Pharmacology Biochemistry and Behavior

Current treatments for opioid use disorder (OUD) have major barriers to access. As such, researching new potential therapies for OUD is important to public health. Previous research has implicated glucagon-like peptide-1 (GLP-1) receptor agonists in decreasing the use of addictive substances by animals. In this study, female Wistar rats (N=32) were surgically implanted with jugular catheters and trained to self-administer fentanyl at a fixed-ratio 1 (FR1) schedule of reinforcement for 21 sessions under short- (ShA; 1 hour) or long-access (LgA; 8 hours) conditions. Next, the animals received injections of semaglutide (0.1 mg/kg, s.c.) or saline (0.9% NaCl, s.c.) prior to another FR1 session. The animals underwent a progressive ratio (PR) schedule of reinforcement while receiving saline (i.v.) or fentanyl (0.625-10 {micro}g/kg/inf, i.v.) and semaglutide (0.1 mg/kg, s.c.) or saline (s.c.). Next, the animals underwent a semaglutide (0-0.1 mg/kg, s.c.) dose response procedure at FR1 and a single dose of fentanyl (2.5 {micro}g/kg/inf, i.v.). Following drug discontinuation, spontaneous locomotor activity and withdrawal-like symptoms were measured. Semaglutide dose-dependently decreased fentanyl rewards under ShA and LgA conditions (p<0.05). Under a PR, semaglutide significantly decreased breakpoint (p<0.05), suggesting semaglutide decreases motivation to self-administer fentanyl. Semaglutide-treated ShA animals displayed significantly less withdrawal-like behavior (p<0.05) but not LgA animals. Overall, these findings suggest semaglutide may modulate motivation to seek opioid reward and could be useful in the development of pharmacotherapies to address OUD.

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.

BackgroundIntravenous self-administration (IVSA) of opioids by rats has been shown frequently to exhibit no sex differences, in many cases a higher intake of females, and only rarely higher rates in males. A diversity of methodological parameters (opioid identity, training doses, rat strain, session duration) makes it difficult to identify consistent contributions to these outcomes. ObjectiveTo determine if Heterogeneous Stock (HS) rats derived from 8 founder strains differ by sex in the IVSA of opioids. MethodsMale and female Heterogeneous Stock (N=7-8 per sex) rats were permitted to self-administer heroin (20 {micro}g/kg/infusion) in 2 hour sessions under a Fixed Ratio 1 schedule of reinforcement. After acquisition, animals completed sessions in which different infusion doses of heroin (0, 15, 30, 60, 120 {micro}g/kg/infusion), oxycodone (0, 30, 60, 150, 300 {micro}g/kg/infusion) and fentanyl (0, 0.625, 1.25, 2.5, 5.0 {micro}g/kg/infusion) were assessed. Next, animals were evaluated on doses of heroin (15, 30, 60, 120 {micro}g/kg/infusion), oxycodone (30, 60, 150, 300 {micro}g/kg/infusion) and fentanyl (0.625, 1.25, 2.5, 5.0 {micro}g/kg/infusion) under a Progressive Ratio schedule. Anti-nociceptive effects of heroin (0.56-2.4 mg/kg, s.c.) were examined with a warm water tail-withdrawal assay. ResultsFemale HS rats consistently self-administered more infusions of opioids, including heroin during acquisition, all three opioids during FR-1 dose substitution and of oxycodone and fentanyl in the PR procedure. Male rats were moderately more sensitive to the anti-nociceptive effects of heroin. ConclusionsFemale rats drawn at random from a genetically diverse population self-administer opioids at higher rates than their male counterparts.

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

BackgroundEvidence suggests steeper accelerating opioid-related overdose, and non-medical use rates, in middle aged men in recent years compared with younger cohorts. Little is known about whether this is driven by age-related differences in the effects of opioids compared with socio-cultural factors driving non-medical consumption. Rodent models can be useful for dissociating biological from psychosocial factors, however, only minimal evidence exists on the effects of opioids in middle-age rats. ObjectiveTo determine if the anti-nociceptive and rewarding effects of opioids differ between adult and middle-age rats. MethodsFemale and male Wistar rats were obtained in early adulthood and examined across 4 to 11 months of age for nociceptive responses to heroin (0-1.56 mg/kg, s.c.) using a warm-water tail withdrawal assay. Subgroups (N=8 per group) were initiated on intravenous self-administration (IVSA) of heroin at either 5 months or 12 months of age. ResultsAnti-nociceptive effects of heroin did not differ across age. Female rats that initiated IVSA in early adulthood or middle-age obtained significantly more infusions of heroin than male rats of the same age during acquisition, and in dose-substitution under a FR1 schedule. Male, but not female, rats that initiated IVSA in middle age self-administered less heroin then rats that initiated in early adulthood; this was observed in acquisition and in dose-substitution. DiscussionThis study shows that opioid reward is diminished in middle aged male rats. It also found that middle age rats can be used effectively to model opioid-related outcomes, including drug seeking using the IVSA procedure.

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.

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

RationaleThe progression of psychostimulant abuse is associated with a shift from recreational to habitual use (R2H-shift). Because this R2H-shift can be modeled using behavioral economics, we developed a novel Behavioral Economic model for the Analysis of Self-administration Time-curve (BEAST) to obtain R2H-shift variable(s). The relationship(s) between R2H-shift variables and drug intake (under normal and/or punishment conditions) is/are unknown. Our goal was to determine if the R2H-shift variable and intake variables obtained during the initial self-administration training phase were related to 1) drug intake at that time, and subsequent drug intake under 2) normal, 3) punishment, 4) post-punishment, and 5) price-constrained conditions. MethodLong Evans rats self-administered methamphetamine (METH, males n = 16, females n = 14), sucrose (males n = 22, females n = 22) and/or saline (males n = 3, females n = 10) under FR1 for 6 h per day for 20 days to obtain 1) followed by the assessment of subsequent drug intake under different conditions (2-5 above). We obtained all variables referenced above. We determined the relationships between all variables (multivariate analysis). ResultsThere were no sex differences detected in the METH and sucrose studies. For METH and sucrose, prior drug intake levels could predict drug intake under normal/punishment but not under price-constrained conditions. The R2H-shift variable could predict drug intake under a consumption-price curve but could not predict intake under normal/punishment conditions. ConclusionsWhile related to economic demand, the recreational-to-habitual shift rate was unrelated to drug intake levels (under normal and punishment conditions).

Chronic pain and nicotine use frequently co-occur, and individuals with chronic pain often experience greater difficulty quitting. Therefore, we examined nicotine withdrawal behaviors and analgesic-like effects in pain-naive and chronic pain conditions. Adult male and female rats underwent chronic constriction injury or sham surgery. After pain establishment, rats received twice-daily subcutaneous nicotine (0.3 or 0.7 mg/kg) or saline for 14 days. 24 h after the final injection, withdrawal was assessed, including physical signs and anxiety-like behavior. Depressive-like responses were evaluated at 72 h. Pain sensitivity and nicotines analgesic-like effects were assessed throughout. Chronic pain increased physical signs of withdrawal in both sexes, with greater effects in females. It also induced anxiety-like behavior in controls of both sexes. In rats with comorbid chronic pain and withdrawal, anxiety-like behavior was further enhanced in males, whereas females showed variable responses across assays, with increases or decreases depending on the test. Chronic pain induced depressive-like behavior in males but not in females. During withdrawal, depressive-like responses in males with chronic pain were not greater than those in the chronic pain alone group, while chronic nicotine exposure reduced depressive-like behavior in females. Nicotine produced acute analgesic-like effects that diminished over time in both pain-naive and chronic pain conditions, indicating tolerance. In pain-naive rats, repeated nicotine exposure induced mechanical hypersensitivity. Chronic pain intensified nicotine withdrawal severity in a nicotine concentration- and sex-dependent manner. These findings highlight the importance of considering pain status and sex when developing effective cessation strategies, particularly for individuals with comorbid chronic pain. SummaryChronic pain exacerbates nicotine withdrawal severity. Chronic nicotine exposure induces pain hypersensitivity and tolerance to analgesic effects. These effects vary by nicotine concentration and sex.

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.

Sweetened alcoholic beverages are thought to contribute to developing Alcohol Use Disorder by increasing palatability. One monosaccharide, glucose, readily enters the brain more than fructose and directly impacts the activity of central neurons. The objective of this study is to determine the impact of glucose versus fructose on alcohol drinking patterns in female and male rats. Rats drank alcohol cocktails (1.25%-10%) containing either glucose or fructose (10%) in 4-hour sessions. We sought to parse orosensory effects from post-ingestive central effects by analyzing drinking microstructure. We compared measures of palatability and post-ingestive feedback between early and later in the session when brain levels of alcohol and glucose are different. We found that rats of both sexes drank more low alcohol glucose cocktails than cocktails containing fructose by volume and by overall calories. When considering the dose of alcohol, glucose potentiated alcohol intake by shifting the dose-response curve leftward compared to similar fructose cocktails. We found that drinking patterns associated with palatability remained stable for both types of cocktails over the entire drinking session. In contrast, post-ingestive behavior related to brain mediated satiety or positive feedback showed a greater influence of the session time, as well as a greater interaction with sex. Overall, our results suggest that glucose and alcohol interact to impact central regulation of cocktail drinking. This highlights that the type of sugar within cocktails interacts and ultimately have different effects on brain regulated alcohol drinking.

BackgroundIndividual vulnerability to addiction is driven by neuroadaptations within dopaminergic circuits. G protein-coupled receptor kinases (GRKs), specifically GRK2 and GRK3 regulate D2 receptor (D2R) signaling and trafficking, but their role in amphetamine (AMPH)-induced locomotor sensitization remains unclear. This study aimed to determine whether GRK2/3 inhibition alters locomotor sensitization, and its associated molecular correlates across striatal regions. MethodsAdult rats (n = 39) were assigned to saline, acute AMPH, or repeated AMPH groups and received intraperitoneal administration of vehicle or the GRK2/3 inhibitor Cmpd101 (1.0 mg/kg intraperitoneally, i.p.). Locomotor activity was assessed under basal and injection conditions to evaluate sensitization. Protein levels of D2R, GRK2, and GRK5 were quantified across striatal regions using Western blot and analyzed with linear mixed models. ResultsCmpd101 did not significantly affect the acute hyperlocomotor effects of AMPH or the expression of AMPH sensitization. At the molecular level, Cmpd101 had no effect on D2R levels and produced selective, region-dependent changes in GRK2 and GRK5. Notably, GRK2/3 inhibition altered the relationship between protein expression and the degree of locomotor sensitization in a region-specific manner, rather than inducing consistent changes in absolute protein levels. ConclusionsGRK2/3 inhibition by Cmpd101 produces region-specific molecular effects and reshapes protein-behavior relationships without significantly altering locomotor sensitization. These findings support a model in which GRKs act as context-dependent modulators of dopaminergic signaling rather than direct drivers of behavioral output.

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are chronic psychiatric disorders that have overlapping symptomology and risk factors, including altered motivation and impulsive behavior. Inescapable exposure to a predator odor stressor (2,3,5-Trimethyl-3-Thiazoline (TMT)) produces PTSD-like symptomology in rats. Individual differences in stress-coping behaviors such as freezing and defensive digging during TMT exposure can predict long-term differences in alcohol-related behaviors and altered neurobiology. Here, we sought to evaluate the relationship between stress coping behavior during TMT exposure and different aspects of decision making. In Experiment 1, male and female rats were trained on an adjusting-amounts delay discounting task, and delay discounting curves were established before and >2 weeks after TMT exposure. In Experiment 2, female rats were trained to self-administer alcohol and sucrose in a concurrent choice procedure. Lever responses and preference for alcohol over sucrose were evaluated before and >2 weeks after TMT exposure, and then motivation for competing reinforcers was evaluated using progressive ratios. Active coping (digging) during TMT exposure was correlated with increased post-TMT impulsive choice (Experiment 1), reduced sucrose lever responses both before and after TMT exposure (Experiment 2), and reduced sucrose lever breakpoint (Experiment 2). Additionally, TMT-exposed rats had increased motivation for both alcohol and sucrose self-administration when available concurrently (Experiment 2). Overall, these findings suggest that behavior prior to and during a stressful experience can predict susceptibility to negative effects on decision making, which may help future studies identify the neurobiology underlying risk for aberrant reward-related behaviors after a traumatic event.

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.

BackgroundAlcohol use disorder (AUD) is linked to increased neuroinflammation. Alcohol (ethanol) may activate toll-like receptors, which leads to the release of inflammatory molecules that could influence AUD-related behaviors, such as increased alcohol intake. Activation of toll-like receptor 3 (TLR3) by Polyinosinic:polycytidylic acid (Poly(I:C) or PIC) is associated with escalation of alcohol consumption in male, but not female F1 hybrid mice from reciprocal crosses between FVB/NJ (FVB) and C57BL/6J (B6) strains. Little is known about the underlying mechanisms of these sex-specific behavioral effects. In this study, we investigated the effects of TLR3 activation by PIC on temporal profiles of several pro- and anti-inflammatory molecules in the blood and brain of FVB/B6 F1 hybrid male and female mice at multiple time points. We hypothesized that TLR3 - dependent immune profiles would differ between males and females, which may, at least in part, explain the observed differences in drinking behavior. MethodsMale and female FVB/B6 F1 hybrid alcohol-naive mice were injected intraperitoneally with PIC (10 mg/kg) or saline. Blood and perfused brain tissues from the prefrontal cortex (PFC) and striatum were collected at 6-, 24-, and 48-hours post-injection. The expressions of Ccl2, Ccl5, Tnf, Il-6, Il-1{beta}, Ifng, Ifnb1, and Mmp9 genes were analyzed using qPCR. Protein levels of a subset of these molecules and IL-17r/a, IL-4, and IL-10 were measured in striatal samples from the same animals using ELISA. ResultsActivation of TLR3 by PIC triggered time-dependent, sex- and tissue-specific responses in immune genes and their proteins. PIC induced a time-dependent increase in expression of majority of the genes peaking at the 6 hr time point. Temporal immune profiles for pro-inflammatory chemokines, Ccl2 and Ccl5 differed between males and females in the PFC and striatum, suggesting possible sex-specific effects of these molecules on behavior. Protein levels of CCL2, CCL5, and IL-6 increased in the striatum of both sexes and correlated strongly with gene expression, with females showing somewhat higher protein fold changes. MMP-9, a key regulator of blood-brain barrier (BBB) permeability and synaptic plasticity, showed an increase in protein levels, but not mRNA levels in striatum. This pattern suggests altered blood-brain barrier (BBB) permeability, although this would require further investigation. ConclusionOur results revealed distinct TLR3-dependent immune gene and protein expression profiles in blood and brain between males and females and suggested different roles for these molecules in regulating alcohol consumption. We identified CCL2, CCL5 and MMP-9 as target molecules for investigating sex-specific behavior in the immune modulation of alcohol consumption.

"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.

RationalePrenatal alcohol exposure (PAE) can result in Fetal Alcohol Spectrum Disorder (FASD), which consists of a group of diagnosable medical conditions that can include an increased risk for anxiety disorders and/or alcohol misuse, and sensory issues, such as increased mechanical sensitivity. ObjectiveThis study investigated how a single moderate PAE on gestational day 12 (G12) alters anxiety-like behavior, ethanol (EtOH) intake, and mechanical sensitivity across the lifespan of Sprague Dawley rats. MethodsPregnant dams were exposed to vaporized EtOH or room air (control) for 6 hours (BECs [~]108 mg/dL). Testing in male and female offspring began at three different ages: juveniles ([~]postnatal day (P) 25), adolescents ([~]P45) and adults ([~]P80). ResultsThe greatest PAE effects were observed in adolescent animals, with alterations in anxiety-like behaviors demonstrated in the light-dark box and elevated plus maze. Additionally, adolescent female animals consumed more sweetened EtOH compared to males. However, PAE adolescent animals consuming less sweetened EtOH compared to their counterparts, which was also observed in adult PAE females. Interestingly, this effect is reversed in juvenile and adolescent males when tested with unsweetened EtOH, with juvenile females consuming more EtOH also. Finally, PAE and air animals exhibited increased mechanical sensitivity following post-natal EtOH consumption across all ages. ConclusionThese data demonstrate that there are age- and sex-specific effects of PAE on anxiety-like behaviors, EtOH intake, and mechanical sensitivity that are more distinct in adolescent animals.