRK2 and 3 (GRK2/3) in novelty-seeking behavior and striatal dopamine dynamics. Experimental Approach Individual novelty-seeking trait was assessed in Sprague Dawley rats using an open-field test. D2R, GRK2, and GRK5 protein levels were measured in synaptosomal DS samples using Western Blot. Pharmacological inhibition of GRK2/3 was assessed using Cmpd101. We tested the effect of systemic Cmpd101 administration on contextual novelty discrimination and local Cmpd101 perfusion on baseline and stimulus-evoked extracellular dopamine (DA) levels in the DS. Key Results Locomotor activity in a novel environment inversely correlated with the synaptosomal GRK2 and D2R protein levels in the DS. Systemic Cmpd101 treatment modulated the locomotor discrimination index in a novelty phenotype-dependent manner. Moreover, local Cmpd101 perfusion in the DS elicited a sustained elevation in basal extracellular dopamine levels and significantly blunted subsequent stimulus-evoked DA release, both effects independent of novelty-seeking phenotype. Conclusion and Implications Together, our findings link GRK2/3 signaling to individual differences in novelty-seeking and striatal dopaminergic function. These results suggest a potential relevance to drug addiction vulnerability.

Objective To evaluate the association of early addiction consultation with withdrawal pharmacotherapy and complications in hospitalized patients with alcohol withdrawal. Methods Retrospective single-center cohort of adult hospitalizations with alcohol withdrawal (diagnosis code or CIWA-Ar [≥]10 within 24 hours) from July 2020 to January 2026. Exposure was an addiction consult service order within 24 hours of arrival, compared with no or later consultation. Primary outcomes were intensive care unit (ICU) transfer after the first 24 hours and delirium tremens or withdrawal seizure not present on admission. Process outcomes included phenobarbital and thiamine administration within 24 hours and benzodiazepine dose in the first 48 hours. Secondary outcomes included length of stay and 30-day death or return to hospital. Results Of 4,663 hospitalizations, 1,379 (29.6%) received early consultation. After adjustment, early consultation was associated with lower odds of ICU transfer (odds ratio [OR] 0.73; 95% CI 0.54-0.99) and fewer episodes of delirium tremens (risk difference -1.09 per 100; -1.45 to -0.73), with no significant difference in withdrawal seizure. Early consultation was associated with phenobarbital (OR 1.57; 1.32-1.86) and thiamine (OR 1.23; 1.07-1.41) within 24 hours, higher 48-hour lorazepam-equivalent dose (ratio of geometric means 1.44; 1.32-1.57), shorter stays (ratio 0.92; 0.88-0.97), and lower odds of 30-day death or return to hospital (OR 0.78; 0.66-0.91). Conclusions Early addiction consultation was associated with earlier, more intensive withdrawal pharmacotherapy, lower odds of ICU transfer, and fewer episodes of delirium tremens, although the complication associations were sensitive to cohort definition.

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.

Importance: Preventing substance use disorder (SUD) is a top public health priority, but clinical practice currently lacks validated prognostic tools to identify risk before the onset of heavy use/problems. Objective: To determine whether models can predict SUD using genetic, behavioral, and environmental factors and to evaluate performance across substance types (alcohol, cannabis, tobacco), sex, and genetic similarity groups. Design: This prognostic study developed and validated a multi-domain risk model using data from four longitudinal cohorts (The National Longitudinal Study of Adolescent to Adult Health, Avon Longitudinal Study of Parents and Children, The Collaborative Study on the Genetics of Alcoholism, and FinnTwin12) with follow-up from early adolescence into young adulthood. Setting: Population-based and family-based samples from the United States, United Kingdom, and Finland. Participants: The analysis included participants with available genomic and behavioral data. The cohorts represented a spectrum of risk, ranging from nationally representative community samples to high-risk populations. Exposure(s) (for observational studies): Predictors included polygenic scores for substance use-related phenotypes and 10 established early-life behavioral and environmental risk factors. Main Outcome(s) and Measure(s): The outcomes were a diagnosis of moderate-to-severe SUD based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria. Model performance was evaluated using the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and positive and negative predictive value. Results: A total of 16,362 participants were included in the study (54.7% female; mean [SD] age 26.73 [3.36]). Genetic similarity group composition was 70.4% European, 16.8% African, 9.8% admixed American, 2.2% East Asian, and 0.7% South Asian. SUD prevalence was 22.0%. The integrated model achieved an AUC of 0.78 for predicting any SUD, with a sensitivity of 69% and a positive predictive value of 45%. Performance was comparable for specific substances (AUC range 0.74-0.82) and across genetic similarity groups (AUC range 0.61-0.82). The model demonstrated higher AUC in females compared with males. Conclusions and Relevance: Findings highlight the potential utility of an SUD prediction model to inform clinicians and patients about risk for developing a SUD.

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.

Age of first alcohol use is associated with alterations in brain development and an increased risk of problematic substance use later in life. However, it remains unclear if neurobiological alterations associated with first-use age are present in young adulthood and how these alterations may be associated with different patterns of adult substance use. In this study, we employed SuperBigFLICA, a semi-supervised multimodal imaging data fusion approach, to identify joint structural morphometric and functional MRI (fMRI) brain profiles present in adulthood that are most predictive of age of alcohol first-use in the Human Connectome Project-Young Adult cohort (n=622). Our model achieved moderate performance (r=0.22 in external validation), identifying multimodal structure-function profiles predictive of first-use largely involving frontal regions. We then utilized Canonical Correlation Analysis to determine whether these multimodal brain profiles were differentially linked to patterns of adult substance use behaviors. We identified three significant brain modes (Mode 1: r = 0.2750, p = 0.014; Mode 2: r = 0.2249, p = 0.005; Mode 3: r = 0.1568, p = 0.029) associated with distinct profiles of alcohol and cannabis use. Together, we identified multimodal brain patterns in young adults that predict age of first use of alcohol and demonstrated that resulting multimodal structure-function brain profiles capture distinct multivariate patterns of adult substance use, providing a more integrated view of substance use neurobiology.

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.

Chronic heavy alcohol use shifts brain metabolism from glucose toward acetate utilization. Sudden decreases in acetate following alcohol cessation in individuals with alcohol use disorder (AUD) may create an energetic imbalance that contributes to craving and withdrawal. A recent randomized study found that, compared with a standard American (SA) diet, a ketogenic diet (KD) during alcohol detoxification reduced withdrawal severity and craving and increased ketones and glutamate in the dorsal anterior cingulate cortex. These findings suggest that ketones may support metabolic adaptation during alcohol withdrawal, but the effects of altered brain metabolism on neural dynamics and alcohol cue reactivity remain unknown. Here, we used a control theoretic approach to test whether shifts in cerebral energy substrate utilization influence large-scale network dynamics through altered information flow. We analyzed fMRI cue-reactivity scans collected after one, two, and three weeks of inpatient treatment during presentation of alcohol, food, and neutral cues. Whole-brain effective connectivity was estimated using Shannon transfer entropy applied to whole brain atlas-aligned fMRI time series. Assuming neural activity flow along effective connections, we identified distinct shifts in the state-transition landscape in participants receiving the KD compared with the SA diet. Increases in serum {beta}-hydroxybutyrate (BHB) were associated with changes in state-transition landscape organization, which were in turn associated with reduced cue-induced alcohol craving. These findings link metabolic adaptation to large-scale neural dynamics and clinically relevant behavior and suggest that cerebral energy substrate availability may shape behavior by altering the organization of large-scale neural dynamics.

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.

Approximately 30% of U.S. adolescents have consumed alcohol, and earlier onset is associated with faster escalation and greater risk of adult alcohol use disorder. Predicting not only who will initiate drinking but also when could enable more timely and targeted prevention. Existing machine-learning approaches have largely classified drinking status or predefined trajectory classes, requiring fully observed outcomes and often excluding adolescents who remain abstinent at last follow-up. Many also rely on neuroimaging or small, hand-selected feature sets, limiting scalability. We developed a stacked-encoder survival framework for predicting drinking-onset timing from baseline clinical data alone. The framework compresses 1,582 clinical features using an unsupervised denoising autoencoder and re-embeds them with FocalTab, a TabPFN encoder fine-tuned with focal loss; the encoders are then frozen for survival modeling using a neural Cox or random survival forest head. Among 661 NCANDA adolescents who were non-drinkers at baseline, 495 initiated drinking within six years. The stacked representation outperformed single-encoder and raw-feature models, achieving an Uno C-index of 0.7654 and time-dependent AUC of 0.8274. Focal-loss encoding outperformed cross-entropy encoding, while SHAP identified alcohol expectancies, substance access, and community prevention engagement among the most influential predictors. Unsupervised clustering further identified five biotypes with a monotonic gradient in mean time to onset (4.30 to 2.86 years) and differential limbic and thalamic neuroimaging phenotypes. These findings support the feasibility of scalable, clinically based prediction of drinking-onset timing and data-driven adolescent risk stratification.