Addiction Biology

Background: Substance use disorders (SUDs), including alcohol and drug dependence, and smoking, pose a public health threat with their high prevalence and comorbidity with other diseases, and contribution to mortality. SUDs are highly correlated, and their genetic background is shared to some degree. Objectives: We aimed to investigate the genetic associations of previously reported loci for a wide range of SUDs in an unstudied Ukrainian population. Methods: We collected data from 595 individuals (339 women, 253 men), including 321 participants from two rehab centres. Based on clinical review and questionnaire data we defined drug dependence, alcohol dependence, alcohol abuse, binge drinking, smoking, opiate, amphetamine, cannabis, and hallucinogen use, along with several intermediary alcohol use and smoking variables considering the amount of use and the level of dependence. We genotyped COMT-rs4680, ADH1B-ADH1C-rs1789891, and HTR2A-rs6313, and applied logistic and ordered logistic regression assuming an additive inheritance model, controlling for the recruitment group, other substance uses, age, and sex, in the association analyses. Results: We replicate (P<0.05) the associations at COMT-rs4680 with smoking status (OR[95% CI]=1.56[1.01-2.41], P=0.047) and heaviness (1.37[1.04-1.80], P=0.026), and at ADH1B-ADH1C-rs1789891 and HTR2A-rs6313 with alcohol dependence (1.69[1.03-2.76], P=0.038 and 0.66[0.47-0.92, P=0.016], respectively). Furthermore, we provide evidence for an association at HTR2A-rs6313 with hallucinogen use (0.58[0.35-0.98], P=0.040). Conclusion: In this study on multiple SUDs we shed light on the genetic background of SUDs in Ukrainians and provide further evidence that variation at COMT is mainly associated with smoking, at ADH1B-ADH1C with alcohol-related variables, whereas HTR2A is a more general SUD-associated locus.

Regular cannabis use has been associated with alterations in reward-related neural processes, yet findings remain inconsistent and the relationship between neural activity and behavioural performance is not fully understood. The present study aimed to characterise neural and behavioural correlates of reward processing in regular cannabis users (CU) compared with matched non-users (NU) using the Monetary Incentive Delay Task (MIDT). Firstly, we assessed behavioural performance through reaction times, accuracy and monetary earnings to determine whether potential neural alterations were reflected in task performance. Secondly, focusing on reward-related brain regions, we examined group differences in BOLD functional MRI activity during anticipation and outcome phases separately for monetary win and loss conditions. Finally, we explored the association between behavioural performance and neural activation. Our findings indicate that regular cannabis use is associated with altered engagement of key nodes within the mesocorticolimbic circuit during both anticipatory and outcome phases of reward processing, accompanied by impaired behavioural performance. Particularly, compared with NU, CU showed (I) lower striatal activity during anticipation of monetary win and higher ventral striatum and frontal pole activity during anticipation of monetary loss; (II) greater VTA activation during outcome of successful monetary win and loss avoidance and lower frontal pole activity during outcome of unsuccessful loss avoidance; (III) impaired behavioural performance, reflected in lower monetary rewards and a trend towards slower reaction times and reduced accuracy; (IV) disrupted brain-behaviour coupling. Results from this study may help inform future research on the neurobiological mechanisms underlying changes in reward function and the resultant behavioural consequences of cannabis use.

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

BackgroundIndividual vulnerability to addiction is driven by neuroadaptations within dopaminergic circuits. G protein-coupled receptor kinases (GRKs), specifically GRK2 and GRK3 regulate D2 receptor (D2R) signaling and trafficking, but their role in amphetamine (AMPH)-induced locomotor sensitization remains unclear. This study aimed to determine whether GRK2/3 inhibition alters locomotor sensitization and its associated molecular correlates across striatal regions. MethodsAdult rats (n = 39) were assigned to saline, acute AMPH, or repeated AMPH groups and received intraperitoneal administration of vehicle or the GRK2/3 inhibitor Cmpd101 (1.0 mg/kg intraperitoneally, i.p.). Locomotor activity was assessed under basal and injection conditions to evaluate sensitization. Protein levels of D2R, GRK2, and GRK5 were quantified across striatal regions using Western blot and analyzed with linear mixed models. ResultsRepeated AMPH exposure induced locomotor sensitization in a subset of animals, but Cmpd101 did not significantly modify locomotor activity or sensitization expression. At the molecular level, Cmpd101 had no effect on D2R levels and produced selective, region-dependent changes in GRK2 and GRK5. Notably, GRK2/3 inhibition altered the relationship between protein expression and locomotor sensitization in a region-specific manner, rather than inducing consistent changes in absolute protein levels. ConclusionsGRK2/3 inhibition by Cmpd101 produces region-specific molecular effects and reshapes protein-behavior relationships without significantly altering locomotor sensitization. These findings support a model in which GRKs act as context-dependent modulators of dopaminergic signaling rather than direct drivers of behavioral output.

Craving is a hallmark feature of substance use disorders (SUDs) and a major risk factor for relapse, yet reliable biomarkers that enable individual-level prediction remain scarce. Here, we applied connectome-based predictive modeling (CPM) to resting-state functional magnetic resonance imaging (fMRI) data in a transdiagnostic sample of individuals with cannabis, opioid, or tobacco use disorder (n = 78). Using CPM, we identified a distributed functional brain network that reliably predicted self-reported craving. Computational lesion analyses revealed key contributions from the right medial orbitofrontal cortex, right dorsal posterior cingulate cortex, and left lateral medial frontal gyrus. Importantly, the craving network generalized across two independent datasets. In alcohol-dependent patients (n = 41), the identified craving network, along with its positive and negative subnetworks, predicted distinct cognitive and motivational components of craving. In a second external dataset of smokers (n = 28), the craving network predicted both nicotine craving after abstinence as well as intra-individual changes in craving between sated and craving states. Together, these findings provide evidence for a robust, transdiagnostic craving signature in SUDs. Future work should assess the networks predictive utility for longitudinal outcomes such as relapse risk and treatment response.

Substance Use Disorders (SUDs) constitute a major and rising public health concern. In addition, there is a growing appreciation that different classes of addictive substances are likely to lead to qualitatively different types of SUDs requiring differing treatment and relapse prevention strategies to be most effectively managed. Biological temperament, particularly on the internalizing - externalizing axis, is well established to influence addiction susceptibility. Externalizing behavior has long been understood to predispose individuals to addiction through novelty-seeking, sensation-seeking and impulsivity, while internalizing behavior provides an alternate pathway into addiction via increased occurrence of comorbid disorders (anxiety, depression). Here, we utilize a selectively bred rat model of internalizing vs externalizing temperament (bred High Responders, representing genetically mediated externalizing behavior and bred Low Responders, representing internalizing behavior) to examine differences in the acquisition of self-administration of the prototypical psychostimulant cocaine and the prototypical opioid heroin (diacetylmorphine). We found that, as predicted, cocaine and heroin drove different patterns of acquisition in the two different bred lines of rats. Further, this was influenced by temperament in complex ways. Notably, in females the "telescoping effect" for opioid addiction-like behavior was primarily specific to externalizing temperament. These findings highlight the impact and interaction of many factors, including drug class, temperament, and sex, on the acquisition of drug-taking behavior. Additionally, these findings indicate that sex differences in addiction vulnerability may be influenced in part by biological temperament.

BackgroundPersistent automatic approach tendencies toward alcohol cues that resist goal-directed control are a key feature of harmful alcohol use, yet the causal neural mechanisms underlying this imbalance remain poorly understood. Converging evidence implicates the frontoparietal network (FPN) in actively regulating alcohol approach-avoidance behavior, but whether its constituent nodes make dissociable causal contributions has not been established. MethodsIn a within-subject, active-sham counterbalanced design, inhibitory continuous theta burst stimulation (cTBS) was applied to right dorsolateral prefrontal cortex (rDLPFC) and right posterior parietal cortex (rPPC) in separate groups of non-clinical alcohol users (rDLPFC: n = 29; rPPC: n = 28), followed by an Alcohol Approach-Avoidance Task. ResultsActive rDLPFC cTBS selectively slowed down alcohol push responses, whereas rPPC suppression produced a bidirectional action-specific shift in response to alcohol cues, where pull responses accelerated, and push slowed simultaneously. Suppression of either node shifted automatic tendencies toward greater alcohol approach through mechanistically distinct routes. ConclusionThese dissociable profiles indicate that rDLPFC is causally necessary for effortful top-down avoidance control, while rPPC supports the priority-based selection of alcohol cue-driven actions. These findings provide the first node-specific causal evidence for functional specialization within the FPN in the context of automatic tendencies towards alcohol. Alcohol avoidance emerges as an active, prefrontal-dependent process, whereas priority-based regulation emerges as a parietal-dependent process, together indicating rDLPFC and rPPC as mechanistically independent targets for intervention in maladaptive alcohol approach behavior.

In rats, cue-induced opioid craving intensifies (incubates) during abstinence from opioid self-administration and then remains high for a prolonged period. The prolonged plateau models persistent vulnerability to cue-induced craving and relapse in humans recovering from opioid use disorder. However, a very significant contributor to relapse vulnerability in these individuals is the presence of negative affective states that can persist for months to years, far beyond physical dependence. The goal of this study was to determine if the incubation of craving model recapitulates this aspect of relapse vulnerability. We began by comparing rats trained to self-administer oxycodone using a regimen leading to persistent elevation of cue-induced craving (6 h/d x 10 d) and rats trained to self-administer saline. We assessed somatic withdrawal signs in early abstinence and conducted behavioral tests modeling negative affect (open field, social preference, sucrose preference, and elevated plus maze) in late abstinence. Some somatic withdrawal signs were greater in oxycodone rats on abstinence day (AD)1, but cumulative scores did not differ between groups on AD1-3. On AD41-46, no group differences were found in behavioral tests modeling negative affect. To compare early and late abstinenceperiods, a second cohort of rats self-administered saline and oxycodoneand then received two cue-induced seeking tests (AD1 and AD40; oxycodone rats exhibited incubation of craving) and two series of negative affect tests (AD2-7 and AD41-48). While some time-dependent changes in affect were observed within each group, they were suggestive of reduced anxiety-like behavior in oxycodone rats. Finally, because rats are single-housed during our incubation studies, we compared drug-naive rats after 8-9 weeks of single vs pair housing and found no difference in behavioral tests modeling negative affect. We conclude that the persistence of elevated cue-induced craving observed after a standard opioid incubation regimen is not accompanied by negative affective states, probably due to lower drug intake during the intravenous regimen compared to non-contingent escalating dose regimens typically used to study withdrawal signs. This does not negate the utility of the incubation model for studying cue-induced opioid craving and its neurobiological basis.

Substance use disorders run in families, yet the mechanisms underlying intergenerational transmission remain unclear. We investigated indirect genetic effects, pathways through which parental genotypes influence offspring phenotypes via the family environment, for alcohol use disorder (AUD), nicotine dependence (ND), and related quantitative outcomes, and aimed to identify family environmental factors through which such effects may operate. Using transmitted and non-transmitted polygenic scores (PGS) constructed for problematic alcohol use, tobacco use disorder, and general addiction liability, we analyzed 5972 European-ancestry adult offspring with at least one genotyped parent from the population-based Lifelines cohort (Netherlands). Offspring outcomes included lifetime DSM-5 AUD diagnosis, AUD symptom count, maximum drinks in 24 hours, Fagerstrom Test for Nicotine Dependence score, and cigarettes per day. AUD findings were meta-analyzed with data from the Brisbane Longitudinal Twin Study (N = 1368; Australia). We also examined parent-of-origin effects and mediation by parental substance use and socioeconomic status using structural equation modeling. Transmitted PGS robustly predicted all AUD and ND outcomes ({beta} = 0.07-0.16; OR = 1.20 for AUD diagnosis). Non-transmitted PGS, indexing indirect genetic effects, were negligible for all clinical syndrome outcomes. The only significant indirect genetic effect was on cigarettes per day ({beta} = 0.03, p = 0.01), mediated by parental smoking behavior but not socioeconomic status. These findings indicate that intergenerational transmission of risk for AUD and ND is driven primarily by direct genetic effects, with modest indirect genetic effects on smoking quantity. Larger samples and cross-trait analyses are needed to further elucidate these mechanisms.

The misuse of opioid medications is a significant health issue in the United States. Very few studies have investigated the effect of opioids on perineuronal nets (PNNs), scaffold-like structures that surround neurons and are involved in the regulation of plasticity-dependent mechanisms such as development, learning and memory, and acquisition of addiction-like phenotypes. Regulation of PNNs in the orbitofrontal cortex (OFC) during periods of drug intoxication or withdrawal is widely unknown. In this study, male Wistar rats were injected with fentanyl (0.125 mg/kg, s.c.) or 0.9% saline twice daily for 7 days and once on day 8 (7continuous days following by 3 days of abstinence) or twice daily for 15 days (5 continuous days followed by 2 days of abstinence for more than 3 weeks) and twice on day 16. Antinociception was evaluated using the tail immersion test immediately before and 30 minutes after injections. Whole-brain coronal slices were collected, and immunohistochemistry was used to identify Wisteria Floribunda Agglutinin (WFA)-positive PNNs and parvalbumin (PV)-expressing cells. Results confirmed that repeated fentanyl injections induced tolerance to the antinociceptive effects, which normalized following acute abstinence periods. WFA intensity decreased following 8 days of injections. Analyses confirmed significant correlations between PV+ density and tail withdrawal latency following 8 days of fentanyl injections. These data confirm that repeated fentanyl injections modulate both WFA+ and PV+ expression in the rodent brain and antinociceptive tolerance in a duration-dependent manner. Overall, these data suggest that perineuronal nets may mediate opioid-induced behavioral effects, such as antinociceptive tolerance, following repeated administration and abstinence in rats.

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

Nicotine use disorder shows heterogeneity in treatment response, potentially reflecting differences in underlying neural circuitry, particularly in the presence of depression. We examined real-time neural dynamics during nicotine inhalation in two chronic users - one with depression and one without - using simultaneous hippocampal recordings from responsive neurostimulation (RNS) electrodes and scalp EEG. Oscillatory activity and hippocampal-cortical connectivity were analyzed in relation to mood and craving. Oscillatory activity tracked mood in the non-depressed individual but was attenuated or reversed in the depressed individual, suggesting reduced reward-related neural responsiveness. In contrast, both participants showed reduced alpha hippocampal-cortical connectivity following nicotine use, suggesting a shift from reward-seeking to reward and relief processing. These findings support a network-based framework of nicotine-driven neural dynamics and provide preliminary evidence that depressive status may modulate these processes. Although limited to two cases, this work highlights the potential for identifying neurophysiological subtypes of nicotine users and informs future efforts toward personalized treatment approaches.

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

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

IntroductionCannabis is increasingly used for pain management, with many patients reporting relief from chronic pain that did not respond to conventional treatments. However, cannabis is also associated with unwanted side effects including psychomimetic effects and the potential of developing a cannabis use disorder. To circumvent the central nervous system effects, we investigated whether a peripherally restricted cannabinoid receptor (CB1) agonist, PrNMI [(4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3yl]ethyl}morpholine] attenuated pain hypersensitivity associated with nerve injury and profiled its abuse potential. Materials and MethodsMice with chronic constriction injury (CCI) of the sciatic nerve developed hypersensitivity to mechanical stimulation. Paw withdrawal thresholds were assessed following administration of PrNMI (i.p. 0.3 mg/kg and 0.6 mg/kg) or vehicle in CCI and sham mice. The conditioned place preference model was used to measure drug-reward to 0.6 mg/kg i.p. PrNMI in CCI and sham-injury control animals. We further assessed abuse potential to determine if PrNMI (0.5 mg/kg) would reinstate drug-seeking behavior in mice trained to self-administer intravenous fentanyl (10 g/kg/infusion). ResultsPrNMI administration transiently increased paw withdrawal thresholds in mice with CCI-induced allodynia in a dose-dependent manner. PrNMI conditioning did not produce a conditioned place preference in mice with either CCI or sham injury. Mice who had learned to self-administer fentanyl and went through extinction training did not reinstate drug-seeking behavior when administered PrNMI. DiscussionThe systemic CB1 receptor agonist PrNMI demonstrated analgesic benefit in alleviating mechanical allodynia associated with chronic constriction injury of the sciatic nerve without increasing addiction related behaviors associated with the establishment of addiction.

Opioid and methamphetamine use disorders (OUD and MUD) are characterized by enduring neural adaptations within brain reward circuitry, yet the cell-type-specific post-transcriptional mechanisms underlying these changes remain poorly understood. While microglia are essential for maintaining central nervous system homeostasis and modulating neuroinflammatory responses to drugs of abuse, their alternative splicing (AS) programs have not been defined in the context of addiction. This study characterized the microglial AS landscape in the mouse dorsal striatum during morphine and methamphetamine intravenous self-administration (IVSA), as well as following a 21-day period of abstinence. Analysis of RNA-sequencing data using rMATS and DEXSeq revealed that both drugs significantly dysregulate core splicing machinery, with skipped exons (SE) emerging as the most prevalent splicing event. Notably, morphine exposure induced a robust persistent splicing signature, comprising 736 exonic regions in 221 genes that remained altered through abstinence, whereas methamphetamine-induced changes were primarily reversible. Functional annotation predicted that approximately 27.5% of these events induce frameshifts, potentially impacting critical microglial pathways such as autophagy (Wdr81), chromatin remodeling (Chd4, Kmt2c), and RNA processing (Hnrnpl, Mbnl2, Tia1). These findings identify previously unrecognized post-transcriptional neuroimmune mechanisms and suggest that persistent splicing dysregulation in microglia may contribute to the long-term pathophysiology of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/716002v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1c30f01org.highwire.dtl.DTLVardef@10e16d7org.highwire.dtl.DTLVardef@1fd80dforg.highwire.dtl.DTLVardef@17c80f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

BackgroundExternalizing liability is a strong risk factor for early substance initiation, but the neurobiological pathways linking polygenic risk to initiation remain incompletely characterized. MethodsUsing the ABCD Study, we implemented a four-stage framework linking an externalizing polygenic risk score (extPRS) to baseline multimodal neuroimaging-derived phenotypes (IDPs) and longitudinal substance initiation (alcohol [primary], nicotine, cannabis, and any substance). First, we screened extPRS-IDP associations using covariate-adjusted linear models (age, sex, ancestry principal components, site/scanner variables; modality-specific covariates where applicable) and controlled multiple testing using false discovery rate (FDR) procedures. Second, we estimated direct extPRS associations with time-to-initiation using Cox proportional hazards models. Third, we fit joint Cox models including extPRS and each discovery-significant IDP, retaining outcome-IDP associations after within-outcome FDR correction. Fourth, we conducted mediation analyses for prioritized outcome-IDP pairs using an extPRS [-&gt;] IDP mediator model and an initiation model including both extPRS and IDP, estimating indirect (ACME) and direct (ADE) effects via bootstrap with multiple-testing control. ResultsAmong 10,608 participants, higher extPRS was associated with earlier initiation across outcomes, with the largest effects observed for nicotine and cannabis and a modest but significant effect for alcohol. Stage 1 identified thousands of extPRS-associated IDPs that were highly concordant across robustness specifications. Stage 3 prioritized outcome-specific IDPs associated with initiation beyond extPRS, with the number of retained IDPs varying across sensitivity settings (site-clustered vs. HC3 standard errors; SES covariates on/off) but showing a replicated core set across models. In Stage 4, mediation analyses showed that indirect effects of extPRS through IDPs were small in magnitude (ACME {approx} 10-4) and accounted for less than 2% of the total effect, while direct effects (ADE {approx} 0.02-0.05) remained strong across outcomes. FDR-significant mediation signals were observed only for alcohol and any-substance initiation, whereas no mediation effects survived multiple testing correction for cannabis or nicotine. Across outcomes, direct genetic effects were substantially larger than mediated effects, indicating that genetic liability operates primarily through direct pathways rather than through baseline brain measures. ConclusionsExternalizing polygenic liability is broadly associated with substance initiation, with robust and consistent direct effects across substances. Although specific frontal structural and microstructural phenotypes show statistically significant mediation signals, their contribution is small, suggesting that baseline brain measures explain only a limited proportion of genetic risk. This framework provides a scalable approach to prioritize neurobiological pathways linking genetic liability to early substance initiation while highlighting the dominant role of direct genetic effects.

"Dry Hitting" is a unique phenomenon of e-cigarette use that has been shown to produce toxic chemical degradants and byproducts. Although it is widely understood that nicotine exposure during adolescence impacts neurobiological and behavioral function, little is known about how dry hitting may impact users. We hypothesized that subjects repeatedly exposed to nicotine dry hit vapor would exhibit distinct behavioral responses compared with saturated nicotine vapor and would differentially alter the expression of perineuronal nets (PNNs) in the rodent brain. Using a customized system of e-cigarette vapor inhalation, adolescent male Wistar rats (PND 31-40) received vaporized nicotine (30 or 60 mg/mL; [~]2.5-3 mL/cage), nicotine with dry hits (60 mg/mL; 1.75-2 mL/cage), or propylene glycol (PG) vehicle for 30 minutes over 7 daily sessions. Locomotor activity, antinociception, and elevated plus maze testing were used to assess behavioral response to drug intoxication and tolerance. Immunohistochemistry was used to identify Wisteria Floribunda Agglutinin (WFA)-positive PNN structures in the amygdala and insular cortex. Rats exposed to dry hits exhibited behavioral responses (locomotor sensitization, antinociception) similar to those of rats exposed to saturated nicotine vapor, but spent more time in the open arms of the elevated plus maze. Immunohistochemical analyses confirmed significantly greater WFA intensity in the central nucleus of the amygdala, but not the basolateral amygdala or insular cortex, of rats exposed to dry hits. Overall, these data confirm the impact of dry hit vapor on behavioral responses and perineuronal net expression in rats during adolescence.

Adolescent binge drinking is a strong predictor of alcohol use disorder and related mental health outcomes in adulthood, which may be due to disruptions in myelination during this dynamic period of brain development. White matter expansion in frontal regions during adolescence is essential for mature decision-making and stress regulation, yet the cellular mechanisms by which alcohol disrupts this process remain poorly understood. We used multi-label immunofluorescence and confocal microscopy to visualize proteins in oligodendrocyte lineage cells and myelin ensheathment of axons in the anterior cingulate cortex (Cg1) and corpus callosum (CC) following four weeks of episodic voluntary binge drinking using the Drinking-in-the-Dark model in adolescent male and female C57BL/6NJ mice beginning on postnatal day 28. Contrary to our initial hypothesis that alcohol targets early-stage oligodendrocyte precursor cells (OPCs), binge drinking selectively depleted mature oligodendrocytes expressing aspartoacylase (ASPA) in the Cg1 and CC of male mice, but not females. This enzyme is essential for lipid biosynthesis and myelin production, and this cell-specific loss was accompanied by significant hypomyelination of axons only in males. These findings identify a later maturational stage of oligodendroglial development as a sex-dependent target of alcohol, advancing our mechanistic understanding of prefrontal myelin deficits in adolescent drinking. Furthermore, ASPA emerges as a potential therapeutic target for alcohol use disorder and demyelinating diseases, with differential vulnerability across sex carrying important implications for adult neurodevelopmental outcomes.