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.

RationaleThe opioid epidemic continues to be driven by synthetic opioids, particularly fentanyl, yet the long-term behavioral manifestations of withdrawal remain poorly characterized. Women exhibit unique vulnerabilities to opioid use disorder, including greater susceptibility to withdrawal-related symptoms and relapse, suggesting that defining the behavioral adaptations that emerge during prolonged fentanyl abstinence may identify mechanisms underlying female-specific relapse vulnerability. Thus, we used an oral fentanyl drinking model to longitudinally examine nociceptive, affective, and exploratory behaviors across chronic exposure and abstinence in female mice. MethodsAdult female C57BL/6J mice underwent a modified 5-week Drinking-in-the-Dark paradigm with 4-hour daily access to fentanyl (30 {micro}g/mL) or water. Somatic withdrawal was assessed following naloxone-precipitated withdrawal. Thermal and mechanical nociception, sucrose preference, exploratory behavior, acoustic startle, nest building, and locomotor activity were evaluated during fentanyl exposure and throughout one month of abstinence. ResultsFemale mice exhibited robust naloxone-precipitated somatic withdrawal, confirming physiological effects of opioid drinking. Chronic fentanyl exposure produced transient thermal hyperalgesia during weeks 2-4 of drinking that resolved by the final week, but re-emerged after 30 days of abstinence. After 30 days of abstinence, we also found increased mechanical sensitivity. During early abstinence, fentanyl-exposed mice exhibited increased sucrose consumption and greater exploration of anxiogenic environments without alterations in general locomotion. Increased exploratory behavior persisted into prolonged abstinence and was partially rescued in mice who previously received naloxone. Additionally, fDID mice exhibited impaired nesting behavior, while prior naloxone-precipitated withdrawal improved nest-building performance. ConclusionsWe demonstrate that chronic opioid exposure, precipitated withdrawal trials, and duration of abstinence interact to shape protracted withdrawal phenotypes in female mice that extend well beyond drug exposure. These results provide insight into persistent withdrawal symptoms that may shape relapse vulnerability using a novel translationally relevant framework.

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

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.

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.

RationaleCannabis withdrawal contributes to relapse in individuals with cannabis use disorder, yet preclinical studies have largely focused on withdrawal induced by injected cannabinoids rather than inhaled cannabis, which remains the most common route in humans. The behavioural effects of chronic exposure to vapourized cannabis flower and resulting withdrawal after cessation of exposure remain poorly characterized. ObjectivesTo determine the behavioural effects of chronic vapourized high-THC cannabis flower exposure on cannabinoid tetrad, somatic withdrawal and behavioural transition networks in rats following both chronic vapour exposure and administration of the cannabinoid receptor 1 (CB1) receptor antagonist SR141716A (rimonabant). MethodsTwo studies were conducted using adult male Sprague Dawley rats. The first study (N = 16) exposed rats to either air or vapourized high-THC cannabis flower three times a day for seven days using a Volcano vapourizer, followed by intraperitoneal administration of the CB1 antagonist SR141716A (3 mg/kg). The second study (N = 24) included two air controls and two cannabis groups, with one of each receiving either saline or SR141716A. Behavioural assessments included triad measurements to confirm the cannabis effect, along with withdrawal assessment via a sucrose preference test and somatic signs 30 minutes following rimonabant administration. ResultsRepeated cannabis vapour exposure produced reduced locomotor activity, hypothermia, and increased tail-flick latency. Rimonabant administration precipitated withdrawal characterized by increased total withdrawal scores and somatic signs, including blinking, body shakes/tremors, and grooming-related behaviours. Behavioural network analyses revealed substantial reorganization of behavioural transition structure during both chronic cannabis exposure and withdrawal. Chronic cannabis exposure was associated with reduced network modularity, a condensed behavioural repertoire, and altered behavioural centrality measures. At the same time, precipitated withdrawal further increased the influence of exploratory behaviours, particularly sniffing, and reduced the network prominence of locomotor-associated behaviours, such as walking, beyond that detected using conventional behavioural measures alone. ConclusionChronic exposure to vapourized cannabis flower followed by CB1 receptor antagonism produces reliable withdrawal symptoms in rats. Behavioural network analyses further reveal that cannabis exposure and withdrawal are both associated with widespread reorganization of behavioural dynamics, suggesting that withdrawal alters not only individual behaviours but also the structure of behavioural transitions. These findings establish a translational model of cannabis withdrawal using inhaled cannabis flower vapour and identify behavioural network analysis as a sensitive approach for characterizing withdrawal-related behavioural states.

Treatment-resistant depression and anxiety remain major challenges in psychiatry, particularly in female patients, who are disproportionately affected yet remain underrepresented in preclinical ketamine research. The present study investigated short- and long-term anxiolytic effects of acute subanesthetic ketamine administration in female Wistar-Kyoto (WKY) rats, a validated genetic model of treatment-resistant affective dysfunction. Subjects received a single intraperitoneal injection of saline vehicle or racemic ketamine (5, 10, or 15 mg/kg), followed by acoustic startle response (ASR) testing 24 hours and 7 days later. Oxidative stress was assessed using 8-oxo-2'-deoxyguanosine (8-oxo-dG) immunofluorescence in the basolateral amygdala (BLA), prefrontal cortex (PFC), and hippocampus, alongside analysis of parvalbumin-positive (PV+) interneurons. Ketamine treatment produced dose- and time-dependent behavioral effects with 10 mg/kg eliciting the strongest delayed anxiolytic-like response at 7 days, while 15 mg/kg showed more immediate behavioral effects at 24 hours. While ketamine did not alter PV+ cell count, it significantly increased oxidative stress markers globally in the BLA and prelimbic region of the PFC and specifically in the PV+ interneurons in the BLA. The findings suggest that ketamine's therapeutic effects in female WKY rats may involve region-specific modulation of stress circuitry and oxidative signaling rather than gross interneuron loss. Overall, the study provides evidence for sex-dependent and temporally dynamic effects of ketamine in a translational model of treatment-resistant anxiety and depression.

Background and PurposeSerotonergic psychedelics affect behavior and physiology, but the relationships among these effects remain poorly understood. In rodents, the head-twitch response is used as a measure of psychedelic-like activity, yet it does not capture changes in physiological state or the performance of learned behaviors. Here, we investigated the acute effects of the 5-HT2A receptor agonist TCB-2 across several behavioral and physiological measures and examined how these effects were modified by pretreatment with the 5-HT2A receptor antagonist volinanserin. Experimental ApproachMice were tested in head-fixed and freely moving conditions. During a learned auditory trace-conditioning task, we measured licking, pupil area, eye position, and blinking. We measured locomotor activity in an open field and quantified head-twitch responses using a DeepLabCut-based method. To examine the contribution of 5-HT2A receptors, mice were pretreated with the 5-HT2A receptor antagonist volinanserin. Key ResultsTCB-2 caused pupil constriction without detectable changes in eye position or blinking when administered alone. TCB-2 also reduced licking at the highest dose, but the cue-locked temporal pattern of licking remained evident. In freely moving mice, TCB-2 reduced locomotor activity and produced a dose-dependent increase in head-twitch responses. Volinanserin partially attenuated TCB-2-induced pupil constriction and reduced head-twitch responses under some conditions, but it did not consistently prevent the other effects of TCB-2. Conclusions and ImplicationsTCB-2 produced distinct effects across physiological and behavioral measures rather than a uniform disruption of behavioral function. Pronounced pupil constriction and head-twitch responses occurred without detectable changes in eye position or blinking, while the temporal organization of conditioned licking was retained despite a reduction in its magnitude. The incomplete and variable effects of volinanserin preclude definitive conclusions about the receptor mechanisms underlying each response. Combining automated head-twitch detection with physiological and task-related measurements provides a broader framework for comparing the pharmacological profiles of serotonergic compounds. What is already knownO_LIClassical psychedelics produce characteristic effects primarily through serotonin 5-HT2A receptor activation. C_LIO_LIHead-twitch responses capture only one dimension of psychedelic-like drug action. C_LI What this study addsO_LITCB-2 reduced locomotion and licking while preserving the cue-locked pattern of conditioned licking. C_LIO_LIPupil constriction occurred without detectable changes in eye position or blinking. C_LI Clinical significanceO_LIMultidimensional phenotyping can distinguish the physiological and behavioral profiles of serotonergic compounds. C_LIO_LIComplementary measures may improve preclinical evaluation of emerging serotonergic therapeutics. C_LI

RationaleAdolescence is a period of heightened vulnerability to nicotine reinforcement. While zebrafish are a valuable model for investigating drug reward, standard conditioned place preference (CPP) assays typically test subjects in isolation. In this highly social species, solitary testing may act as an environmental stressor that confounds behavioral readouts. ObjectivesThis study examined how social context during testing (isolated vs. grouped) affects experimental attrition, behavioral stability, and nicotine CPP expression in late juvenile zebrafish. MethodsZebrafish housed in groups of four were tested either individually (isolated) or in their housing groups (grouped) during daily 20-minute sessions. Following baseline preference assessments, subjects underwent six days of conditioning pairing their initially non-preferred compartment with fish water or nicotine (0.5, 1.6, or 5.0 {micro}mol/L). Place preference, locomotion, and thigmotaxis were assessed on a drug-free test day. ResultsIsolated testing reduced distance traveled, decreased swimming speed, and increased time spent near tank walls, indicating heightened anxiety-like behavior. Experimental attrition was significantly higher in isolated (38.9%) than grouped (2.5%) subjects. Grouped subjects developed significant place preference at 1.6 and 5.0 {micro} mol/L nicotine, whereas preference was not detectable in isolated subjects. ConclusionsSolitary testing acts as a stressor that increases experimental attrition and masks place preference. Conversely, testing in the presence of conspecifics stabilizes behavior and facilitates the detection of nicotine reward in late juvenile zebrafish.

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.

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.

Background: Hyperbolic tapering is an increasingly recognized approach for discontinuing serotonin reuptake inhibitor (SRI) antidepressants that involves non-linear dose reductions with equal stepwise reductions in serotonin transporter (SERT) occupancy to mitigate withdrawal symptoms. Its theoretical basis is the hyperbolic relationship between SRI dose and SERT occupancy reported in radioligand imaging studies. Hyperbolic tapering implicitly assumes that changes in SERT occupancy approximate changes in biologic effect and withdrawal risk. Because SERT occupancy plateaus across the therapeutic dose range of SRIs, this framework predicts relatively small biologic effects and withdrawal risk within this range. However, SERT occupancy influences serotonergic activity only indirectly via its effects on extracellular serotonin concentrations, and the relationship between these two variables is poorly characterized. Methods: We developed a two-pathway clearance model derived from mass-action kinetics to evaluate the steady-state relationship between SERT occupancy and extracellular serotonin concentrations under chronic SRI treatment. Results: Our analysis indicates that serotonin concentrations increase hyperbolically as transporter occupancy increases, suggesting that biologically meaningful differences in serotonergic signaling persist across the therapeutic dose range of SRIs despite plateauing occupancy. Conclusions: Our model predicts a hyperbolic relationship between SERT occupancy and extracellular serotonin concentrations, suggesting that changes in occupancy may not map proportionally onto serotonergic effect. These findings provide a potential mechanistic explanation for dose-dependent clinical effects of SRIs despite plateauing transporter occupancy and generate testable hypotheses regarding antidepressant tapering strategies. Empirical validation is warranted.

Serotonin (5-HT) 2A receptors (5-HT2AR) modulate corticolimbic circuits regulating fear extinction. Although activation of these receptors has been shown to facilitate fear extinction, the behavioral consequences of 5-HT2AR antagonism during extinction is not well defined. Here, we examined the systemic effects of two 5-HT2A receptor antagonists, the mixed 5-HT2A/2C antagonist MDL 11,939 (Glemanserin) and the selective 5-HT2A antagonist MDL 100,907 (Volinanserin) on fear extinction in adult C57BL/6J mice. Prior to drug administration, mice assigned to future treatment groups acquired comparable conditioned freezing responses during delay fear conditioning. Twenty-four hours later, acute administration of MDL 11,939 (1.0 mg/kg) or MDL 100,907 (0.01 mg/kg) increased freezing to the first conditioned stimulus (CS) presentation on Extinction Day 1, indicating enhanced expression of conditioned fear. However, acquisition of fear extinction differed between the respective cohorts of mice treated with the two 5-HT2AR antagonists. Repeated administration of MDL 11,939 significantly impaired extinction, as evidenced by increased freezing across extinction trials and an increased number of trials required to reach extinction criterion. In contrast, MDL 100,907 has reported affinity for did not significantly alter extinction under either acute or repeated dosing conditions. Because MDL 11,939 has reported affinity for 5-HT2C receptors, we tested potential contributions of 5-HT2C receptor antagonism in a separate cohort of mice using two doses of the selective 5-HT2C antagonist, SB 242084. Neither dose affected conditioned fear expression, extinction learning, or trials required to reach extinction criterion. Together, these findings demonstrate ligand-specific and dose-dependent effects of 5-HT2AR antagonism on fear extinction and suggest that distinct intracellular receptor signaling pathways may differentially regulate extinction-related behavior.

Cigarette smoking remains one of the most important preventable causes of premature death worldwide. However, the mechanisms sustaining nicotine dependence and relapse are incompletely understood, particularly the role of impulsive action in persistent tobacco use. Dopamine D1 and nicotinic acetylcholine receptors both regulate nicotine-related behavior, but whether they contribute differently to nicotine-motivated responding and nicotine-induced impulsive action is unclear. The present study examined the effects of D1 receptor blockade and stimulation in male and female rats trained to self-administer nicotine intravenously in a Go/No- Go task. Nicotine was available during Go periods but not during No-Go periods, and impulsive action was measured as the percentage of active lever responses during No-Go periods. The D1 receptor antagonist SCH23390 reduced nicotine intake and active lever responding during Go periods and affected the percentage of active lever responses during No-Go periods. However, SCH23390 also reduced inactive lever responding, so its effect on impulsive action could not be separated from a general reduction in operant output. In contrast, the D1 receptor agonist A77636 reduced nicotine intake and Go-period responding without affecting No-Go responding, indicating that D1 receptor stimulation reduced nicotine-motivated responding without altering impulsive action. Rats showed greater No-Go responding during nicotine self-administration than during saline self-administration, indicating that nicotine increased impulsive action rather than general operant responding. The non-selective nicotinic antagonist mecamylamine reduced nicotine-motivated responding and decreased No-Go responding, indicating reduced impulsive action. These findings suggest that nicotinic acetylcholine receptor signaling plays a major role in nicotine-induced impulsive action, whereas D1 receptor signaling contributes more clearly to nicotine-motivated responding. D1 receptor agonism may reduce nicotine-motivated behavior without affecting nicotine-induced impulsive action.

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.

Opioid Use Disorder (OUD) remains a prominent threat to global health. Genetic background influences the susceptibility of developing OUD, although specific genetic factors remain elusive. Rodent models that differ in susceptibility to escalation and dysregulation of opioid use are valuable tools to facilitate discovery of genetic pathways. Phenotypes associated with the development of OUD were compared in seven classic inbred rat strains (M520/N, WKY/NCrl, F344/NCrl, F344/Stm, LEW/Crl, LEW/SSNHsd, LE/Stm) from the Hybrid Rat Diversity Panel (HRDP). A two-phase self-administration paradigm was utilized to assess characteristics of the acquisition of oxycodone self-administration during daily 2-h sessions, and the escalation of oxycodone use during daily 12-h sessions. Genetic background influenced the acquisition of oxycodone self-administration as indicated by differences in the initiation of responding for oxycodone during each session and different amounts of oxycodone intake. We observed that escalation of oxycodone intake between-sessions was strain dependent, and the within-session distribution of oxycodone intake was strongly influenced by strain. The M520/N strain engaged in a unique pattern of intake, characterized by rapid initiation of oxycodone responding during the acquisition phase and a significant burst-like responding during escalation. Strain-dependent sex differences were also observed in several acquisition and escalation metrics. Of interest, burst responding was more prevalent in females of the M520/N strain compared to males. Together, these data indicate that genetic background influences not only overall oxycodone intake, but specific within- and between-session metrics that capture patterns of consumption across the substance use trajectory.

Chlorpyrifos is a widely used organophosphate pesticide that exerts its primary toxic effect through inhibition of acetylcholinesterase (AChE). Although the acute neurotoxicity of chlorpyrifos is well characterized, the lasting biochemical, behavioral, and epigenetic consequences of sub-chronic exposure remain poorly understood, particularly when considering sex-specific differences. Therefore, we exposed male and female C57BL/6J mice to either peanut oil (n=19), low chlorpyrifos exposure (1 mg/kg/day; n=19), or high chlorpyrifos exposure (10 mg/kg/day n=10) repeatedly for 21 days via subcutaneous injection. Blood AChE activity, behavior, and hippocampal DNA methylation were measured across groups. During exposure, AChE activity decreased in both males and females but only returned to baseline after behavioral testing in females exposed to low chlorpyrifos levels. Behavioral tests also revealed a sex-specific phenotype, with females in the low exposure group exhibiting reduced forced swim test immobility and a significant time by exposure interaction in open field habituation. No significant behavioral effects were observed in males. Significant DNA methylation changes were observed at 3,538 CpG sites in male and female mice after high exposure. Sex-specific analyses revealed two female-specific differentially methylated CpGs after high exposure. Pathways enriched for differentially methylated genes included several related to synaptic remodeling, cholinergic synapse, and various endocrine systems. These findings demonstrate that repeated high chlorpyrifos exposure leads to persistent cholinergic disruption and DNA methylation changes. However, the female-specific behavioral changes seen are independent of AChE activity and widespread DNA methylation changes, suggesting additional mechanisms, present only in females, may underlie behavioral sensitivity to chlorpyrifos. New and NoteworthySub-chronic chlorpyrifos exposure in adult mice produced dose-dependent blood AChE suppression and widespread hippocampal DNA methylation changes in both sexes, with pathway enrichment including cholinergic synapse and endocrine systems. Behavioral effects were subtle, with females in the low exposure group showing reduced forced swim immobility and altered locomotor habituation. Notably, the female-specific behavioral changes seen are independent of AChE activity and DNA methylation changes, suggesting novel mechanisms may underlie female behavioral sensitivity to chlorpyrifos.

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.

The need for improved treatments for chronic pain has driven increased interest in cannabis-based therapeutics. Peripheral dorsal root ganglion (DRG) neurons, including nociceptors, express cannabinoid receptors (CB1 and CB2), suggesting that modulation of DRG excitability may provide an effective strategy for peripheral analgesia. Here, we investigated the effects of cannabidiol (CBD), {Delta}9-tetrahydrocannabinol (THC), terpene mixtures as well as cannabis plant extracts on neuronal excitability in small-diameter mouse DRG neurons using whole-cell current-clamp electrophysiology and assessed potential synergistic interactions. Both CBD and THC produced a concentration- and time-dependent inhibition of rheobase-evoked action potential firing, which were reversible in the presence of bovine serum albumin (BSA), both with similar estimated IC50 values of 5 M (. Terpene mixtures, as well as individual terpenes (linalool, {beta}-pinene, and myrcene), similarly reduced neuronal firing. Co-application of CBD with THC or terpenes enhanced inhibition, consistent with synergistic interactions and the known "entourage effect." Application of WIN55,212-2 (WIN), a non-selective cannabinoid receptor agonist, in the presence of CBD also accelerated the time-dependent inhibition of neuronal firing. The inhibition of firing by the CB2-selective inverse agonist JTE-907 indicated the presence of CB2 receptors on DRG neurons. Plant extracts from the Cannabis sativa leaves also reversibly inhibited neuronal firing. CBD and a terpenes mixture produced modest effects on hERG channels, whereas plants extracts had negligible effects. Collectively, these findings demonstrate that phytocannabinoids and terpenes suppress peripheral sensory neuron excitability via receptor-dependent and indirect mechanisms, supporting their potential as non-opioid analgesics. Their synergistic interactions suggest that multi-component formulations may enhance analgesic effects.