Alcohol, Clinical and Experimental Research

Background: Compulsive alcohol use despite negative outcomes is a defining characteristic of alcohol use disorder. Rats exposed to long-term intermittent alcohol access (IAA) demonstrate sustained motivation for ethanol despite presence of the bitter additive quinine, offering a useful preclinical model of compulsive alcohol use. However, little is known about the role of habenular circuitry in the development of this phenotype. Here, we employed chemogenetic techniques targeting basal forebrain (BF) input to the lateral habenula (LHb) to probe the involvement of this neural circuitry in aversion-resistant alcohol consumption. Methods: Following long-term IAA or control conditions, male and female Long-Evans rats underwent surgery for the expression of designer receptors in BF-to-LHb projections. We then excited this pathway in rats with IAA history, or inhibited this pathway in rats with more limited ethanol history, before testing consumption of unadulterated and quinine-adulterated ethanol as well as unadulterated and quinine-adulterated sucrose. Results: Long-term IAA elevated ethanol drinking in all rats and aversion-resistant ethanol preference in males. Chemogenetic activation of BF-to-LHb neurons in rats with IAA history produced different effects in males and females: excitation enhanced ethanol intake in females, but reduced ethanol preference in males, regardless of quinine adulteration. Activation also led to a relative insensitivity to quinine-adulteration of sucrose when compared to controls, particularly in females. Chemogenetic inhibition in rats with limited prior ethanol exposure did not alter either ethanol or sucrose consumption with or without quinine. Conclusions: Our results suggest a differential role for BF-to-LHb circuitry in ethanol drinking based on sex, and a potential role for this circuitry in the sensitivity to quinine in the context of natural reward consumption.

Alcohol use disorder (AUD) is a widespread psychiatric condition, yet the molecular mechanisms underlying its development remain poorly understood. While prior studies have largely focused on protein-coding genes, long non-coding RNAs (lncRNAs) remain underexplored in AUD. Malat1, a highly abundant and evolutionarily conserved lncRNA, is elevated in post-mortem brain tissue of human AUD subjects and rodents chronically exposed to ethanol; however, its causal contribution to AUD-relevant behaviors remains unknown. Using CRISPR/Cas9 genome editing, we generated two complementary global Malat1 knockout models to assess its role in alcohol intake and related phenotypes. Constitutive knockout selectively attenuated acute functional tolerance rate and every-other-day two-bottle-choice alcohol intake in females. These results were supported by an inducible adult conditional global knockout model, which reduced ethanol consumption in females without altering taste preference. Together, our findings provide the first causal evidence that Malat1 regulates alcohol consumption in a sex-specific manner, supporting further investigation into its underlying mechanisms in AUD.

Alcohol drinking motives vary among individuals and shape experiences and beliefs about alcohol, influencing the processing of alcohol-related cues. In real-life settings, these cues are contextually rich, amplifying the role of such individualized drinking motives on cue processing. However, previous literature has primarily relied on images of alcohol, which lack contexts and differ significantly from real-life. Here, aiming to investigate real-life craving, we examined the role of alcohol drinking motives in craving in response to naturalistic alcohol-drinking videos. We asked fifty-three problematic alcohol users to speak about their reasons for drinking alcohol to capture unique alcohol drinking motives of each individual. Participants also underwent functional MRI while watching fifteen alcohol-drinking videos, and reported their subjective level of craving and self-relatedness for each video. Behavioral data analysis revealed that individuals with greater alcohol use severity tended to report greater cue-induced craving, but only when they reported that a video was related to themselves. Inter-subject representational similarity analysis showed that participants with similar alcohol drinking motives, reflected in shared drinking reasons and similar self-relatedness to the videos, exhibited synchronized craving-related neural responses during video-watching. Notably, these shared neural processes mediated the link between similar drinking motives and similar self-reported craving levels across participants. Together, our findings highlight the crucial role of alcohol drinking motives in shaping cue-induced alcohol craving, and provide deeper insights into craving in real-world contexts.

Alcohol demand is commonly assessed using hypothetical alcohol purchase tasks (APTs), from which individual demand curves are constructed and yield multiple indices of reinforcing value. Procedurally, APTs can confer participant burden, and existing brief alternatives cannot produce demand curves or derived indices. Thus, we evaluated a novel, adjusting APT that efficiently and idiographically assesses alcohol demand while preserving the benefits of a full task. Adults reporting past-six-month alcohol use (n=897) completed either the adjusting or full APT, the former utilizing a binary-search-style algorithm to administer six prices from the full APT's price set based on level of alcohol demand. The adjusting APT reduced item burden by 49% and produced well-fitting individual demand curves. Average demand intensity and elasticity estimates did not differ significantly by modality, whereas Omax and breakpoint estimates were significantly higher on the adjusting APT, though only by $3 each. All demand indices from both APTs were positively associated with alcohol use and problems, with similar magnitude by modality. Results provide support for the adjusting APT as a brief measure of alcohol demand that retains demand-curve-based indices of reinforcing value.

Alcohol use disorder demonstrates ~50% heritability, much of which remains unexplained by genetic sequence alone. Chronic alcohol exposure before conception changes offspring phenotypes through epigenetic mechanisms that are still being elucidated. Preconception ethanol exposure studies have focused on paternal exposure, neglecting maternal and biparental exposure. To address this, we exposed adult male and female mice to five cycles of chronic intermittent ethanol vapor interleaved with two bottle choice ethanol drinking and mated them to produce male and female F1 offspring with paternal, maternal, or biparental preconception ethanol exposure or controls. Whole blood and medial prefrontal cortex from adult, ethanol-naive offspring underwent RNA-sequencing. We also analyzed previously unpublished RNA-sequencing data from male and female preimplantation embryos derived from preconception ethanol-exposed sires. Here, we report transcriptomic patterns of preconception ethanol exposure that depend on the exposed parent, offspring sex, and tissue which suggest metabolic and immune dysfunction in offspring.

BackgroundAdolescence is characterized by profound neurodevelopmental changes that shape large-scale brain network organization and may confer vulnerability to risk-taking behaviors, including alcohol use. While cross-sectional and prospective studies have examined functional connectivity (FC) alterations before and after consumption, there is little evidence of how networks evolve during adolescence. MethodsThe present longitudinal study investigated electrophysiological FC trajectories during alcohol initiation using resting-state magnetoencephalography (MEG). 61 alcohol-naive adolescents (mean age at baseline = 14.4) were assessed and re-evaluated two years later (mean age = 16.4). ResultsAt baseline, stronger FC in theta (4-8 Hz), alpha (8-12 Hz), and high-beta (20-30 Hz) bands predicted greater alcohol consumption at follow-up, replicating previous findings. Longitudinal analyses with linear mixed-effects models revealed significant stage x SAUs interactions across all three frequency bands. Adolescents with low-to-moderate alcohol use showed normative increases in FC over time, consistent with typical neurodevelopmental maturation. In contrast, heavier drinkers exhibited stabilization or reduction of FC, suggesting a divergence from normative trajectories. Notably, theta-band hyperconnectivity persisted after alcohol initiation and remained positively associated with current alcohol consumption, particularly across anteroposterior connections. ConclusionThese findings indicate heterogeneous neurodevelopmental trajectories associated with alcohol use severity. Elevated pre-consumption connectivity, especially in the theta band, may reflect a vulnerability marker rather than solely a consequence of alcohol exposure. Overall, results highlight the importance of considering individual variability in brain maturation when examining adolescent substance use and suggest that early hyperconnectivity may signal increased risk for heavier alcohol involvement.

Adolescent use of alcohol, nicotine, and marijuana remains a major public health concern in the United States. Early identification of youth at elevated risk is critical for prevention before use begins or escalates. We developed and evaluated a longitudinal machine learning framework to predict alcohol, nicotine, and marijuana use at the next observed assessment wave. Data came from the Adolescent Brain Cognitive Development (ABCD) Study Release 6.0. The models incorporated predictors from multiple domains, including demographics, friends, family and community context, mental health, physical health, and prior substance-related behaviors. To reduce information leakage across individuals, we implemented a leakage-aware stacked ensemble. This ensemble combined diverse base learners through out-of-fold predictions and an elastic-net meta-learner. Across all three substances, the lagged stacked ensemble outperformed the cross-sectional stack and all single base learners. Adolescents identified as highest risk showed substantially higher observed rates of substance use than would be expected under random screening. Feature-importance analyses showed that the full longitudinal models were strongly influenced by developmental timing and prior-use history. Analyses restricted to current-wave features revealed distinct substance-specific risk patterns beyond prior-use history and developmental timing. Bootstrap stability analyses identified top-ranked features showing consistent positive predictive relevance across resampled adolescents. These findings suggest that longitudinal, leakage-aware machine learning can generate substance-specific risk estimates to support targeted prevention and screening in adolescent populations.

Background and aimsAlcohol-associated hepatitis (AH) is characterized by excessive inflammation and blunted antiviral interferon (IFN) responses. We hypothesized that specific gut microbiome-derived metabolites could selectively enhance interferon signaling while limiting NF-{kappa}B mediated inflammation, thereby restoring immune balance in AH. Our goal is to identify microbiome-derived metabolites that differentially regulate the NF-{kappa}B and IFN signaling pathways. Methods and resultsWe used human monocytic THP1-Dual cells, which secrete reporters for NF-{kappa}B and IFN signaling, to model innate immune responses and screened a library of 152 gut microbiome-derived metabolites. From the metabolite screen, 4-hydroxyphenylacetic acid (4-HPAA) emerged as a unique immunomodulator: in LPS-challenged cells, 4-HPAA selectively increased IFN signaling with minimal NF-{kappa}B activation. 4-HPAA was evaluated in vivo using a NIAAA-model, with 4-HPAA supplementation (0.4mg/ml) added to the diet. In the NIAAA-model, dietary 4-HPAA did not induce liver injury and was associated with enhanced interferon-stimulated gene expression. Simultaneously, 4-HPAA reduced pro-inflammatory markers such as Il1{beta}, Ly6g and F4/80 compared to the group exposed to ethanol alone. Metabolomic profiling of mouse cecal contents revealed 4-HPAA supplementation counteracted ethanols metabolic effects, selectively reducing triglyceride-associated lipids that had accumulated with ethanol feeding. Conclusions4-HPAA enhances interferon signaling and antiviral gene induction while dampening NF-{kappa}B-driven inflammation in the presence of LPS, both in vitro and in vivo. In an acute-on-chronic alcohol injury model, 4-HPAA attenuated hepatic inflammation, reduced immune cell recruitment, and activated antioxidant defenses, reflecting a shift toward a more hepatoprotective effect. 4-HPAA treatment was associated with reduced pro-inflammatory markers and modest attenuation of ethanol-induced liver injury. Additionally, 4-HPAA reversed ethanol-induced lipid-dysregulation, particularly triglyceride accumulation, highlighting its metabolic benefit in alcohol-fed mice. In conclusion, 4-HPAA rebalances immune and metabolic pathways by enhancing IFN signaling, suppressing NF-{kappa}B inflammation, and reversing alcohol-induced hepatic injury and lipid accumulation.

Background: Individuals with attention-deficit hyperactivity disorder (ADHD) are at increased risk for cannabis misuse, with increasing prevalence among young adults. Existing evidence suggests that cannabis can have therapeutic effects on ADHD symptoms, and continued use may be partly driven by perceived improvements in symptom-related deficits. To investigate the neural evidence for these associations, we integrated functional neuroimaging and Allen Human Brain Atlas transcriptomic data to assess neural correlates of ADHD in regions targeted by cannabinoids as predictors of cannabis use. We hypothesized that greater ADHD symptoms would lead to higher cannabis use frequency through associations of ADHD symptoms with functional deficits in cannabinoid receptor type 1 (CB1R; encoded by the CNR1 gene) expressing brain regions. Methods: We tested 466 college students (ages 18-19) with varying ADHD symptom severity and cannabis use, self-reported at baseline and three yearly-follow up questionnaires. ADHD-related neural deficits were tested in a subset of 144 participants using an fMRI stop-signal task at baseline. Growth mixture modelling categorized participants with similar cannabis use into three latent classes. The covariance between the CNR1 gene expression map and differences in stop-signal task activation were tested as a mediator linking ADHD symptoms and cannabis use. Results: Greater ADHD symptoms significantly predicted reduced activation within CNR1-expressing regions, which predicted higher-use cannabis class membership. Conclusions: Our results add support for the self-medication hypothesis for higher rates of cannabis use among individuals with greater ADHD symptoms, which may be mechanistically linked through CB1R-enriched attention and inhibitory networks, highlighting neural targets for prevention and treatment.

Purpose: {beta}-hydroxybutyrate (BHB), a ketone body and alternative cerebral energy substrate, can be measured in vivo using J-difference edited proton magnetic resonance spectroscopy (1H-MRS). Oral ketone supplementation with substrates such as the ketone monoester (R)-3-hydroxybutyl-(R)-3-hydroxybutyrate (KME) and 1,3-butanediol (BD) have gained attention as a mechanism to elevate circulating BHB and induce ketosis without dietary restrictions. Elevated brain ketone availability is of growing therapeutic interest as a strategy to support neuronal energetics in conditions such as epilepsy, neurodegenerative disease, and alcohol use disorder (AUD). However, both pathways introduce BD into the bloodstream, which crosses the blood-brain barrier. Critically, BD exhibits a spectral signature that closely resembles the prominent BHB peak in JDE-MR spectroscopic imaging (MRSI), identified in a pilot AUD study. Methods: Two separate JDE-MRSI acquisitions tailored for BHB and BD editing were implemented, exploiting frequency separation between the BHB (4.14ppm) and BD (3.95ppm) coupling partners of the observed 1.2ppm resonance to independently quantify each metabolite. Results: Brain BD concentrations (0.25-0.58mM) were comparable to or exceeded corresponding BHB concentrations (0.20-0.27mM) in all volunteers after consumption of a single dose of the KME, indicating that BD constitutes a major fraction of the signal conventionally attributed to BHB. Combined BHB+BD concentrations (~0.45-0.85mM) were consistent with brain BHB values reported in prior studies employing similar doses of the KME, indicating that those measurements likely reflect a combined BHB+BD signal. Conclusions: Separate quantification of the two metabolites is important for interpreting brain ketone studies and for understanding the full pharmacology of KME supplementation.

20% of Americans are at risk for environmental circadian rhythm disruptions (CRD) due to shift work, leading to substantial negative health outcomes. However, females are especially affected with greater vulnerability for substance use (SU) and adverse outcomes associated with pregnancy, including for offspring at birth and later in life. In mice, prenatal CRD (pCRD) recapitulates these risks, but it is unknown whether pCRD affects SU in mature offspring. To investigate this, C57BL/6J dams were disrupted by reversing the light/dark cycle during gestation. Following pCRD, reward- (cocaine conditioned place preference, intravenous self-administration) and mood-related behaviors (open field, elevated plus maze, light/dark box, forced swim) were measured in adult offspring. Adult female offspring of dams exposed to CRD developed an anhedonic-like phenotype with decreased food self-administration, cocaine intake and reinforcing properties of cocaine. Opposingly, pCRD male offspring showed a SU-like phenotype with increased cocaine preference, higher order food self-administration and cocaine reinforcement. Interestingly, these divergent behavioral outcomes were not specific to reward. While female pCRD mice showed increased anxiety-like behavior, pCRD males showed decreased anxiety/increased risk-taking behavior, as well as decreased immobility in the forced swim test. Rhythms in corticosterone were also sex-specifically affected by pCRD. These results suggest that pCRD may predispose individuals to distinct psychiatric disorders based on sex with mood disorders developing in females and SU disorders developing in males. By better understanding how disrupted rhythms during pregnancy affect behavior in adulthood, we can develop novel therapeutic approaches for SU and mood disorders in adults.

Importance Prenatal alcohol exposure (PAE) is associated with lasting cognitive and neurodevelopmental deficits and can quadruple risk for depression later in life. However, it remains unknown whether there are specific trimesters when PAE is more strongly associated with longitudinal trajectories of internalizing symptoms - an indicator of depression risk - across childhood and adolescence. Objective To investigate how PAE timing and dosage are associated with internalizing symptom trajectories from ages 4 to 16.5 years. Design, Setting and Participants We analyzed prospective data from the Avon Longitudinal Study of Parents and Children (ALSPAC), an ongoing longitudinal birth cohort from the United Kingdom. Internalizing symptom trajectories were estimated for 6,409 participants. Primary analyses were conducted on 2,254 participants with complete data on PAE in all three trimesters, covariates, and trajectories. Main Outcomes and Measures We used growth mixture modelling to identify latent trajectories of depressive symptoms measured using the internalizing symptom scale from the Strengths and Difficulties Questionnaire (SDQ) at seven occasions between ages 4 to 16.5 years. Prospective alcohol consumption during each trimester were categorized into three PAE dosages: unexposed (0 drinks/week), low (1-7 drinks/week) and high (7+ drinks/week). Results We identified five distinct depressive symptom trajectories: stable low (75.9% of participants), moderate childhood peak (11.2%), progressive increase (5.57%), high early childhood (4.73%), and early adolescent peak (2.61%). PAE in the second (relative risk [RR]=2.08, 95% CI=1.15-3.76) and third trimesters (RR=1.83, 95% CI=1.05-3.21), as well as total PAE burden across pregnancy (RR=1.33, 95% CI=1.06-1.68) increased risk for the progressive increase trajectory, versus the stable low trajectory. High PAE in the second (RR=2.71, 95% CI=1.41-5.21) and third (RR=2.27, 95% CI=1.27-4.05) trimesters drove elevated risk for this trajectory. PAE in the first trimester or at low dosages showed no associations with depressive symptom trajectories. Negative control analyses of paternal drinking also found no associations. Conclusions and Relevance Our results highlight the second and third trimesters as potential sensitive periods for the impact of PAE on rising depressive symptoms from childhood to adolescence. Ultimately, these findings could inform the design of prevention programs, and facilitate targeted interventions to youth at elevated risk for depression.

Cocaine use disorder (CUD) remains a major public health concern with no FDA-approved pharmacotherapy, underscoring the need to define the cellular and molecular adaptations produced by voluntary cocaine taking. The nucleus accumbens (NAc) is a key substrate for cocaine reinforcement and drug-seeking behavior, but interpretation of the functional role of its cellular heterogeneity in these behaviors is limited by past bulk transcriptomic studies. Here, we used single-nucleus RNA sequencing to profile the NAc of male and female rats that self-administered intravenous cocaine for 10 consecutive days versus yoked saline controls. After quality control, we analyzed 36,766 nuclei spanning major neuronal, glial, and vascular cell populations. Pseudobulk differential-expression analyses identified 478 cocaine-associated cell type-specific transcriptional changes that were concentrated in discrete medium spiny neuron (MSN) subclasses and astrocytes. D1 Ebf1+ MSNs showed the largest transcriptomic response, accounting for [~]40% of all differential-expression events, followed by D2 Stk32a+ MSNs, astrocytes, and D1 Ppm1e+ MSNs. These responses were largely cell type-specific, indicating that cocaine self-administration engages multiple molecular programs rather than a uniform accumbens-wide transcriptional signature. Immediate-early gene module-score analyses further revealed cocaine-associated activation states in select neuronal and non-neuronal cell populations, including D1 Ebf1+ MSNs, Drd3+ neurons, Sst+ interneurons, astrocytes, and oligodendrocytes. Gene-set, pathway, and upstream-regulator analyses nominated synaptic organization, axon guidance, RAS/MAPK signaling, NMDA receptor-associated signaling, and CREB-related transcriptional regulation as candidate mechanisms of cocaine-evoked plasticity. Together, these data provide a cell type-resolved resource for understanding how voluntary cocaine taking alters the rat NAc transcriptome and identifies discrete neuronal and glial cell populations for future mechanistic studies using preclinical CUD models.

miRNA-based transcriptomic analysis of extracellular vesicles (EVs) provide a promising strategy for identifying non-invasive biomarkers and understanding complex pathological mechanisms. Recently, however, urinary extracellular vesicles (uEVs) have emerged as a valuable window into molecular alterations. Despite the high morbidity and mortality associated with alcohol use disorder (AUD), the molecular mechanisms underlying its sex-specific differences remain poorly understood. To address this, we characterize for the first time the uEV miRNome in AUD, revealing its sexually dimorphic profile. We employed uEVs from actively drinking AUD patients of both sexes who did not have advanced liver disease, alongside matched controls. Deep sequencing revealed 14 differentially expressed miRNAs in females (e.g., hsa-miR-197-3p, hsa-miR-19b-3p, hsa-miR-505-3p, hsa-miR-625-5p, and hsa-miR-27a-5p) and 6 in males (e.g., hsa-miR-1290, hsa-miR-1246, hsa-miR-450a-5p, and miR-590-5p). Notably, whereas hsa-miR-4787-5p was consistently overexpressed in uEVs from both sexes, it was absent in plasma-derived EVs, highlighting the specificity of the urinary compartment. Remarkably, the miRNA signatures we uncovered reflect the multiorgan impact of AUD. For instance, hsa-miR-1290 and hsa-miR-197-3p point to alcohol-related liver injury and systemic inflammation, whereas hsa-miR-19b-3p and hsa-miR-1246 signal neuroinflammation and neuronal stress. A subset, including hsa-miR-1290, hsa-miR-1246, and hsa-miR-27a-5p, has been implicated in cancer contexts. Collectively, these findings support the uEV miRNome as a promising sex-informed molecular signature of AUD with biomarker and mechanistic relevance.

Objective: To develop and validate clinically relevant polygenic scores (PGS) for alcohol (AUD), cannabis (CanUD), opioid (OUD), tobacco (TUD), and polysubstance use disorders (polySUD) across African (AA), European (EA), and Latinx (LA) ancestry populations. Methods: Using multiple genome-wide association study summary statistics and PGS methods, substance use disorder PGS were developed and evaluated in Indiana Biobank samples (IB, N: 1,356-24,989), then top-performing PGS were validated in All of Us Research Program samples (AOU, N: 62,389-209,952). Case and controls were defined using ICD-9/10 codes. All participants were aged 18 years or older (>=21 years for AUD controls). Clinical relevance was defined as an odds ratio (OR) >=2 for individuals with the highest PGS determined based on disorder prevalence compared to everyone else. Results: In EA and LA, all PGS achieved clinically relevant performance in both IB and AOU (ORs: 2.00-9.10; P <= 3.87E-4). In AA, PGS met this threshold in IB (ORs: 2.02-2.71; P <= 2.20E-4) but not in AOU (ORs: 1.28-1.56; P <=0.03). Overall, OUD PGS showed the strongest associations in most analyses, followed by CanUD and polySUD. Generally, compared to female PGS, male PGS had higher or comparable ORs, but the differences were not significant except AUD PGS in AOU LA. Conclusions: PGS demonstrated clinically meaningful risk prediction for substance use disorders in EA and LA, supporting the feasibility of future clinical implementation for population-level screening. However, reduced performance in AA underscores the urgent need for more genetic studies in that population.

Background: Dopamine (DA) is a neurotransmitter critically involved in food-related reinforcement learning. While mesolimbic DA reward-associated signaling in the nucleus accumbens has been widely investigated, far less is known about DA function in the hippocampus (HPC), a brain region traditionally known for its role in episodic and spatial memory processes that has recently been associated with appetite and food intake control. Methods: Here we investigated dorsal HPC DA signaling dynamics in rats using fiber photometry to detect changes in DA binding (via GRAB-DA sensors) before, during, and after a meal consumption in food-restricted rats. Pharmacological studies targeting HPC dopamine 2 receptors (D2R) assessed the functional role of HPC DA signaling in food intake and meal-related memory processes. Results: HPC DA binding was significantly elevated in the post-meal relative to the pre-meal state following standard chow consumption. This effect was replicated after consuming a high fat diet or liquid sucrose, but not a low-calorie sweetener. These post-meal DA signaling elevations are dependent on nutrient consumption, as HPC DA binding levels were unaffected by intraperitoneal administration of glucose or the satiation hormone, cholecystokinin, in otherwise fasted rats. Direct HPC D2R agonists administration reduced food intake, whereas HPC D2R blockade after a meal reduced the latency to the next meal and impaired spatial memory for meal location without affecting spatial memory for object location. Conclusions: Collective results identify HPC DA-D2R signaling as a candidate neurobiological mechanism through which nutrient consumption promotes meal-related episodic memory formation, and by extension, reduces subsequent food intake.

The ongoing opioid epidemic underscores the need for scalable and translational preclinical models of voluntary opioid intake and dependence. We therefore sought to establish and validate a voluntary two-bottle choice drinking-in-the-dark (DID) model of oral opioid intake in mice and to determine relationships between experimental parameters and behaviors during and after withdrawal. Male and female C57BL/6J mice were given daily access to two bottles during the dark phase for 24 drinking sessions over 5 weeks. Control mice received two bottles containing water. Experimental mice received one water bottle and one bottle containing oxycodone (0.1-1 mg/mL) or fentanyl (10-100 {micro}g/mL) under varying session durations and concentrations. On the final day, physical dependence was assessed using naloxone-precipitated withdrawal and then a behavioral battery to assess negative affect was performed in the following week. Mice voluntarily consumed both oxycodone and fentanyl without taste adulteration and maintained drug preference across most concentrations. Oxycodone intake produced minimal withdrawal symptoms. In contrast, fentanyl intake resulted in naloxone-precipitated withdrawal that was modulated by session duration and concentration. Four-hour sessions produced stronger withdrawal than two-hour sessions at equivalent concentrations. Escalating high-concentration fentanyl exposure revealed emerging sex differences, with females exhibiting greater intake and withdrawal at higher concentrations. Affective behavioral assays following withdrawal revealed minimal persistent alterations in any cohort. These findings establish key parameters for a scalable voluntary fentanyl model that produces dose- and session-dependent physical dependence in male and female mice. This paradigm provides a cost-effective and straightforward platform for future investigations of opioid use and dependence.

RationaleAppetitive Pavlovian cues can drive maladaptive reward seeking via stimulus-reward memories. Disrupting memory reconsolidation offers a potential strategy to reduce their influence, but evidence for {beta}-adrenergic blockade with propranolol is inconsistent across behavioural paradigms, particularly relative to NMDA receptor antagonism. ObjectivesWe tested whether propranolol disrupts reconsolidation of appetitive sucrose memories in a discriminative goal-tracking paradigm, and compared its effects with those of the most commonly used NMDA receptor antagonist, MK-801. MethodsAdult Lister hooded rats underwent discriminative Pavlovian conditioning. Thirty minutes before a brief memory reminder (non-reinforced or reinforced), rats received systemic drug treatment or saline control. In study 1, MK-801 (0.1 mg/kg) was administered to male rats. In study 2, propranolol (10 mg/kg) was administered to equal numbers of male and female rats. Goal-tracking was tested drug-free at 1 and 8 days. ResultsIn study 1, MK-801 impaired subsequent discriminated responding at test. These effects were observed not only when reminder was non-reinforced as in previous successful demonstrations, but also with reinforced reminder. In study 2, Propranolol also impaired subsequent goal-tracking, regardless of reminder type, and the effects were consistent across sexes. ConclusionsPropranolol can disrupt reconsolidation of appetitive goal-tracking memories to a similar extent as MK-801 under conditions that promote memory destabilisation. These findings demonstrate that {beta}-adrenergic blockade can impair appetitive memory reconsolidation in a goal-tracking paradigm, challenging prior null findings and revitalising the potential for propranolol-based interventions in maladaptive reward-seeking behaviours.

Prenatal alcohol exposure (PAE) is a major cause of neurodevelopmental disorders, yet most children are diagnosed late or misdiagnosed. Neuroplacentology suggest that placental factors released into maternal and/or umbilical cord blood contribute to fetal brain development. Consistently, a preclinical inter-organ transcriptomic database revealed that PAE disrupts the expression ratio of angiogenic and inflammatory factors suggesting an angio-inflammatory response. This study aimed i) to assay, by multiplex immunoassay, angiogenic and inflammatory factors in maternal and umbilical cord blood from alcohol-consuming women and ii) to perform a maternofetal analysis according to neonatal sex. Afterwards, dysregulated factors from mothers who gave birth to females or males were submitted to STRING and ShinyGO analyses. Results showed that PAE differently altered the distribution profiles of dysregulated angiogenic and inflammatory factors in maternal and umbilical cord blood. Moreover, sex-specific differences were observed, with 36% of dysregulated proteins specific to males, 48% to females, and 16% common to both. STRING analysis revealed robust functional protein-protein interactions linking together inflammatory and angiogenic clusters while the ShinyGO analysis identified enriched pathways related to vascular shear stress. These findings provide the first maternofetal analysis of combined angiogenic and inflammatory factors from alcohol-consuming mothers.

The locus coeruleus noradrenaline (LC-NA) system is a key regulator of arousal, attention, and reward learning. Noradrenaline plays a critical role in impulse control, and recent evidence indicates the importance of noradrenaline signaling in cost-benefit decision making once choice strategies are established. However, whether the LC causally shapes the acquisition of decision strategies, and how this contribution may differ across sexes, remains unclear. We addressed these questions by chemogenetically inhibiting catecholaminergic neurons within the LC of adult tyrosine-hydroxylase Cre (TH::Cre) rats (n=69; 35 females) throughout acquisition of the cued rat gambling task (crGT), a probabilistic decision making paradigm that incorporates salient audiovisual reward-paired cues and simultaneously measures motor impulsivity. LC inhibition accelerated the development of risky choice strategies early in training in both males and females, reflected by impaired adoption of the most advantageous option and increased preference for risky options. Trial-by-trial analyses reveal that LC inhibition promoted switches in choice strategy following safe wins, while reducing switches away from risky options after both wins and losses. LC inhibition therefore seemed to encourage the repetition of actions that resulted in more uncertain outcomes. LC inhibition also selectively enhanced motor impulsivity in females, particularly early in training. These results provide causal evidence that the LC system guides the formation of optimal decisional strategies, while exerting sex-specific control over impulsive action.