Pharmacology Biochemistry and Behavior

Background: The nicotine analog 6-methyl nicotine (6-MN) has appeared in commercial e-cigarette liquids, and other products, spurring interest in determining the extent to which it conveys similar effects to those of nicotine. Objective: To determine if 6-MN acts like nicotine to decrease body temperature, decrease nociception, suppress wheel activity and reinforce operant behavior when delivered by vapor inhalation using an Electronic Nicotine Delivery System (ENDS; "e-cigarette") approach in a rat model. Methods: Male and female (N=8 per sex) young adult Sprague-Dawley rats were evaluated for rectal temperature and nociceptive responses (warm water tail-withdrawal) to the inhalation of vapor from (-)-6-MN or (-)-nicotine in concentrations ranging from 5-30 mg/mL in the propylene glycol vehicle. Rats were then assessed for the reinforcing effects of nicotine and 6-MN using a vapor self-administration procedure and the rate suppressing effects of nicotine and 6-MN on wheel activity following injection. Results: Inhalation of nicotine or 6-MN for 30 minutes decreased the rectal temperature and increased tail-withdrawal latency of female and male rats in a concentration-dependent manner. The magnitude of the effects of 6-MN and nicotine were similar at similar vapor concentrations. Operant responding for 6-MN vapor was increased by pre-treatment with the antagonist mecamylamine. 6-MN was more potent than nicotine at suppressing wheel activity after injection. Conclusions: 6-MN induces effects very similar to those of nicotine, at a similar potency when inhaled and at a slightly increased potency when injected.

RationalePsychedelic compounds such as psilocybin have attracted growing interest for their potential therapeutic effects in psychiatric disorders, with improvements in cognitive flexibility proposed as a possible mechanism of action. However, the effects of psychedelics on cognitive flexibility remain poorly understood. ObjectiveThis study aimed to examine the acute and post-acute effects of psilocybin (0.1, 0.3, 1 mg/kg) and lysergic acid diethylamide (LSD, (0.02, 0.04, 0.08 mg/kg) on cognitive flexibility in male rats. MethodsThis was tested using two variants of the probabilistic reversal learning task (PRLT): a touchscreen-based operant task and a more ethological foraging-based task. ResultsIn the touchscreen PRLT, acute psilocybin disrupted task engagement, with animals completing fewer trials and showing increased trial initiation latency, although psilocybin also showed a trend toward faster initial rule acquisition. However, psilocybin did not significantly alter the number of rule changes achieved, a canonical measure of cognitive flexibility, or feedback sensitivity. LSD similarly produced limited acute effects, although the highest dose reduced lose-shift probability, suggesting decreased sensitivity to negative feedback under some conditions. Post-acute effects of psilocybin were minimal in both PRLT variants and, where LSD effects were observed these occurred across different doses and timepoints without a consistent pattern. ConclusionsOverall, these findings suggest that serotonergic psychedelics do not robustly enhance reversal learning in these paradigms and that apparent learning effects may reflect transient disruptions in task engagement rather than improvements in cognitive flexibility. These results also highlight potential limitations of these PRLT paradigms for detecting psychedelic-induced changes in cognitive flexibility in rodents.

Tobacco use is the leading preventable cause of death worldwide. Anxiety increases the risk for smoking, and smoking in turn increases the risk for anxiety disorders. There is therefore a need to identify interventions that reduce anxiety, in general and in the context of smoking, without producing sedation. Kava (Piper methysticum), a natural product with a long history of indigenous use, has been shown to have anxiolytic and calming effects and reduce nicotine withdrawal. The current study examined whether kava without the hepatotoxic flavokavains A and B (AB-free) could reduce anxiety-like behavior in mice repeatedly treated with nicotine. Male and female C57BL/6NCrl mice received either a control diet or an AB-free kava-supplemented diet and underwent two blocks of nicotine treatments. Mice underwent a first block of five every-other-day injections of nicotine (0.5 mg/kg) or saline, with open field testing after each injection, followed one week later by a nicotine challenge. A second block of injections was given using the same injection schedule, followed by a second challenge one week later, and two weeks afterward mice received a final challenge in a novel open field. During the first treatment block, AB-free kava significantly increased center time overall, an effect most pronounced in saline-treated animals, and increased locomotor activity, while nicotine decreased both measures. During the second challenge, nicotine reduced center time but not locomotor activity, and AB-free kava increased center time in saline-treated animals only. During the final challenge, nicotine reduced both measures, whereas AB-free kava increased center time regardless of nicotine treatment, and kava-treated animals also showed a near-significant increase in center entries. These results suggest that AB-free kava reduces anxiety-like behavior without inducing sedation but does not prevent nicotine-induced suppression of exploratory behavior.

Adults aged [≥]65 years are increasingly using cannabis products. However, controlled pharmacokinetic and pharmacodynamic data on {Delta}9-tetrahydrocannabinol (THC) in this population are sparse, and remain limited to oral/oromucosal formulations. To characterize the acute pharmacokinetic and pharmacodynamic effects of oral and vaporized THC in healthy adults aged [≥]65, we conducted a two-arm, randomized, double-blind, placebo-controlled trial in which 20 participants (mean age 70.0, SD: 5.1 years) received oral (placebo, 5 mg, or 10 mg) or vaporized THC (placebo, 2 mg, or 4 mg) across three eight-hour sessions separated by [≥]72 hours. Outcomes included plasma pharmacokinetics, subjective drug effects, reinforcement value, cognitive performance, heart rate (HR), blood pressure (BP), and adverse events (AEs). Oral THC was associated with delayed, lower THC exposure (Tmax 60-90 min; Cmax 2.6-6.2 ng/mL), with 11-OH-THC concentrations approximately matching parent-THC; slow-rising subjective effects; no change in reinforcement value; no significant change in HR or BP; and no AEs. Vaporized THC was associated with rapid, THC-dominant exposure (Tmax 3 min; Cmax 24.6-53.8 ng/mL) and minimal 11-OH-THC concentrations; rapid-onset subjective effects; increased reinforcement value at 4 mg; and significant HR elevation peaking within 5 min, without significant BP change. Cognitive performance did not differ from placebo at any oral or vaporized THC dose. At vaporized THC 4 mg, two participants experienced five AEs. Oral and vaporized THC produce route-specific pharmacokinetic and pharmacodynamic profiles in adults aged [≥]65, including an increase in reinforcement value only after vaporization, and should therefore not be treated as interchangeable in risk assessment for older adults.

Fear conditioning studies have historically relied on freezing as the primary measure of conditioned fear despite evidence that defensive responding is behaviorally diverse and sexually dimorphic. The endogenous opioid system, particularly mu-opioid receptor (MOR) signaling, is known to regulate fear learning and conditioned analgesia, yet its role in alternative fear-related behaviors and sex differences remains unclear. Here, we investigated the effects of systemic naloxone administration prior to auditory fear conditioning on freezing, darting, shock responsivity, and ultrasonic vocalizations (USVs) in male and female rats. Adult Sprague Dawley rats received naloxone (5 mg/kg, i.p.) or saline prior to conditioning and underwent fear recall testing 24 hours later. Naloxone produced sex- and behavior-specific effects across conditioning and recall. During conditioning, naloxone increased freezing in males during baseline and early tone presentations, while females exhibited reduced shock-response velocity and increased post-shock freezing. Naloxone did not significantly alter darting or USV production during conditioning. During recall, freezing behavior did not differ across groups. Naloxone-treated females, however, exhibited a distinct alarm-calling pattern, with fewer callers overall but increased call output among those that vocalized. These findings suggest that MOR antagonism differentially alters distinct components of fear expression in a sex-dependent manner and support the idea that freezing and alarm calling may reflect separable aspects of fear processing.

BackgroundConcurrent cocaine and alcohol use is one of the most prevalent forms of polysubstance consumption and is associated with poorer clinical outcomes than cocaine use alone. However, the regional molecular adaptations induced by combined exposure remain poorly understood. Here, we used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to characterize peptide/protein alterations in addiction-related brain regions following cocaine and cocaine-alcohol self-administration. MethodsYoung adult male and female Wistar rats underwent intravenous self-administration of saline, cocaine (1 mg/kg/infusion) or cocaine plus ethanol (1 mg/kg cocaine and 133 mg/kg ethanol per infusion), followed by extinction of drug-seeking behaviour. Coronal brain sections containing the anterior cingulate cortex (ACC) and ventral hippocampus (vHPC) were analysed by MALDI-IMS. Differential molecular features were identified using an exploratory statistical approach (FDR q < 0.20) and subsequently subjected to MS/MS analysis. ResultsThe ACC exhibited a substantially greater number of treatment-associated molecular alterations than the vHPC, suggesting a higher regional susceptibility to cocaine-induced molecular remodelling. Several molecular features were shared between the cocaine and cocaine-alcohol groups, indicating persistent cocaine-driven neuroadaptations. In contrast, additional signals were selectively associated with combined cocaine-alcohol exposure, while others present after cocaine alone were absent following alcohol co-exposure, supporting a modulatory effect of alcohol on specific cocaine-induced molecular responses. Overall, combined exposure generated a distinct regional molecular profile rather than simply reproducing the effects of cocaine alone. ConclusionsThis exploratory study demonstrates that MALDI-IMS enables the identification of region-specific peptide/protein alterations associated with cocaine and cocaine-alcohol exposure while preserving their spatial distribution within the brain. These findings highlight the ACC as a particularly responsive region and provide a framework for future studies aimed at validating molecular pathways involved in cocaine-alcohol polysubstance use.

Long-term opioid therapy is limited by analgesic tolerance and opioid-induced hyperalgesia, but the roles of genetic background, sex, and drug exposure remain unclear. We used 20 inbred strains from the Hybrid Rat Diversity Panel to examine thermal sensitivity, oxycodone analgesia, tolerance, and hyperalgesia-like changes following voluntary intravenous oxycodone or saline self-administration. Rats underwent tail-immersion testing before self-administration (Pre-SA) and after self-administration (Post-SA). Oxycodone analgesia was assessed using the percent maximum possible effect time course and the corresponding area under the curve. Pre-SA thermal sensitivity differed across strains and between sexes, and Pre-SA oxycodone analgesia also differed across strains. Oxycodone self-administration produced a sex-dependent increase in thermal sensitivity that was most evident in males. During Post-SA testing, oxycodone self-administering rats showed reduced analgesic responsiveness compared with saline controls, and the magnitude of this difference varied across strains. Within-strain Pre-SA-to-Post-SA comparisons identified tolerance-like reductions in several strains. Across strains and sexes, oxycodone self-administering rats showed a greater Pre-SA-to-Post-SA reduction in analgesic responsiveness than saline controls, consistent with analgesic tolerance. Total oxycodone intake was not associated with tolerance at either the strain-mean or individual-animal level. Heritability estimates were higher for thermal sensitivity and analgesia (H2 {approx} 0.28-0.40) than for changes in thermal sensitivity and tolerance (H2 {approx} 0.18-0.27). These findings demonstrate strain variation in thermal sensitivity and oxycodone analgesia, sex-dependent hyperalgesia-like effects, and reduced analgesic responsiveness following voluntary oxycodone intake.

Background: As cannabis policy changes in the United States, secondhand cannabis smoke (SCS) is increasingly common, including within families. However, prevalence of exposure and clinical correlates over time in adolescents are not fully understood. Objectives: (1) To estimate the prevalence of SCS and personal cannabis use in US-based teens exposed to SCS, and (2) examine the cognitive trajectories of adolescents exposed to SCS compared to non-exposed peers. Methods: Data from the Adolescent Brain Cognitive Development (ABCD) Study was used. Participants (n=11,316 of full cohort with follow-up data; n=776 with self-reported family SCS exposure) attended yearly visits from ages 11-17, completing substance use interviews, toxicological testing, and the NIH Toolbox Cognitive battery. Youth with SCS but no personal cannabis use (n=419; 47% female) were matched on prenatal substance exposure, family substance use history, and sociodemographics to non-SCS exposed and non-cannabis-using youth with a 1:2 ratio (Controls n=838). Linear mixed-effects models assessed cognitive performance by SCS*age interactions, accounting for random effects of subject and family. Covariates included sex and alcohol, nicotine, and other substance use. Secondary models analyzed performance by cumulative waves of reported SCS exposure interacting with age. Results: Of the full cohort, 6.9% (n=776) reported exposure to SCS. Of these individuals, 46% endorsed lifetime personal cannabis use by age 17, relative to 20% of non-SCS exposed youth (OR=3.83[95%CI:3.29,4.44]). Within matched participants, SCS*age demonstrated a significant interaction on attention and inhibitory control ({beta}=-0.32, p=.028), with SCS demonstrating reduced improvement over time. More waves of exposure were also associated with worse performance over time ({beta}=-0.39, p=.057). Discussion: Almost half of those who had been exposed to SCS endorsed personal cannabis use. Cognitive findings were domain specific, similar to findings in secondhand tobacco: SCS exposed youth showed restricted improvement in attention and inhibitory control by age 17. Public health and policymakers should make efforts to curb youth SCS exposure, given the potential for risk which has not been fully explored to date.

Background: Individuals receiving buprenorphine treatment for opioid use disorder (OUD) remain at high risk for treatment discontinuation and return to opioid use. Transcranial magnetic stimulation (TMS) has shown efficacy in reducing craving and substance use in other substance use disorders, but its application in OUD remains limited and the neural mechanism underlying its therapeutic effects is poorly understood. Determining the feasibility and generalizability of TMS in patients receiving buprenorphine - the most commonly prescribed medication for OUD - is therefore critical. This protocol aims to address these issues in a clinical trial of weekly TMS sessions for OUD. Methods: We will enroll up to 120 individuals with OUD taking buprenorphine in a randomized, single-blind, sham-controlled trial of left dorsolateral prefrontal cortex (DLPFC)-targeted intermittent theta burst stimulation (iTBS). Participants will receive active or sham iTBS weekly (2 sessions of 1800 pulses each applied once per week x 8 weeks, 16 sessions total) with pre- and post-iTBS assessments (10, 12, 20 weeks) of craving, opioid use, and treatment retention. A subset of individuals will undergo optional pre- and post-iTBS neuroimaging. The study will be conducted at an academic medical center and a private outpatient TMS clinic. Aims: Our primary aim is to determine whether 16 sessions of active iTBS applied to the left DLPFC results in reduced craving and opioid use, and higher treatment retention, relative to sham. In a secondary aim, we will also examine whether iTBS-related changes in craving are associated with changes in functional connectivity between the left DLPFC and both the dorsal striatum and anterior cingulate cortex. Discussion: By evaluating the feasibility and efficacy of a weekly TMS protocol that aligns with routine care and focuses on patients maintained on buprenorphine, this study addresses key limitations of prior TMS research in OUD. Furthermore, the inclusion of neuroimaging will help characterize the neural mechanisms underlying TMS-related changes in craving. Trial registration: This clinical trial is registered at ClinicalTrials.Gov; ID NCT07457489; date of registration: 03/02/2026.

Autistic individuals may be at an increased risk of hazardous drinking compared to non-autistic counterparts: potential motivations include facilitated social interactions and self-medication of co-occurring difficulties, and possible risk factors include being older and female. However, research remains limited and centred around clinical samples. Given the diversity of the autistic community, it is important to understand the intricacies of alcohol use to inform appropriate support. Eighteen autistic adults took part in semi-structured interviews about their drinking experiences. Data were analysed using reflexive thematic analysis. Three main themes were created (Autistic experiences, Managing expectations and coping by drinking and Recommendations for support). Autistic experiences was used to denote the ways in which participants described their own autistic features influenced their relationship with alcohol. Managing expectations and coping by drinking was chosen to reflect the pressures felt by participants to show up in social relationships and the co-occurring difficulties many of them managed using alcohol. Therapeutic preferences for alcohol services were captured under Recommendations for support. As expected, participants used alcohol to facilitate social interactions and self-medicate. However, additional nuances uncovered may provide clinical utility and highlight the need for further research in other demographics within the community.

Background: In the United States, several states have restricted sales of flavoured tobacco products, including popular menthol- and mint-flavoured Oral Nicotine Pouches (ONP). In response, tobacco companies introduced "unflavoured" ONP containing odorless synthetic cooling agents. Since these, in turn, have become targets of legislative bans, the tobacco industry may seek out flavourants with other sensory effects to increase the appeal of "unflavoured" ONP. Methods: Online merchants were searched for "unflavored" ONP marketed to consumers in jurisdictions with flavour bans. Sensory effects of aqueous extracts from identified "heat", "spicy" and "unflavoured" ONP were analyzed by Ca2+ microfluorimetry in HEK293 cells expressing the human heat/chili pepper flavourant (capsaicinoid) receptor, hTRPV1. ONP were analyzed for capsaicinoids and sweeteners by Liquid Chromatography/Mass Spectrometry (LC/MS). Results: A new category of "heat" or "spicy" ONP was identified, including products marketed as "unflavoured". Extracts from all these ONP robustly activated TRPV1, with "unflavoured" Lucy Heat the most potent. Chemical analysis demonstrated that Lucy Heat contained the synthetic capsaicinoid nonivamide at high levels (~675 microgram/pouch), while others contained mixtures of capsaicinoids (5-25 microgram/pouch) combined with other characterizing flavours (tropical, fruit). All tested ONP contained sweeteners. Conclusions: The tobacco industry continues to probe regulatory loopholes by claiming that newly introduced capsaicinoid flavourants and sweeteners in ONP do not represent characterizing flavours. This is contradicted by industry and regulatory determinations assigning characterizing flavour properties to these additives. The toxicological health risks of repeated capsaicinoid exposures due to ONP use, in combination with nicotine and other constituents, need to be assessed.

Animals experience stressors in a variety of contexts that result in activation of neuroendocrine and cellular stress responses. Release of stress hormones can disrupt or restore redox homeostasis, and the resulting changes in oxidative states, physiology and behavior vary by an individuals stress coping style. However, oxidative stress can also directly modulate neuroendocrine stress signaling. To what extent individual differences in brain antioxidant levels alter behavioral stress levels is not well understood. The present study investigated how N-acetylcysteine amide (NACA), an antioxidant and glutamate-modulating compound, regulates stress behavior across zebrafish (Danio rerio) with different stress coping styles (proactive, reactive). Following 24-hour exposure to NACA or control conditions, we quantified individual and composite stress behaviors using a Light-Dark Test (LDT). As expected, both proactive fish and NACA-treated fish showed significantly lower stress behaviors compared to reactive and control animals, respectively. Notably, stress-reducing effects of NACA were only seen in those with a reactive stress coping style. Overall, our data suggest that antioxidant mechanisms (e.g., glutathione system) may be key in facilitating the distinct behavioral and physiological responses to stressors that characterize alternative stress coping styles. The results underscore how individual differences in stress coping style and redox state can influence behavioral responses to stress.

Understanding the neurobiological systems that regulate alcohol cue-induced craving is of utmost importance for the development of novel intervention strategies for alcohol use disorders (AUDs). However, although a human experimenter is required to conduct alcohol self-administration studies in the lab, the cues associated with the experimenter are seldom if ever factored into the experimental design. Thus, although we have learned much to date about alcohol cue-induced behavior and neurobiology, and in particular about discrete cues presented many times throughout a single daily alcohol self-administration session, we know relatively little about how responses to alcohol availability cues might predict subsequent alcohol consumption. For the current experiment, mice were exposed daily to auditory cues that preceded 2 hours of alcohol or water access using drinking-in-the-dark (DID) methods. An additional control group experienced cues but were not otherwise manipulated. Importantly, cues were initiated remotely from outside the animal facility, avoiding the experimenter being the first cue predicting ethanol availability. Head direction, location in the home cage, and movement velocity were the primary variables on interest. Surprisingly, during the cue period, there were no significant differences between groups in any of these measures, despite meaningful alterations over days. However, we observed many significant correlations between behaviors and drinking variables. First, we observed significant positive associations between ambulatory velocity during cues and subsequent total alcohol (R2=0.14; p<0.0001) and total water (R2=0.12; p=0.0002) consumption, but only in females. We also observed a significant positive relationship (R2=0.25; p<0.0001) between the amount of time oriented toward the sipper port during the auditory cues and the average rate of subsequent alcohol consumption (i.e. front-loading), but only in females. In males, head direction was found to be positively associated with subsequent total water consumption (R2=-0.21; p<0.0001), but not alcohol (R2=-0.01; p=0.2267). We also observed a significant negative relationship (R2=-0.15; p<0.0001) between proximity to the sipper during the cue period and subsequent total 2-hour alcohol intake in males. Although these associations were modest in strength, they suggest potential sex-specific behavioral predictors of alcohol consumption that are regulated by different neural dynamics.

Exposure to chemical cues released by predator or pathogen can evoke anxiety or fear responses in prey/host animals such as fight, flight or freeze both at behavioral and molecular levels. Freezing is a fundamental anxiety response when fighting or fleeing arent feasible. Despite the potential relevance of freezing as a stress-coping mechanism, its behavioral and molecular underpinnings are not understood yet. At molecular level danger cues are perceived by chemosensory receptors expressed in sensory neurons which may further regulate the animals behavioral responses(Ye et al., 2024){Citation}. 2-nonanone (2-NA) is one of the principal volatile organic compounds secreted by many pathogenic bacteria infecting Caenorhabditis elegans as well as humans and may signal danger to worms. Here, we show that olfactory exposure to threat-associated cue 2-NA induces a reversible fear-like freezing response characterized by immobility and halted feeding in C. elegans. With the application of in silico and behavioral approaches we showed that 2-NA is one of the ligands for an olfactory G-protein Coupled Receptor (GPCR) STR-211 and RNAi knockdown of the receptor leads to a defect in 2-NA induced avoidance behavior in worms. We next discovered that STR-211 is required for immediate behavioral changes in C. elegans during freezing response against 2-NA. The study proposes an environment relevant animal model to mimic human anxiety and fear-like behavior, along with the identification of one of the olfactory GPCRs mediating this behavior. The model may help in understanding the neuromolecular basis of freezing response in human anxiety, contributing towards treatment of mental health disorders.

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.

Addiction is a complex and heritable trait which progresses through several developmental stages, each of which is presumably influenced by multiple partially overlapping genetic factors. Cocaine initially produces rewarding effects, followed by aversive effects including anxiety, craving, anhedonia, and withdrawal. These aversive effects have been suggested to contribute to the etiology of cocaine use disorders (CUD), as repeated exposure is thought to desensitize the rewarding effects and sensitize the aversive effects through a process involving both aberrant reward-based learning and aberrant avoidance-based learning. We examined the genetic basis of aversion learning using both food-based and cocaine-based behavioral assays in outbred Heterogenous Stock (HS) rats. A total of 1,074 HS rats (35.3% male) underwent runway operant cocaine-seeking, food-based progressive ratio and punishment testing, and locomotion testing. These phenotypes were significantly heritable (with h2 estimates as high as 0.307) and identified significant (p < 0.05) genetic loci related to avoidance-based learning including from the punishment task on Chromosomes 2, 3, 5, and 6, and the cocaine-operant runway latency task on Chromosome X. 172 positional candidate genes were identified from significant and suggestive loci, including Cdh10, Cdh12, Cdh18 which have previously been associated with smoking initiation from human GWAS, Adcy3, Cfap206, and Drc1 which are associated with primary neuronal cilia, as well as SNPs associated with novelty-related and social interaction phenotypes in independent samples of HS rats. Our results suggest that these aversion learning phenotypes are themselves complex heritable traits influenced by multiple genetic loci, which may pleiotropically affect other aspects of addiction biology.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundCircadian disruption is strongly associated with alcohol use disorder (AUD), but insight into the underlying brain-region and sex-specific mechanisms is limited. The function of the circadian clock gene Bmal1 within the striatum has been linked to alcohol drinking, yet its role within functionally distinct striatal subregions has not been systematically examined. MethodsWe deleted Bmal1 in medium spiny neurons of the dorsomedial striatum (DMS) or dorsolateral striatum (DLS). Male and female mice were tested for anxiety-like behavior, depressive-like behavior, and motor coordination. Voluntary alcohol intake was measured with an intermittent two-bottle choice paradigm, followed by sucrose preference and quinine-adulterated alcohol tests. To assess hormonal contributions, a subset of female mice underwent ovariectomy before behavioral testing. ResultsDeletion of Bmal1 in the DLS did not alter alcohol intake, alcohol preference, or quinine-adulterated alcohol intake in either sex. In contrast, DMS Bmal1 deletion significantly reduced alcohol consumption and alcohol preference in female mice, with no effect in males. These effects were not accompanied by changes in depressive-like behavior or motor coordination and were not explained by generalized reward changes, as sucrose preference was unaffected. Ovariectomy eliminated the effect of DMS Bmal1 deletion on alcohol intake, indicating dependence on ovarian hormones. ConclusionsThe DMS is a critical site at which Bmal1 regulates alcohol consumption in a sex-specific manner. These findings support an interaction between local circadian mechanisms and ovarian hormones in controlling alcohol drinking and highlight a potential target for sex-specific therapeutics in AUD.

The lateral habenula (LHb) encodes aversive states and negative affect, positioning it as a candidate region for the negative reinforcement that drives alcohol withdrawal. However, little is known about how chronic ethanol exposure affects LHb neuronal function and glial biology during withdrawal. Here, we used chronic intermittent ethanol (CIE) vapor exposure, a well-established model of alcohol dependence that reliably produces somatic and affective signs of withdrawal, to examine LHb physiology and astrocytic markers during acute withdrawal in male and female rats. Whole-cell and cell-attached recordings revealed that withdrawal reduced evoked and spontaneous firing in LHb neurons, with rebound firing following a crossover pattern between males and females. Despite these excitability changes, the overall distribution of firing phenotypes was unchanged, suggesting a shift in gain rather than a reorganization of cell types. Immunofluorescence revealed increased Sox9+ and GFAP labeling in the LHb during withdrawal at the same time point when electrophysiology experiments uncovered impaired astrocytic regulation of glutamate clearance. Together, these findings reveal that withdrawal from chronic ethanol exposure produces neuronal and glial adaptations in the LHb, pointing to impaired glutamate regulation as a candidate mechanism relevant to the negative affective state of alcohol withdrawal. These findings position the LHb as a potential node linking astrocyte-neuron dynamics to withdrawal symptoms and relapse vulnerability in alcohol use disorder.

Opioid addiction treatment is often hampered by the severe dysphoria of opioid withdrawal, but withdrawal treatments are limited by incomplete understanding of brain mechanisms involved. One area frequently implicated in withdrawal symptoms is the central amygdala, whose capsular portion (CeC) is particularly strongly activated during withdrawal. Additionally, a ventral posterior striatal region that resides near CeC, the interstitial nucleus of the posterior limb of the anterior commissure (IPACc), is also activated as strikingly as CeC. However, it is still unknown how these regions are activated, nor whether their activation explains the high intensity of withdrawal dysphoria. Using RNAscope, we found that c-fos expression is induced in the parabrachial nucleus (PB), a key glutamatergic afferent of CeC, after precipitated morphine withdrawal. Chemogenetic inhibition of PB glutamatergic neurons (VG2PB) nearly eliminated withdrawal-induced CeC c-Fos, without affecting IPACc c-Fos, indicating these two nuclei are activated by distinct sources. Furthermore, VG2PB inhibition markedly reduced somatic (jumping) and modestly reduced affective (place avoidance) withdrawal behavior. On the other hand, inhibition of CeC-projecting PB neuronal subtypes expressing calcitonin gene-related peptide (CGRP) or mu opioid receptor (MOR) reduced place avoidance without affecting jumping, indicating their specific role in withdrawal aversion. Strikingly, simultaneous inhibition of VG2PB and posterior striatal region containing IPACc robustly reduced withdrawal-induced place avoidance much more than the modest effects of either inhibition alone, suggesting their cooperative action in driving aversion. Our data suggests that PB-CeC circuit and posterior striatal area constitute a cooperative system driving opioid withdrawal aversion.