Addiction Neuroscience

Alcohol use disorder (AUD) is marked by substantial heterogeneity in drinking behaviors and health outcomes, underscoring the need for preclinical models that capture interindividual variability. We recently developed open-source capacitive lickometer systems for high-resolution monitoring of mouse fluid intake. Using LIQ PARTI and LIQ HD, we found substantial individual differences in alcohol intake that varied across sex and housing status in C57Bl6/J mice. Here, we conducted a secondary analysis of this continuous access ethanol drinking data to quantify behavioral variability in group and singly housed mice. We introduce a fluid "meal" pattern analysis that integrates drinking across ethanol and water sippers to define discrete drinking episodes. Using this approach, we observed sex- and housing-dependent reorganization of drinking structure across group and single-housed settings, with group-housed male mice exhibiting fewer but faster liquid meals. To further characterize multidimensional drinking patterns, we applied principal component analysis to meal variables and identified a "distributed meal" phenotype defined by increased meal number, reduced meal size, earlier onset of drinking, and higher ethanol preference. Considering factors that influence behaviors in a social environment, we next examined whether social hierarchy was associated with these patterns using a tube test dominance assay. Social rank was unrelated to ethanol and meal measures; however, offensive dominance behavior positively correlated with principal component scores in males. Together, these findings demonstrate that high-resolution, longitudinal analysis of ethanol drinking reveals distinct behavioral phenotypes that are associated with key components of social behaviors, providing a potential framework for understanding heterogeneity in AUD-related drinking. HighlightsO_LILIQ PARTI and HD enable high-resolution analysis of ethanol drinking patterns. C_LIO_LIFluid meal analysis captures sex- and housing-dependent drinking structures. C_LIO_LIBehavioral phenotyping reveals individual differences beyond total ethanol intake. C_LIO_LIPCA identifies a meal phenotype associated with male offensive dominance behavior. C_LI

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.

BackgroundAlcohol use disorder is characterized by continued alcohol use despite negative consequences, also known as aversion-resistant drinking. Alcohol related cues can invigorate motivation to seek and consume alcohol. It is currently unknown whether alcohol related cues can invigorate drinking despite negative consequences. Materials and MethodsLong-Evans rats were trained in a discriminative stimulus (DS) task with cues predicting the available of alcohol reward. They were then tested in the task for aversion-resistant drinking by measuring consumption of alcohol adulterated with increasing concentrations of quinine. As a control for an environment free of reward-related cues, rats were also tested for aversion-resistant drinking in the home cage. ResultsWe found that rats displayed robust aversion-resistant drinking in the DS task. When we compared alcohol consumption in the task with home cage consumption, we found that rats were more aversion-resistant in the task than in the home cage. We also found that individual differences in aversion-resistant drinking were correlated within behavioral context (i.e. home cage or DS task) but not between the home cage and the DS task. ConclusionsWe found that aversion-resistant drinking is invigorated during cue-induced alcohol seeking relative to free drinking without explicit cues. Home cage quinine-sensitivity is unrelated to quinine-sensitivity in the presence of cues. This suggests that cues motivate drinking despite negative consequences in a way that is unique from aversion-resistance driven by drinking history. While many behavioral measures use cues and ultimately test aversion-resistant drinking, this is the first explicit test of cue-evoked aversion-resistant drinking.

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: Alcohol-involved drinking episodes vary in whether they involve hazardous alcohol consumption alone, near-miss sexual risk, or sexual risk behavior, but the within-event mechanisms underlying this variability remain unclear. Methods: Guided by syndemic theory, we conducted a qualitative event-level analysis using modified grounded theory among adults in the San Francisco Bay Area who reported hazardous alcohol consumption, defined as an Alcohol Use Disorder Identification Test score [≥]16. In-depth interviews elicited narratives of recent heavy drinking episodes and yielded 64 discrete drinking events across 22 participants. We focused on 35 events with evidence of within-event interaction between biopsychosocial and contextual factors. Using constant comparison, we identified escalation pathways, characterized interruption, and examined how events diverge into three outcomes: hazardous alcohol consumption only, hazardous alcohol consumption with near-miss sexual risk (when risk was plausible but not enacted), and hazardous alcohol consumption with sexual risk behavior. Results: Two primary escalation pathways emerged. Dose-driven escalation involved cumulative alcohol or substance exposure that progressively impaired awareness and self-regulation. Meaning-driven escalation involved prioritizing connection, intimacy, or belonging despite awareness of risk. Time-driven continuation extended exposure across contexts and amplified both pathways. Hazardous alcohol consumption-only events more often followed dose-driven pathways, whereas events involving sexual risk behavior more often followed meaning-driven pathways. Near-miss events occurred across both pathways and illustrated how interruption before the escalation constraint point, when the capacity to modify behavior became reduced, could redirect escalation before sexual risk behavior occurred. Across events with similar levels of intoxication narratives, outcomes diverged according to when the interruption occurred and whether it altered escalation. Conclusion: Hazardous drinking episodes diverge into different outcomes based on escalation pathways and the timing and effectiveness of interruption. Early and effective interruption before the escalation constraint point may represent a key target for harm-reduction strategies to prevent progression to sexual risk behavior.

We previously reported, using in-vivo fiber photometry, that operant responding reinforced by access to a peer (social self-administration) is associated with phasic dopamine increases in nucleus accumbens (NAc) core following lever insertion (reward-availability cue) and gradual increases preceding lever-pressing. Here, we sought to replicate these findings and determine whether dopamine signals (1) generalize to responding for high-carbohydrate palatable food, (2) show opposite patterns during negative reinforcement (shock avoidance/escape), and (3) depend on whether reinforcers are experienced alone or together. We trained rats (n=11; 6 females) to lever-press for access to a same-sex peer (15 s/trial) and palatable food (45-mg pellet/trial), followed by shock avoidance/escape (0.18-0.26 mA). After training, we expressed the dopamine sensor GRAB-DA2m and implanted optic fibers into NAc core. We measured dopamine activity during sessions with either one- or three-reinforcers. During social self-administration, dopamine activity showed phasic increases following lever insertion and gradual increases preceding lever-pressing; responses were moderately greater during sessions with all three reinforcers. Palatable food self-administration showed a similar pattern, but responses were approximately twofold greater during single-reinforcer sessions. During shock avoidance/escape, dopamine activity showed phasic decreases at warning onset, lever insertion, and shock onset; responses were also greater during single-reinforcer sessions. Results suggest that NAc core dopamine signaling distinguishes positive from negative reinforcement and is modulated by reinforcer availability. Compared with single-reinforcer sessions, dopamine responses during food self-administration and shock avoidance/escape were reduced during sessions with all three reinforcers, whereas responses during social self-administration modestly increased. Significance statementNucleus accumbens (NAc) dopamine is critical for processing the valence of positive reinforcers, whereas its role in negative reinforcement is less well understood. Here, we extended our prior work on operant social self-administration to determine whether these findings generalize to food reinforcement, show the opposite pattern during operant negative reinforcement (shock avoidance/escape), and depend on whether reinforcers are experienced alone or together. Results suggest that NAc core dopamine activity differentiates positive and negative reinforcement and is modulated by the presence of other available reinforcers. Specifically, dopamine responses during food self-administration and shock avoidance/escape were reduced when all three reinforcers were available together compared with when each reinforcer was experienced alone, whereas responses during social self-administration showed the opposite pattern.

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.

Quantifying decision-making in experimental settings that mimic real-world conditions may provide insights into mechanisms underlying addiction. This study developed a computational model of opioid-seeking behavior. Out-of-treatment persons who regularly used heroin were stabilized on buprenorphine 8mg/day to minimize opioid withdrawal. Across programmatically-linked studies, three experimental conditions presented differing money vs. opioid unit amounts that could be earned per trial ($2 vs. 1-mg hydromorphone, n=23; $2 vs. 2-mg hydromorphone, n=36; $4 vs. 2-mg hydromorphone, n=24), controlling other factors. Progressive ratio schedules on each choice option required increasing effort across trials to earn the same amount. Trial-level outcomes were decision latency and choice on each option, and session-level outcomes were drug-money latency and breakpoint difference scores. A Markov computational model was used to predict the probability of choosing the same option as the previous trial (vs. switching). Model inputs included effort discrepancy (between earning the same vs. other commodity on next choice) and logarithm of the ratio of decisional speed (current vs. previous choice). Participants who more rapidly chose hydromorphone vs. money made more consecutive drug choices and expended greater effort earning hydromorphone. First-trial hydromorphone choice predicted continued effortful opioid-seeking. Participants repeated choices on 80% of trials; the model accurately predicted stick vs. switch behavior on 93% of trials. Participants typically repeated choices when faced with lower effort discrepancies and higher hydromorphone dose (2-mg vs. 1-mg). In conclusion, a Markov computational model accurately predicted effortful behavior in a choice paradigm that mimics real-world decisions between opioid and nondrug reinforcers.

BackgroundSex differences in psychostimulant-related behaviors are often attributed to biological sex; however, individual variability may also strongly influence behavioral outcomes. The new MISSING (Mapping Intrinsic Sex Similarities as an Integral quality of Normalized Groups) model identifies mixed-sex behavioral groups in which differences are driven primarily by individual variability rather than sex. The goal of this study was to validate the MISSING model for psychostimulant/sucrose self-administration. MethodsLong Evans rats self-administered methamphetamine (METH, male n = 25, female n = 32, 0.1 mg/kg/infusion, FR1, 6h per day for 20 days), sucrose (male n = 20, female n = 22, one-20 mg pellet/delivery, all other conditions being equal) and saline (male n = 3, female n = 10, other things being equal). We developed a new Quantitative Structure of Curve Analytical (QSCAn) model (using exponential-plateau and linear fit) for the assessment of individual drug self-administration time curve profiles irrespective of biological sex. We analyzed our data using regression analysis and ANOVA. ResultsQSCAn identified three distinct self-administration profiles (consisting of both sexes), which we named exponential-plateau negative (EP-), exponential-plateau positive (EP+), and undefined (EP0). There were no differences in self-administration profiles when we compared males and females within the same group. Differences between sexes (when observed) were due to mismatched comparisons (males from one group versus females from a different group). ConclusionsOur study reinforces the MISSING model for psychostimulant and sucrose self-administration by indicating that differences between males and females (when observed) may not necessarily be driven by biological sex. Significance StatementCurrent approaches often interpret variability in psychostimulant self-administration between males and females primarily through the lens of biological sex. However, this framework may overlook meaningful behavioral phenotypes shared across sexes. The present quantitative model suggests that individual patterns of behavior may better account for variability than sex alone, particularly in behaviors not strongly driven by sex-hormone-dependent mechanisms such as drug self-administration. By classifying animals according to behavioral profiles rather than biological sex, this approach may foster the identification of clinically and biologically relevant phenotypes underlying psychostimulant reinforcement.

RationaleAlcohol use disorder is defined by drinking alcohol despite knowledge of negative consequences, often referred to as aversion-resistant drinking (ARD). The dorsomedial (DMS) and dorsolateral striatum (DLS) are necessary for goal-directed and habitual action selection, respectively. Leading hypotheses posit that once drug use becomes compulsive, DMS dependence degrades while DLS dependence increases. This shift may be mediated by changes in synaptic weights from glutamatergic inputs. ObjectivesUsing a combination of western-blot, micro-injections, and ex-vivo electrophysiology, we investigated the role of -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors AMPAR, which drive glutamatergic transmission, during quinine-adulterated alcohol (QuA) drinking in the DMS and DLS across the development of ARD. ResultsWe found that AMPAR subunit composition and function change in the DMS across the development of ARD whereby, calcium permeable (CP) - AMPARs drive behavior. Western blots revealed a negative relationship between DMS GluA1 and QuA drinking in aversion-sensitive mice and positive relationships between DMS or DLS GluA1/A2 ratios and QuA drinking in ARD mice. DMS CP-AMPAR antagonism caused an increase in QuA drinking suggesting that CP-AMPARs in the DMS prevent ARD. Ex-vivo electrophysiology of DMS spiny projection neurons (SPNs) revealed that ARD mice had a greater rectification index than aversion-sensitive mice indicating that SPNs in the DMS express more CP-AMPARs following the development of ARD. ConclusionsThese data provide evidence that repeated alcohol binges alter DMS CP-AMPAR activity, where initial DMS activity acts to prevent ARD but after repeated binges that result in ARD, DMS SPNs recruit CP-AMPARs.

Alcohol craving and consumption fluctuate across the reproductive cycle in females with alcohol use disorder (AUD), suggesting that estrogen signaling contributes to disease vulnerability. Here, we investigated the role of estrogen receptor alpha (ER; Esr1; ESR1) in alcohol seeking using complementary mouse and human approaches, as this receptor was previously identified as a risk factor for AUD. Female mice were characterized in an IntelliCage-based multidimensional AUD paradigm that stratifies individuals into AUD-prone and AUD-resistant phenotypes. Transcriptomic profiling of the amygdala revealed that Esr1 is a top transcription factor for differentially expressed genes in mice drinking alcohol, and the estrogen signaling pathway was deregulated specifically in AUD-prone mice. Although alcohol exposure did not alter overall Esr1/ER mRNA or protein abundance, both transcript and protein levels positively correlated with cue-induced alcohol seeking, indicating that inter-individual variation in ER signaling predicts relapse-like behavior. Causal manipulations confirmed a functional role of ER. Local knockdown of Esr1 in the basolateral amygdala reduced excitatory synaptic transmission, attenuated alcohol motivation, cue-induced seeking, and relapse drinking, and impaired cue-associated memory recall without affecting anxiety-like behavior. Similarly, ovariectomy decreased amygdala ER expression, altered synaptic protein markers, and reduced alcohol-seeking behaviors, supporting regulation by endogenous ovarian hormones. Extending these findings to humans, ESR1 gene polymorphisms (rs6902771, rs11155819 and rs6557171) were associated with the probability of alcohol binge drinking and alcohol consumption days as well as craving and loss of control in real world in a longitudinal clinical cohort, while ESR1 mRNA blood levels were increased in women with AUD diagnosis. Together, these convergent molecular, circuit, behavioral, and genetic data identify ER signaling in the amygdala as an important modulator of alcohol-seeking behavior induced by alcohol cue and relapse vulnerability, highlighting estrogen pathways as potential therapeutic targets and markers for AUD.

Dysregulation of the dynorphin (DYN) / kappa-opioid receptor (KOR) system is heavily implicated in symptoms of alcohol use disorder (AUD) including negative affective-like states that can drive maladaptive behavioral regulation. Substantial efforts have been made towards understanding the neurobiology of DYN / KOR dysregulation; however, the role of dynorphinergic islands of Calleja within the ventral striatum remain poorly understood. Presently, adult male Wistar rats were trained to self-administer 10% alcohol, exposed to either air or alcohol vapor for eight weeks, and alcohol self-administration and 22-kHz ultrasonic vocalizations (USVs) assessed during acute withdrawal. Subsequently, brains were extracted during acute withdrawal and DYN A-like immunoreactivity was measured in the ventral striatum. Alcohol vapor-exposed rats demonstrated increased alcohol consumption and 22-kHz USVs compared to air-exposed controls. Vapor-exposed rats additionally demonstrated increased DYN A-like immunoreactivity in the islands of Calleja. Moreover, the average DYN A neuron size positively correlated with the number of 22-kHz USVs in vapor exposed animals, but not in air-exposed controls. The present findings identify the islands of Calleja as a novel DYN-associated region that may be recruited during alcohol dependence with enhanced DYN plasticity in the islands of Calleja contributing to affective dysregulation in AUD and other neuropsychiatric conditions.

Food addiction (FA) has been proposed as a phenotype sharing features with substance use disorders. Despite increasing recognition of food addiction as a behavioral phenotype with features overlapping substance use disorders, little is known about its prevalence or clinical significance among individuals with alcohol use disorder (AUD). Objective: To examine the prevalence of FA and to evaluate demographic, psychological, and alcohol-related correlates in individuals with AUD. Design, Setting, and Participants: This cross-sectional analysis included 743 adults with AUD who were either treatment seeking (Tx) (n = 534) for AUD and were enrolled in an inpatient program at the National Institutes of Health Clinical Center or not treatment-seeking (non Tx) (n = 209). Main Outcomes and Measures: FA symptoms were assessed using the Yale Food Addiction Scale, with >=2 symptoms categorized as FA in this report. Multivariable logistic regression models adjusted for age, education, and income were conducted separately within each cohort. Results: Among 743 adults with AUD, 238 (32.1%) met criteria for FA symptoms, with similar prevalence among Tx (32.6%) and nonTx (30.6%) participants despite marked differences in clinical characteristics. Across both cohorts, FA was independently associated with higher body mass index, greater psychological distress, and greater alcohol dependence severity. Childhood trauma and poorer sleep quality were additionally associated with FA among treatment-seeking participants, whereas alcohol-related measures differed according to treatment status. Conclusions and Relevance: FA was common among adults with AUD and was associated with greater psychological, behavioral, and metabolic burden regardless of treatment-seeking status. These findings suggest that FA identifies a clinically meaningful subgroup of individuals with AUD who may benefit from more comprehensive assessment and integrated treatment approaches.

Cannabis use disorder (CUD) is a chronic relapsing disorder characterized by compulsive drug seeking, persistent drug use despite adverse consequences, and a high risk of relapse. Although the nucleus accumbens (NAc) is a central hub in the neural circuitry underlying addiction, the specific glutamatergic inputs regulating vulnerability to cannabinoid addiction remain poorly understood. Here, we investigated the contribution of two major limbic glutamatergic projections to the NAc, the dorsal hippocampus (dHPC) to NAc and basolateral amygdala (BLA) to NAc pathways, using a validated mouse model of WIN55,212-2 intravenous self-administration combined with projection-specific chemogenetic inhibition. Male C57BL/6J mice received combinatorial viral vector delivery of inhibitory hM4Di DREADDs selectively targeting either the dHPC to NAc or the BLA to NAc pathway. Chronic pathway inhibition was achieved by continuous administration of deschloroclozapine through osmotic minipumps during the development of cannabinoid addiction-like behavior. Animals were evaluated using a multidimensional behavioral paradigm assessing the three-core addiction-like criteria of persistence of drug seeking, motivation, and compulsive-like behavior, as well as craving-related behaviors and phenotypic vulnerability traits. Chronic inhibition of either the dHPC to NAc or the BLA to NAc pathway significantly increased the proportion of mice developing an addiction-like phenotype. Both manipulations enhanced persistence of drug seeking during periods of drug unavailability, identifying persistence as a shared behavioral consequence of disrupting glutamatergic signaling to the NAc. In contrast, the two pathways differentially regulated other addiction-related behaviors. Inhibition of the dHPC to NAc pathway increased motivation to obtain WIN55,212-2, impulsivity, reward sensitivity, and resistance to extinction, whereas inhibition of the BLA to NAc pathway selectively enhanced cue-induced drug seeking. Neither manipulation altered compulsive-like responding, locomotor activity, or body weight. These findings demonstrate that distinct glutamatergic afferents to the NAc differentially regulate vulnerability to cannabinoid addiction-like behavior while converging on persistence as a common circuit-level mechanism. Our results establish the NAc as an integrative hub coordinating complementary contextual and emotional information during the transition to cannabinoid addiction and provide a circuit-based framework for understanding the neural mechanisms underlying Cannabis Use Disorder.

Cocaine use disorder remains a critical public health concern with limited treatment options. Astrocytes are increasingly recognized as active regulators of neurotransmission and are emerging as important contributors to addiction neurobiology. Here, we examined how chemogenetic activation of astrocytic Gq signaling within the ventral tegmental area (VTA) influences cocaine-associated behaviors in drug-naive and cocaine-experienced rats. Using a subthreshold dose of cocaine in a conditioned place preference (CPP) paradigm, we found that VTA astrocyte Gq activation facilitated the acquisition of cocaine CPP in drug-naive rats but suppressed the development of cocaine CPP in cocaine-experienced rats. Additionally, chemogenetic activation of VTA astrocyte Gq signaling suppressed voluntary cocaine intake in a self-administration paradigm. Together, these findings demonstrate that VTA astrocyte Gq signaling shapes cocaine-associated behaviors in a drug history-dependent manner, highlighting VTA astrocytes as a potential therapeutic target in cocaine use disorder.

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.

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

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

Neuroimmune signaling is increasingly implicated in alcohol use disorder (AUD). Microglia, the brains resident immune cells, signal in part through the adaptor protein myeloid differentiation primary response 88 (MyD88), a key mediator of innate immune responses. Here, we investigated whether microglial-specific MyD88 signaling regulates voluntary alcohol consumption in adulthood, as whole-body loss of MyD88 was previously shown to increase drinking. We further determined if alcohol altered parvalbumin-expressing interneurons (PVIs) and microglia within the pre-frontal cortex, based on our previously described role for MyD88 signaling on perineuronal net (PNN) deposition on PVIs in several brain regions, and the well characterized role of inhibitory signaling in alcohol use disorders. Loss of microglial-MyD88 had minimal effects on voluntary alcohol intake and anxiety-like behaviors. Alcohol exposure did not modify observed MyD88-dependent changes in PVIs/PNNs, despite altering microglial morphology in the male prefrontal cortex independent of genotype. The addition of an early life endotoxin challenge was sufficient to induce an increase in adult alcohol consumption in both MyD88-deficient and control males. However, injection of saline alone also induced an increase in adult drinking in MyD88-deficient males. These findings suggest that microglial-MyD88 signaling does not strongly regulate alcohol intake under baseline conditions in a one-bottle, voluntary binge-drinking paradigm, however there may be a role for microglial-MyD88 signaling in modulating the impact of developmental environmental contexts, such as stress, in later-life male drinking behavior. This work highlights the importance of developmental context, such as stress or inflammatory history, in understanding underlying microglia signaling mechanisms in conferring AUD risk.

Alcohol misuse leads to a range of health complications and induces various metabolic perturbations that impacts multiple physiological systems, including the cardiovascular system, liver, and gut microbiota. However, limited research has been reported on these metabolic profile changes, particularly using models of alcohol dependence such as after chronic intermittent ethanol (CIE) vapor exposure. This study investigated CIE-induced metabolomic alterations of CIE were investigated using fecal, liver, and serum samples of adult male and female C57BL/6J mice following 72 hr withdrawal. Significant metabolite changes were observed in both fecal and liver extracts and these changes were sex-specific. Both liver and fecal metabolites had systematic changes, while blood serum influences were limited after CIE. Female fecal samples showed higher metabolite perturbations than male samples according to PCA studies. The female samples showed significant butyrate downregulation and acetate upregulation, which are critical microbial products as beneficial microbe cell energy sources and influence intestinal absorption in the host. In addition, the female fecal samples showed significant downregulation of branched-chain amino acids including leucine, isoleucine, and valine, while male samples showed downregulation of glucose and taurine, with upregulated phenylalanine and tyrosine. In contrast, in the liver study, phenylalanine and tyrosine were upregulated while taurine was downregulated in females. Both sexes showed downregulation of liver glycine and glucose. These data indicate that CIE induces sex-specific metabolic perturbations in the mouse liver and fecal metabolome, and have implications for guy disturbances and liver damage observed following alcohol dependence. This study provides potential targets for future examination of mechanisms and treatment approaches for alcohol dependence.