Pharmacology Biochemistry and Behavior

The increasing prevalence of cannabis use, including among pregnant women, highlights the critical need for a deeper understanding of prenatal cannabis exposure. This study aimed to develop a standardized cross-species inhalation exposure protocol to administer the principal psychoactive component of cannabis, {Delta}9-tetrahydrocannabinol (THC), to prairie voles (Microtus ochrogaster) and laboratory rats (Rattus norvegicus), and to investigate the distribution of THC in maternal and fetal tissues following prenatal exposure. Using an established e-cigarette system for delivering vaporized THC, we administered THC to pregnant prairie voles and rats. THC concentrations were measured in maternal plasma and fetal brain tissue using LC-MS/MS (Liquid Chromatography coupled with Tandem Mass Spectrometry). In both species, THC levels were compared across groups to evaluate the impact of fetal position on THC uptake. We found that THC readily crossed the placental barrier in both species, resulting in significantly higher concentrations of THC in the fetal brain within the THC-exposed groups compared to the vehicle controls. Interspecies comparison revealed higher THC concentrations in rat fetal brain tissue compared to prairie voles. No significant effects of fetal position on THC levels were found for either species. The findings confirm placental transfer of THC and reveal species-specific patterns of THC distribution. Additional studies were then carried out in voles to compare plasma and brain THC levels in maternal and virgin adult prairie voles. Maternal brain THC concentrations were significantly higher than fetal brain concentrations in prairie voles. Strong positive correlations were observed between plasma and brain THC concentrations in both maternal and virgin adult prairie voles. This study establishes a translational model for investigating prenatal cannabis exposure using an aerosolized administration method in voles compared to established methods in rats. The standardized protocol and results provide a foundation for future research into the developmental consequences of prenatal cannabis exposure and offer crucial insights for informing public health policies and clinical practices in response to the global increase in cannabis use.

Cannabis contains many bioactive compounds, including {Delta}9-Tetrahydrocannabinol (THC) and cannabidiol (CBD), which influence behavior through complex pharmacological interactions with endogenous targets. This study examines whether CBD influences THC-induced changes in motor activity, hypothermia, and antinociception traits across different THC:CBD ratios, sexes, and genetic backgrounds. Traits were measured in C57BL/6J (B6) and DBA/2J (D2) mice of both sexes following baseline intraperitoneal (i.p.) injection of vehicle (VEH) and two consecutive daily doses of VEH or THC (10 mg/kg) alone or in combination with 0.56, 5, or 10 mg/kg CBD (THC:0.56CBD, THC:5CBD, or THC:10CBD, respectively). Motor activity and hypothermia were quantified daily from 0 to 120 min following injection and antinociception was measured daily at 60 min. We found that CBD alters THC-induced changes in motor activity and hypothermia as a function of day, dose, time, sex, and strain. In D2 females, CBD dose-dependently attenuated the hypolocomotor effects of THC immediately following acute injection and enhanced these effects later at 75 min. Following repeated exposure, CBD dose-dependently enhanced THC-induced hypolocomotion in B6 females at 75 min and in D2 males at 30 min while attenuating THC-induced hypolocomotion in D2 females immediately following injection. In D2 females, CBD dose-dependently attenuated THC-induced hypothermia at 15 min and enhanced hypothermia relative to THC at 30 min in D2 males following acute injection. After repeated exposure, CBD dose-dependently enhanced THC-induced hypothermia in B6 females at 15 min and in D2 males from 30 to 120 mins, while attenuating hypothermia in D2 females at 30 min. No significant effects of CBD on antinociception were observed. Our results indicate that CBD can modulate some THC-induced traits acutely and after repeated exposure. Regulation of THC-induced behavioral responses is dependent on CBD dose, genetic background, and sex. A candidate gene search using brain gene expression in recombinant inbred mice revealed greater genetic variation in ion channel genes relative to key metabolic genes, suggesting an underlying pharmacodynamic mechanism. Future research and validation of molecular mechanisms underlying these differences is expected to enhance our understanding of potential health risks and clinical relevance of cannabis and cannabinoid compounds containing THC and CBD.

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.

Tobacco use disorder is a chronic condition characterized by compulsive nicotine use, withdrawal, and relapse following abstinence. Impulsivity contributes to persistent nicotine use and poor cessation outcomes. This study examined whether nicotinic acetylcholine receptor (nAChR) modulators alter impulsive action in a nicotine self-administration Go/No-Go task in male and female rats. Rats acquired intravenous nicotine self-administration and were then trained in a Go/No-Go procedure in which active lever presses were reinforced during Go periods but not during No-Go periods. We then assessed the effects of varenicline (0.1-3 mg/kg), nicotine (0.1-0.6 mg/kg), and the nAChR antagonist mecamylamine (0.5-2 mg/kg) in the Go/No-Go procedure. Varenicline and nicotine pretreatment reduced active responding during both Go and No-Go periods, whereas mecamylamine selectively reduced responding during No-Go periods. Mecamylamine decreased the percentage of active responses during No-Go trials, indicating reduced bias toward the nicotine-associated lever. In contrast, nicotine and varenicline did not alter response allocation, suggesting that their effects reflected nonspecific reductions in responding rather than changes in impulsive action. No sex differences were observed. Substituting saline for nicotine during self-administration did not alter active responding during Go periods, but rats in the saline group had fewer active responses during No-Go periods than rats in the nicotine group. These results show that chronic nicotine self-administration increases impulsive action and that nAChR antagonism, but not agonism or partial agonism, reduces nicotine-related impulsive action. This work supports the utility of the Go/No-Go self-administration task for investigating nAChR-dependent mechanisms underlying nicotine-induced impulsivity.

Endocannabinoid (eCB) signaling is a key regulator of reward-related dopaminergic signaling, particularly in response to drugs of abuse, such as cocaine. To date, our understanding of this mechanism has primarily been limited to male subjects. Prior work establishes that female cocaine users have more adverse outcomes, and female rats show greater sensitivity to cannabinoid type 1 receptor (CB1R) regulation of cocaine self-administration. Therefore, we hypothesize that female rats exhibit enhanced eCB regulation of cocaine-evoked dopamine (DA). We used in vivo fiber photometry recording of the dopamine biosensor, dLight 1.3b, in the nucleus accumbens medial shell (NAcms) in response to cocaine in male and female rats. Rats were pretreated with cannabinoid-targeting drugs to investigate the effects of CB1R inactivation or augmentation of the eCB 2-AG on cocaine-evoked DA. Our results revealed that CB1R inactivation attenuates cocaine-evoked DA in male and female rats, but females showed enhanced sensitivity for CB1R regulation of cocaine-evoked DA. Cocaine-evoked DA was enhanced by augmenting 2-AG levels, and females again showed increased sensitivity to this manipulation. Finally, females show greater cocaine-evoked DA when in a non-estrous cycle compared to estrous, reinforcing that estrous cycle is a determinant of cocaine-evoked DA. These data indicate that females show enhanced eCB regulation of cocaine-evoked DA signaling, underscoring the importance of sex as a biological variable in our understanding of endocannabinoid regulation of drug reward. HighlightsO_LICB1R inactivation attenuates cocaine-evoked DA in NAcms, preferentially in females C_LIO_LI2-AG augmentation via MAGL inhibition enhances cocaine-evoked DA, with female bias C_LIO_LIEstrous phase modulates the dopamine response to a high dose of cocaine in females C_LIO_LIMale and female rats show similar baseline DA and locomotor responses to cocaine C_LI

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.

The use of neuromodulation techniques for the treatment of alcohol use disorder is receiving increasing attention, especially non-invasive approaches, such as repetitive transcranial magnetic stimulation or transcranial direct current stimulation, while the hypothetical use of electroconvulsive therapy remains unexplored. Given our experience inducing electroconvulsive seizures (ECS) for therapeutic purposes in psychopathology rodent models, we evaluated the role of ECS on reducing the increased voluntary ethanol consumption caused by adolescent ethanol exposure in our validated preclinical model. Rats were treated in adolescence with a binge paradigm of ethanol (2 g/kg, i.p.; 3 rounds of 2 days at 48-h intervals; post-natal day, PND 29-30, PND 33-34 and PND 37-38) or saline. Following persistent withdrawal until adulthood, rats were allowed to: voluntarily drink ethanol (20%) by a two-bottle choice test, for 3 days (PND 80-82); treated with ECS (95 mA for 0.6 s, 100 Hz, pulse width 0.6 ms; ear-clip electrodes) or SHAM for 5 days (PND 86-90); re-exposed to voluntarily ethanol exposure (PND 94-96). Brains were collected on PND 97 to evaluate hippocampal markers of ethanol toxicity and/or treatment response (e.g., NeuroD, NF-L, BDNF and NF-L/BDNF ratio). Our results reproduced the increased voluntary ethanol consumption in adult rats induced by adolescent ethanol exposure and demonstrated that ECS could improve this abuse-prone response. Moreover, we suggested a possible role for BDNF in the beneficial effects induced by ECS, especially reducing the neurotoxic ratio NF-L/BDNF. Overall, we provide preclinical evidence for the potential use of ECS as an efficacious treatment for alcohol use disorder.

Opioid addiction and misuse are a serious national crisis that affects public health, as well as social and economic welfare. Mortality due to opioid misuse is further exasperated by the combination of opioids with non-opioid respiratory depressants such as xylazine that are resistant to mu opioid receptor antagonists such as naloxone. This study tested the hypothesis that oxytocin can mitigate the severe opioid induced respiratory depression (OIRD) and mortality induced by high doses of fentanyl or the combination of fentanyl with xylazine. Our results show OXT can improve survival and respiratory function in both male and female rats with opioid induced respiratory depression caused by fentanyl, as well as a combination of fentanyl and xylazine. The improvement in respiratory function by OXT post fentanyl-xylazine was significantly greater than the recovery using only naloxone. Chemogenetic activation of OXT receptor positive neurons in the ventral respiratory group (VRG) provided similar benefits to that of OXT administration in reversing OIRD. These results indicate OXT is a promising therapeutic target for reversing OIRD and the respiratory depression that occurs with the combination of opioids and xylazine, a situation where naloxone is only partially effective. Additional translational benefits of OXT include it can be repurposed as it is already a FDA approved drug for other uses, has a high safety profile, and is unlikely to induce the withdrawal or reversal of analgesia that occurs with naloxone. Key PointsO_LIOxytocin (OXT) improves survival and respiratory function in both male and female rats with opioid induced respiratory depression (OIRD) caused by fentanyl C_LIO_LIOXT also reverses OIRD induced by the combination of fentanyl and xylazine C_LIO_LIThe improvement in respiratory function by OXT post fentanyl-xylazine was significantly greater than the recovery using only naloxone C_LIO_LIChemogenetic activation of OXT receptor positive neurons in the ventral respiratory group (VRG) provided similar benefits to that of OXT administration in reversing OIRD C_LIO_LIThese results indicate OXT is a promising therapeutic target for reversing OIRD and the respiratory depression that occurs with the combination of opioids and xylazine C_LI

Early life adverse (ELA) experiences such as child maltreatment (MALT) are associated with physical and mental illness, including substance use disorders (SUDs), but underlying neurobiological mechanisms remain unclear. This study examined long-term effects of infant MALT on adult brain serotonin (5HT) and dopamine (DA) receptors in corticolimbic regions involved in reward and emotional control, using positron emission tomography (PET) imaging, a translational infant MALT macaque model of cocaine use disorder (CUD) risk and a COC self-administration (SA) paradigm. The study focused on regional serotonin 5HT1A, 5HT2A, and dopamine D2/D3 receptor availability (BP) differences between MALT and Control animals using PET, both at baseline (pre-COC SA) and following chronic COC SA (once they reached a total of 100 mg/kg intake). We also examined whether levels of these neurochemical receptors predicted COC SA measures, including reinforcing effects and potency using fixed-ratio (FR) peak response rates and progressive-ratio (PR) peak breakpoint. Our findings showed long-term effects of infant MALT on 5HT, but not DA, receptors in corticolimbic circuits. Specifically, MALT animals showed lower 5HT1A BP in the anterior cingulate cortex (ACC), medial prefrontal cortex (mPFC), and hippocampus compared to Controls. A MALT by Sex interaction effect was detected in 5HT2A BP in the OFC, with lower levels in MALT than Control males, but not in females. In addition, upregulation of 5HT1A and 5HT2A receptors was detected following chronic COC SA in most PFC subregions, hippocampus, and NAcc, particularly in the Control group. These findings suggest long-term effects of ELA on adult 5HT, but not DA, receptors in corticolimbic regions involved in emotional and reward processes. We also found associations between PET baseline (pre-COC SA) receptor BP data and COC SA measures. In particular, a positive correlation between 5HT1A receptor BP in caudate and peak FR Response Rates, whereas amygdala 5HT1A receptor levels were positively correlated with peak PR breakpoint and negatively correlated with peak FR Response Rates. Overall, these findings suggest an important role of 5HT1A and 5HT2A PFC receptors in early COC-related changes in reward circuitry and of amygdala 5HT receptors on cocaine-maintained behaviors. The dynamic change of these 5HT1A and 5HT2A receptors following chronic COC exposure was blunted in animals with ELA. It would be important to understand the biological consequences of these dynamic changes in 5HT receptors and whether they are associated with other stages of the addiction cycle, for example COC relapse, which could inform future pharmacological interventions that target 5HT receptors for treatment of CUD. Simple SummaryWe studied the long-term effects of early life adversity (ELA) on adult brain dopamine (DA) and serotonin (5HT) signaling in corticolimbic regions involved in emotional and reward regulation. We used specific PET radioligands that bind to the DA D2/D3, 5HT1A and 5HT2A receptors, finding lower levels of 5HT, but not DA, receptors binding potential (BP) in animals that experienced ELA. We also found associations between PET receptor BP measures and reinforcing effects of cocaine in i.v. self-administration paradigms using fixed- and progressive-ratio reinforcement schedules. In addition, a strong upregulation of 5HT, but not DA, receptors was identified following chronic cocaine exposure in prefrontal cortex (PFC). Our findings suggest long-term effects of ELA on adult PFC 5HT1A and 5HT2A receptors. The findings also suggest an important role of 5HT1A and 5HT2A, more so than D2/D3, receptors in early cocaine-related changes in reward circuitry. The early dynamic changes of these 5HT receptors could serve as biomarkers for cocaine use disorder (CUD) and inform future pharmacological interventions.

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.

Xylazine is a 2-adrenergic agonist typically used in as a sedative and analgesic in veterinary medicine. For some years, xylazine has been reported as an additive to fentanyl on the illicit drug market and has been associated with severe side-effects including severe ulcerations and potential amputations at the sites of injection along with an increased risk of respiratory depression and death. We recently reported that xylazine has modest {kappa} opioid agonist activity in vitro and in vivo and asked if other 2-adrenergic agonists had similar off-target activities. To test this hypothesis, we profiled US FDA-approved 2-adrenergic agonists at 320 G protein coupled receptors (GPCRs) to identify potentially deleterious and/or beneficial off-targets. Although all other tested 2-adrenergic agonists were devoid of {kappa} opioid agonist activity, each had a distinct pattern of activity at various GPCRs and differential patterns of signaling bias at 2-receptor subtypes. These findings suggest potential molecular targets for both side-effects and therapeutic activities among known 2-adrenergic agonists.

"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.

Sex differences in responses to chronic stress are implicated in the higher prevalence of major depression and PTSD in females. Evidence of sex differences in endocannabinoid (eCB) physiology suggests that eCB signaling contributes to sexual disparities in fear conditioning and extinction. In adolescent male Sprague-Dawley rats, exposure to chronic-mild-unpredictable stress (CMS) resulted in enhanced trace-fear conditioning that was reversed by CB1 activation (Reich et al, 2013). In the present study, we assessed the effects of CMS and CB1 activation on hippocampal-dependent trace and contextual fear conditioning in adolescent female Sprague-Dawley rats. CMS exposure enhanced trace freezing behavior during memory recall compared to non-stress controls. This effect was not observed in contextually conditioned females. The CB1 receptor agonist, ACEA (0.1 mg/kg), administered prior to trace memory recall, but not prior to acquisition, significantly decreased freezing in both stress and non-stress females. ACEA significantly reduced baseline freezing behavior during trace memory recall in both stress and non-stress rats, however ACEA either 1) did not affect or 2) impaired short and long-term extinction in stress and non-stress females. In contextually conditioned females, ACEA decreased freezing during memory recall, although the effect was more robust in stress rats. ACEA impaired long-term contextual extinction in stress females while facilitating this in non-stress controls. However, ACEA had no effect or impaired short-term contextual extinction in both stress and non-stress groups. The results demonstrate that CMS enhances hippocampal-dependent episodic fear memories but has limited effects on contextual fear conditioning in female rats. These findings have implications in the use of medical cannabinoid treatment of disorders such as PTSD, as well as recreational cannabis use in adolescent/young adult females.

Astrocytes are the most abundant glial cells in the brain and an integrative component of the neural network. Studies have shown that ethanol altered expression of an astrocyte marker, i.e., glial fibrillary acidic protein (GFAP), in two key corticolimbic regions, the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). These regions comprise anatomically and functionally different subregions, i.e., the prelimbic (PL) and infralimbic (IL) cortex of the mPFC, the shell and core subregions of the NAc. However, ethanol effects on GFAP expression within these subregions remain largely unknown. In addition, effects of pharmacological manipulation of astrocytes on alcohol drinking have been understudied. Western blot was conducted to determine GFAP expression in subregions of the mPFC and NAc after chronic ethanol drinking. Fluorocitrate, an astrocyte-specific metabolic inhibitor, was administered to inhibit astrocytes and was tested on ethanol drinking. Ethanol drinking enhanced GFAP protein expression in the PL cortex and NAc core, but not in the IL cortex or NAc shell. Intra-ventricular administration of fluorocitrate reduced ethanol intake and preference, but increased water consumption during choice ethanol drinking. In addition, fluorocitrate did not affect total fluid consumption or basal locomotor activity. These results indicate that chronic ethanol drinking induced GFAP elevation in a subregion-specific manner within the mPFC and NAc, and that metabolic inhibition of astrocytes selectively attenuated ethanol drinking without non-specific effects on water drinking or general activity. Together, these results suggest that astrocytes may play an important role in ethanol drinking. HighlightsO_LIEthanol drinking enhanced GFAP levels in the PL cortex and NAc core. C_LIO_LIFluorocitrate inhibition of astrocytes reduced intermittent ethanol drinking. C_LIO_LIFluorocitrate did not alter total fluid consumption or basal locomotor activity. C_LI

Excessive alcohol drinking is a leading cause of preventable death in the United States. High alcohol consumption and persistent drinking despite adverse events, also known as compulsive drinking, are key criteria that contribute to the development and progression of alcohol use disorder (AUD). There is a clear need to better understand the mechanisms that support these related but distinct behaviors. The serotonin (5-HT) system has been associated with alcohol consumption and risk of alcohol dependence, however given the complexity of this system, there remains much to discover regarding specific alcohol related phenotypes. The current study uses a combination of volitional home-cage drinking and operant conditioning to phenotype mice based on ethanol intake and persistence of alcohol drinking following quinine adulteration, a model to study compulsive drinking. Brain tissue of 10 regions known to be implicated in regulating executive function, reward, and stress was collected, and gene expression of serotonergic receptors, transporters, and enzymes was quantified. Three opioid receptors were included given their well-established roles in alcohol-related behaviors and interactions with the 5HT system. Region-specific gene expression patterns emerged, with serotonergic and opioid receptor expression differentially associated with alcohol drinking phenotype. 5-HT and opioid receptors displayed opposing directionality across regions, consistent with functional heterogeneity within the system. These findings identify region-specific molecular alterations following chronic alcohol that may contribute to individual differences in alcohol drinking phenotypes, highlighting candidate targets for biomarkers of increased alcohol use disorder susceptibility or as interventions aimed at preventing the progression of AUD.

The Reln gene encodes the extracellular glycoprotein Reelin that regulates synaptic plasticity and activity-dependent gene expression with implications in several neuropsychiatric disorders, including substance use disorder. While reduced Reln expression alters responses to psychostimulants and cannabinoid, its role in opioid-related behaviors remains unknown. Here, we examined whether Reln haploinsufficiency modifies behavioral and molecular responses to the synthetic opioid fentanyl. Heterozygous Reeler (Reln+/-) mice and wild-type littermates were assessed using using complementary contingent and non-contingent models of fentanyl exposure, including multi-phase fentanyl intravenous self-administration paradigm, conditioned place preference paradigm, locomotor assay, and dorsal striatal immediate early gene expression. Reln haploinsufficiency did not alter acquisition, extinction, or cue-induced reinstatement during self-administration, indicating stable opioid reinforcement and relapse-like behavior. Progressive ratio testing revealed a sex-dependent effect in which male Reln+/- mice showed reduced motivation for fentanyl compared to male wild-type mice. In contrast, following passive fentanyl exposure, Reln+/- mice exhibited enhanced fentanyl-induced locomotion and increased Fos immunoreactivity in the dorsal striatum, while CPP remained unchanged. Together, these findings demonstrate that Reln haploinsufficiency does not substantially modify opioid reinforcement or cue-driven drug seeking but enhances acute pharmacological sensitivity to fentanyl. These results identify Reln as a modulatory factor in opioid-responsive neural circuits that preferentially influences acute drug-evoked neuronal activation rather than the associative learning processes underlying opioid reinforcement.

RationaleRecent research on habits and skills has produced a wave of new theories regarding the shift in control from medial to lateral regions of the dorsal striatum, and how these regions are implicated in the selected and executed of action sequences. ObjectivesTo examine the comparative effects of muscimol/baclofen inactivation and dopamine D1 and D2 receptor agents in the dorsomedial (DMS) and dorsolateral (DLS) striatum on the performance of skilled action sequences. MethodsInfusions were made in well-trained rats using the five-step nose poke task to isolate the effects on initiation, execution and termination components of skilled action sequences. ResultsDLS inactivation produced sequencing deficits like those observed with pre-training lesions, indicating that the DLS is critical for both the acquisition and performance of sequences. Behaviour was unchanged following DMS inactivation, consistent with models of DMS disengagement following training. Infusions of D1 and D2 antagonists did not alter behaviour, however the D2 receptor agonist quinpirole increased sequence errors at a low dose and reduced sequences at the high dose in the DLS. DLS manipulations impaired sequence initiation and termination as well as reward transitions, while the chunking ballistic response pattern was largely unaltered, indicating that between-but not within-sequence actions rely on the DLS. ConclusionsSkilled action sequencing, including chunk transitions was dependent on DLS and its modulation by D2 receptors, but not on DMS function. Using a novel sequencing task, these results support the dissociable and dopamine-dependent role of the dorsal striatum subregions in performing skilled motor actions.

Preclinical evidence indicates that cocaine exerts acute and chronic effects on astrocyte functioning, which in turn modulate cocaine-related impacts on neural integrity and brain function. However, human evidence for astrocytic involvement in cocaine users (CU) remains limited. Glial fibrillary acidic protein (GFAP) is a marker of astrocyte activation with promising clinical utility in neurological conditions, yet its relevance in the addiction field is unclear. Hence, we investigated plasma GFAP levels in chronic CU (n=41) and cocaine-naive controls (HC; n=34) at baseline and after a 4-month follow-up. GFAP was assessed alongside plasma neurofilament light chain (NfL) levels, a marker of neuroaxonal injury previously associated with cocaine use in the same sample. Contrary to our hypothesis, we found no group differences in plasma GFAP concentrations between CU and HC. Neither cross-sectional nor longitudinal associations between GFAP levels and objective indices of cocaine use (derived from hair testing) were detected. However, exploratory analyses revealed higher plasma GFAP levels among CU with recent cocaine consumption (within the last 7 days), suggesting transient astrocytic responses following acute exposure. Additionally, GFAP and NfL were positively correlated across participants, supporting their functional association. Overall, these findings suggest that while GFAP might not be chronically elevated in CU, it may exhibit transient increases related to recent cocaine use. Further research is warranted to characterize the temporal dynamics and biological significance of these glial responses.

Background: Alcohol use disorder is associated with dysregulated glutamatergic signaling within mesocorticolimbic circuits that govern reinforcement and excessive ethanol intake. Group II metabotropic glutamate receptors (mGlu2/3) act primarily as presynaptic autoreceptors that regulate glutamate release. However, how voluntary alcohol intake alters mGlu2/3 expression within reward circuitry remains unclear. Methods and Results: We examined the effects of operant alcohol self-administration on mGlu2/3 protein expression and assessed the functional impact of group II receptor modulation on binge-like ethanol intake. Male C57BL/6J mice self-administered sweetened ethanol or sucrose under behaviorally matched conditions for 35 days. Immediately after the final session, tissue punches from the nucleus accumbens (NAc), amygdala, and prefrontal cortex were collected for Western blot analysis. Operant ethanol self-administration selectively reduced mGlu2/3 protein expression in the NAc, with no changes detected in the amygdala or prefrontal cortex. Both monomeric and dimeric mGlu2/3 protein levels were reduced, and a composite index revealed coordinated downregulation of receptor expression. In separate cohorts, systemic administration of the mGlu2/3 agonist LY379268 dose-dependently reduced binge-like ethanol intake in a limited-access home-cage drinking model, whereas positive allosteric modulation of mGlu2 receptors with LY487379 was ineffective. Conclusions: These results show that low-dose operant ethanol self-administration produces an ethanol- and region-specific reduction of mGlu2/3 protein expression in the NAc and that pharmacological activation of group II receptors, potentially involving mGlu3-specific receptors, is sufficient to suppress binge-like ethanol consumption. These data identify presynaptic mGlu2/3 dysregulation as a mechanism contributing to ethanol-related behaviors and support group II metabotropic glutamate receptors as therapeutic targets for alcohol use disorder.

Previous research indicates that chronic intraperitoneal (ip) administration of the GABAB receptor agonist baclofen reduces body weight gain in rats without altering daily food intake. The present study was undertaken to extend these observations by investigating the effects of chronic ip administration of the potent GABAB receptor agonist 3-aminopropyl (methyl) phosphinic acid (SKF-97541) on daily changes on body weight and food intake in free feeding rats. The animals were injected ip once daily with SKF-97541 (0.1 mg / kg for 5 days, followed by 0.2 mg / kg for 8 days; Experiment 1) or SKF-97542 (0.4 mg / kg) for 17 days (Experiment 2). Control animals received physiological saline in both experiments. While daily food intake did not differ significantly between groups, the SKF-97541 group exhibited significant reductions in body weight gain compared to controls. These results extend previous findings and show that systemic administration of SKF-97541 suppresses weight gain independently of caloric intake, and lend further support to the hypothesis that GABAB receptor agonists decrease body weight primarily by increasing metabolic rate.