Focus on fentanyl in females: Sex and gender differences in the physiological and behavioral effects of fentanyl.

The prevalence of opioid use disorder and overdose continues to harm the U.S. population and is further exacerbated by the use of the synthetic opioid, fentanyl, and its analogs. Gender differences in the effects of fentanyl are not well understood. The present article reviews evidence for gender and sex differences in the physiological and behavioral effects of fentanyl in humans and animals. Biological sex seems to be a foundational driver in addiction vulnerability and affects mechanisms related to opioid use including fentanyl. Fentanyl has distinct pharmacodynamics and enhanced efficacy relative to other opioids that highlights the need to investigate how females may be uniquely altered by its use. Behavioral and physiological responses to fentanyl are found to differ by sex and gender in many cases, including outputs like affective symptoms, analgesia, tolerance, and withdrawal emphasizing the need for further research about the role of biological sex on fentanyl use.

Paternal alcohol exposure attenuates maintenance and reinstated operant responding for alcohol in the offspring of rats.

BACKGROUND: The heritability of alcohol use disorder is close to 50%, yet common genetic variants account for less than 5% of risk. The missing heritability may reflect environmental exposure in the parents prior to conception. Indeed, paternal alcohol exposure has many behavioral and biological consequences for rodent offspring. We recently found that paternal alcohol exposure attenuated the acquisition of operant alcohol self-administration in offspring of rats of both sexes. Here we test whether this effect extends to other phases of operant self-administration thought to model motivation, craving, and relapse. METHODS: Wistar male rats exposed to alcohol vapors or air for 6 weeks were mated with alcohol-naïve females 8 weeks later. The adult offspring were trained to lever press for alcohol and tested under several conditions: (1) maintenance responding under a progressive ratio schedule, (2) extinction responding due to removal of the alcohol delivery contingency, (3) reinstatement of extinguished responding in the presence of alcohol-associated cues, and (4) reinitiation of lever press responding for alcohol delivery under fixed and progressive ratio schedules. RESULTS: Alcohol-sired offspring showed reduced responding under the progressive ratio schedule and blunted cue-induced reinstatement of extinguished responding. Alcohol-sired offspring also emitted fewer responses during extinction sessions and did not reinitiate responding to the same extent as control-sired rats after alcohol delivery was restored. CONCLUSIONS: Across all conditions, paternal alcohol exposure led to a reduction in the reinforcing effects of alcohol in offspring. These results are consistent with studies conducted with paternal cocaine exposure except that here we find effects in rats of both sexes.

An Immunconjugate Vaccine Alters Distribution and Reduces the Antinociceptive, Behavioral and Physiological Effects of Fentanyl in Male and Female Rats.

Fentanyl (FEN) is a potent synthetic opioid associated with increasing incidence of opioid use disorder (OUD) and fatal opioid overdose. Vaccine immunotherapy for FEN-associated disorders may be a viable therapeutic strategy. Here, we expand and confirm our previous study in mice showing immunological and antinociception efficacy of our FEN vaccine administered with the adjuvant dmLT. In this study, immunized male and female rats produced significant levels of anti-FEN antibodies that were highly effective at neutralizing FEN-induced antinociception in the tail flick assay and hot plate assays. The vaccine also decreased FEN brain levels following drug administration. Immunization blocked FEN-induced, but not morphine-induced, rate-disrupting effects on schedule-controlled responding. Vaccination prevented decreases on physiological measures (oxygen saturation, heart rate) and reduction in overall activity following FEN administration in male rats. The impact of FEN on these measures was greater in unvaccinated male rats compared to unvaccinated female rats. Cross-reactivity assays showed anti-FEN antibodies bound to FEN and sufentanil but not to morphine, methadone, buprenorphine, or oxycodone. These data support further clinical development of this vaccine to address OUD in humans.

Paternal alcohol exposure has task- and sex-dependent behavioral effect in offspring.

BACKGROUND: Endophenotypes for alcohol use disorder are well known and may reflect paternal exposure effects passed down to offspring via epigenetic mechanisms. Previously, we showed that paternal alcohol exposure prior to conception attenuates the acquisition of operant alcohol self-administration. We now test whether paternal alcohol exposure alters their offsprings' behavioral responses to alcohol (endophenotypes) and global DNA methylation levels in reward-related brain regions. METHODS: Adult male rats were exposed to alcohol vapors or air for 6 weeks and mated with alcohol-naïve females 8 weeks later. Adult male and female offspring of the alcohol- and control-sired litters were tested on three behaviors 30 m after gavage with water or alcohol (1.5 g/kg): open field, elevated plus maze, and accelerating rotarod. Global DNA methylation levels in sperm, nucleus accumbens, and prefrontal cortex were examined in male sires and in another group of offspring. RESULTS: Alcohol-sired males showed less anxiety-like behavior in the elevated plus maze that was not affected by alcohol administration. By contrast, alcohol had anxiolytic effects in the open field in male offspring only with no paternal alcohol effect. Neither paternal alcohol exposure nor alcohol administration altered locomotor activity in either sex. Sex-specific effects of paternal alcohol exposure were seen in the rotarod test. Alcohol-sired male offspring showed blunted sensitivity to the alcohol's motor-impairing effects, whereas alcohol-sired female offspring showed enhanced sensitivity. Global DNA methylation was altered in the sperm of alcohol-exposed males, but no changes were seen in their offspring. CONCLUSIONS: Paternal alcohol exposure prior to conception has sex- and task-dependent effects on unconditioned behaviors in their offspring.

Active and Passive Immunization with an Anti-Methamphetamine Vaccine Attenuates the Behavioral and Cardiovascular Effects of Methamphetamine.

BACKGROUND: Methamphetamine use disorder (MUD) is a growing health concern with no FDA-approved treatment. The present series of studies build upon our previous work developing an anti-methamphetamine (MA) vaccine for MUD. We determined the effects of a formulation that included tetanus-toxoid (TT) conjugated to succinyl-methamphetamine (TT-SMA) adsorbed onto aluminum hydroxide (alum) in combination with the novel Toll-Like Receptor-5 agonist, entolimod. METHODS: Mice were vaccinated (0, 3, 6 weeks) with TT-SMA+alum and various doses of entolimod to determine an optimal dose for enhancing immunogenicity against MA. Functional effects were then assessed using MA-induced locomotor activation in mice. Experiments using passive immunization of antibodies generated by the vaccine tested its ability to attenuate MA-induced cardiovascular effects and alter the reinforcing effects of MA in an MA-induced reinstatement of a drug seeking model of relapse in male and female rats. RESULTS: Antibody levels peaked at 10 weeks following vaccination with TT-SMA+alum combined with entolimod (1, 3 and 10 µg). MA-induced locomotor activation was significantly attenuated in vaccinated vs. unvaccinated mice and antibody levels significantly correlated with ambulation levels. Passive immunization decreased mean arterial pressure following MA dosing in rats of both sexes but did not alter heart rate. Passive immunization also attenuated the ability of MA to reinstate extinguished drug-seeking behavior in male and female rats. Results support further development of this vaccine for relapse prevention for individuals with MUD.

Paternal alcohol exposure reduces acquisition of operant alcohol self-administration and affects Bdnf DNA methylation in male and female offspring.

Familial transmission of alcohol use disorder reflects genetic and environmental factors. Paternal alcohol exposure may affect rodent offspring via epigenetic modifications transmitted through the male germ line. While such exposure alters alcohol sensitivity in mouse offspring, no studies examined if it impacts the development of operant alcohol self-administration in rats. We exposed male (sires) Wistar rats to chronic intermittent ethanol in vapour chambers (16 h/day; 5 days/week) or to air for 6 weeks. Eight weeks later, rats were mated with alcohol-naive females. Adult alcohol- and control-sired F1 offspring were assessed in acquisition of alcohol self-administration in which increasing alcohol concentrations (2.5%, 5% and 10%, v/v) were delivered after one lever press (fixed ratio 1 or FR1). Prior to alcohol sessions, rats were trained to lever press for food delivery under an FR1 schedule of reinforcement. DNA methylation levels of the brain derived neurotrophic factor (Bdnf) gene were measured in sperm, nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) in sires and in offspring. Alcohol-exposed sires had lower Bdnf DNA methylation levels in NAc and greater methylation levels in mPFC. Although this pattern was not recapitulated in offspring, alcohol-sired offspring of both sexes did show aberrant Bdnf DNA methylation patterns compared to control-sired offspring. Alcohol-sired offspring self-administered less alcohol (5% and 10%) with no group differences in food responding. Results indicate that paternal alcohol exposure prior to conception protects against alcohol's initial reinforcing effects but the pattern of dysregulated Bdnf methylation in reward-related circuitry did not mimic changes seen in sires.

Immune receptor toll-like receptor 4 contributes to stress-induced affective responses in a sex-specific manner.

Stress activates innate immune Toll-like receptors (TLRs) and enhances susceptibility to depression, a condition that is more prevalent in females. The TLR4 receptor type is involved in inflammatory responses and its expression levels associate with depressive symptoms and their successful treatment. Yet, little preclinical research has examined the role of TLR4 in stress-induced affective responses to determine if these are sex-specific. One group per genotype of male and female Tlr4 knockout (KO) and wild type (WT) rats were exposed to predator odor in a place conditioning apparatus with others exposed to saline. Affective behaviors evaluated included distance traveled and center time in an open-field apparatus, sucrose preference and fluid intake in a two-bottle test, and conditioned place aversion to the odor-paired compartment. Predator odor exposed rats showed conditioned place aversion to the odor-paired compartment, demonstrating predator odor was aversive. Such exposure led to anhedonia (decreased sucrose preference) across genotypes and sex. Predator odor exposure decreased distance traveled, an effect that was greater in KO rats, especially in females. Tlr4 deletion also resulted in sex-specific effects on anxiety-like behavior. Compared to WTs, female KO rats showed lower center time after predator odor exposure whereas genotype did not affect this response in male rats. Across litters, fewer male KO and heterozygous rats and more WT rats were born whereas female rats showed the typical genotype distribution. Results suggest predator odor alters affective behaviors, consistent with the preclinical literature, and deletion of Tlr4 enhances some stress-induced affective responses, often in a sex-specific manner.

Drugs and bugs: Negative affect, psychostimulant use and withdrawal, and the microbiome.

BACKGROUND AND OBJECTIVES: A growing body of literature demonstrates that the human microbiota plays a crucial role in health and disease states, as well as in the body's response to stress. In addition, the microbiome plays a role in psychological well-being and regulating negative affect. Regulation of negative affect is a factor in psychostimulant abuse disorders. We propose a risk chain in which stress leads to negative affect that places an individual at risk to develop or relapse to psychostimulant abuse disorder. Stress, negative affect, and psychostimulant use all alter the gut microbiome. METHODS: This review brings together the literature on affective disorders, stress, and psychostimulant abuse disorders to assess possible modulatory actions of the gut-brain axis to regulate these conditions. RESULTS: Studies reviewed across the various disciplines suggest that the dysbiosis resulting from drug use, drug withdrawal, or stress may cause an individual to be more susceptible to addiction and relapse. Probiotics and prebiotics reduce stress and negative affect. SCIENTIFIC SIGNIFICANCE: Treatment during the withdrawal phase of psychostimulant abuse disorder, when the microbiome is altered, may ameliorate the symptoms of stress and negative affect leading to a reduced risk of relapse to psychostimulant use.

The GABAB receptor positive allosteric modulator ASP8062 reduces operant alcohol self-administration in male and female Sprague Dawley rats.

RATIONALE: Pre-clinical evidence implicates the GABAergic system in mediating the reinforcing effects of alcohol and offers a therapeutic target for alcohol use disorder (AUD). The orthosteric GABAB receptor agonist baclofen decreases alcohol self-administration in animals and alcohol use in humans; however side effects limit its utility. Pre-clinical evidence shows positive allosteric GABAB receptor modulators also decrease alcohol self-administration without untoward side effects. OBJECTIVES: We assessed the impact of the novel GABAB-positive allosteric modulator ASP8062 and baclofen on operant alcohol self-administration and their potential non-specific effects. METHODS: The effects of ASP8062 (1 - 10 mg/kg, PO) and baclofen (0.3 - 3 mg/kg, IP) were evaluated in male and female rats lever pressing for alcohol (10%, w/v) under a fixed ratio 2 schedule of reinforcement. On the fourth consecutive day of vehicle, ASP8062 or baclofen administration, active and inactive lever presses, reinforcers earned, head entries, and estimated alcohol consumed were analyzed. Locomotor activity was assessed in separate groups of rats following dosing. RESULTS: Both ASP8062 and baclofen decreased alcohol self-administration and amount consumed (g/kg) in male and female rats. ASP8062 decreased operant alcohol self-administration to a greater extent in male rats, whereas baclofen was more efficacious in female rats. ASP8062 did not alter locomotor activity in either sex, whereas baclofen (3.0 mg/kg) decreased activity in male rats yet (1.0 mg/kg) increased activity in female rats. CONCLUSIONS: ASP8062 decreases alcohol reinforcement like baclofen but without non-specific effects which are influenced by sex. Results support further development of ASP8062 as a potential treatment for AUD in humans.

Fentanyl conjugate vaccine by injected or mucosal delivery with dmLT or LTA1 adjuvants implicates IgA in protection from drug challenge.

Fentanyl is a major contributor to the devastating increase in overdose deaths from substance use disorders (SUD). A vaccine targeting fentanyl could be a powerful immunotherapeutic. Here, we evaluated adjuvant and delivery strategies for conjugate antigen vaccination with fentanyl-based haptens. We tested adjuvants derived from the heat-labile toxin of E. coli including dmLT and LTA1 by intramuscular, sublingual or intranasal delivery. Our results show anti-fentanyl serum antibodies and antibody secreting cells in the bone-marrow after vaccination with highest levels observed with an adjuvant (alum, dmLT, or LTA1). Vaccine adjuvanted with LTA1 or dmLT elicited the highest levels of anti-fentanyl antibodies, whereas alum achieved highest levels against the carrier protein. Vaccination with sublingual dmLT or intranasal LTA1 provided the most robust blockade of fentanyl-induced analgesia and CNS penetration correlating strongly to anti-FEN IgA. In conclusion, this study demonstrates dmLT or LTA1 adjuvant as well as mucosal delivery may be attractive strategies for improving the efficacy of vaccines against SUD.

Persistence of Operant Responding for Food After Prior Cocaine Exposure in Fischer 344 But Not Lewis Rats.

BACKGROUND AND OBJECTIVES: Chronic cocaine exposure has differential neural effects in Fischer 344 (F344) vs Lewis inbred rats that may explain strain-dependent differences during acquisition vs maintenance of cocaine self-administration. We assessed whether prior cocaine exposure alters operant responding for food across various phases (acquisition, maintenance, extinction, spontaneous recovery, reinitiation) in these strains. METHODS: Lewis and F344 rats (N = 12) were administered three cocaine (15 mg/kg) or saline injections at hourly intervals for 3 consecutive days. Beginning the next day for 24 days, rats had access to operant chambers in which one lever depression resulted in the delivery of a food pellet. Then, four extinction sessions were conducted in which food was no longer available, but other stimulus conditions remained the same. After a 2-day break, spontaneous recovery was assessed over four sessions. Food delivery was then restored for 3 days to test reinitiation followed by a progressive ratio session. RESULTS: Lewis rats acquired the operant faster than F344 rats. F344 rats showed lower maintenance rates than Lewis rats but higher spontaneous recovery responding. Cocaine exposure caused persistence of responding during extinction in F344 but not Lewis rats. All groups reinitiated responding when food was available again and did not differ in final ratios completed under the progressive ratio schedule. DISCUSSION AND CONCLUSIONS: That prior cocaine exposure led to persistence of responding in F344 rats during extinction may reflect heightened contextual conditioning that interferes with the ability to extinguish responding. SCIENTIFIC SIGNIFICANCE: Results have implications for the genetic contribution to relapse-like behaviors. (Am J Addict 2021;00:00-00).

Methamphetamine exposure and its cessation alter gut microbiota and induce depressive-like behavioral effects on rats.

RATIONALE: Methamphetamine is a highly abused psychostimulant drug and its use remains a major public health concern worldwide with limited effective treatment options. Accumulative evidence reveals the influence of gut microbiota on the brain, behavior, and health as a part of the gut-brain axis but its involvement in modulating this substance use disorder remains poorly understood. OBJECTIVE: We sought to determine whether methamphetamine exposure and cessation or withdrawal alter the intestinal gut microbiota as well as characterize cessation-induced behavioral changes. METHODS: Male, Sprague-Dawley rats were administered methamphetamine (2 mg/kg; s.c.) or vehicle (n = 8 per group) twice per day for 14 consecutive days. On various days before, during, and after administration, fecal samples were collected and tests of anxiety- and depressive-like behaviors were conducted. RESULTS: Methamphetamine administration and cessation did not alter the relative abundance of bacteria but significantly changed the composition of gut bacteria through 16S rRNA sequencing. These changes were normalized after 7 days of methamphetamine cessation. Moreover, acute methamphetamine cessation induced depressive-like behavior, with an increase in immobility in the forced swim test but did not alter anxiety-like behaviors in tests of open field test or elevated plus maze. CONCLUSIONS: These findings provide direct evidence that methamphetamine and its cessation cause gut dysbiosis and that the latter associates with depressive-like behavior in rodents. Our observation will contribute to a better understanding of the function of gut microbiota in the process of substance use disorders and guide the choice of target therapeutics.

Sex and Age Effects on Neurobehavioral Toxicity Induced by Binge Alcohol.

Historically, most alcohol neurotoxicity studies were conducted in young adult males and focused on chronic intake. There has been a shift towards studying the effects of alcohol on the adolescent brain, due to alcohol consumption during this formative period disrupting the brain's developmental trajectory. Because the most typical pattern of adolescent alcohol intake is heavy episodic (binge) drinking, there has also been a shift towards the study of binge alcohol-induced neurobehavioral toxicity. It has thus become apparent that binge alcohol damages the adolescent brain and there is increasing attention to sex-dependent effects. Significant knowledge gaps remain in our understanding of the effects of binge alcohol on the female brain, however. Moreover, it is unsettling that population-level studies indicate that the prevalence of binge drinking is increasing among American women, particularly those in older age groups. Although study of adolescents has made it apparent that binge alcohol disrupts ongoing brain maturational processes, we know almost nothing about how it impacts the aging brain, as studies of its effects on the aged brain are relatively scarce, and the study of sex-dependent effects is just beginning. Given the rapidly increasing population of older Americans, it is crucial that studies address age-dependent effects of binge alcohol, and given the increase in binge drinking in older women who are at higher risk for cognitive decline relative to men, studies must encompass both sexes. Because adolescence and older age are both characterized by age-typical brain changes, and because binge drinking is the most common pattern of alcohol intake in both age groups, the knowledge that we have amassed on binge alcohol effects on the adolescent brain can inform our study of its effects on the aging brain. In this review, we therefore cover the current state of knowledge of sex and age-dependent effects of binge alcohol, as well as statistical and methodological considerations for studies aimed at addressing them.

Who's your daddy? Behavioral and epigenetic consequences of paternal drug exposure.

Substance use disorders (SUDs) reflect genetic and environmental factors. While identifying reliable genetic variants that predispose individuals to developing SUDs has been challenging, epigenetic factors may also contribute to the heritability of SUDs. Familial drug use associates with a wide range of problems in children, including an increased risk for developing a SUD. The implications of maternal drug use on offspring development are a well-studied area; however, paternal drug use prior to conception has received relatively little attention. Paternal exposure to several environmental stimuli (i.e. stress or diet manipulations) results in behavioral and epigenetic changes in offspring. The purpose of this review is to determine the state of the preclinical literature on the behavioral and epigenetic consequences of paternal drug exposure. Drug-sired offspring show several developmental and physiological abnormalities. These offspring also show deficits in cognitive and emotional domains. Examining sensitivity to drugs in offspring is a growing area of research. Drug-sired offspring are resistant to the rewarding and reinforcing properties of drugs. However, greater paternal motivation for the drug, combined with high drug intake, can result in addiction-like behaviors in offspring. Drug-sired offspring also show altered histone modifications and DNA methylation levels of imprinted genes and microRNAs; epigenetic-mediated changes were also noted in genes related to glutamatergic and neurotrophic factor signaling. In some instances, drug use resulted in aberrant epigenetic modifications in sire sperm, and these changes were maintained in the brains of offspring. Thus, paternal drug exposure has long-lasting consequences that include altered drug sensitivity in subsequent generations. We discuss factors (i.e. maternal behaviors) that may moderate these paternal drug-induced effects as well as ideas for future directions.

Early life stress and the propensity to develop addictive behaviors.

There is a vast literature on effects of early life manipulations in rodents much of which is aimed at investigating the long-term consequences related to emotion and cognition in adulthood. Less is known about how these manipulations affect responses reflective of alcohol (AUD) and substance (SUD) use disorders. The purpose of this paper is to review the literature of studies that employed early life manipulations and assessed behavioral responses to psychoactive substances, specifically alcohol, opiates, and stimulants, in rodents. While the findings with alcohol are more limited and mixed, studies with opiates and stimulants show strong support for the ability of these manipulations to enhance behavioral responsivity to these substances in line with epidemiological data. Some outcomes show sex differences. The mechanisms that influence these enduring changes may reflect epigenetic alterations. Several studies support a role for altered DNA methylation (and other epigenetic mechanisms) as biological responses to early environmental insults. The chemical changes induced by DNA methylation affect transcriptional activity of DNA and thus can have a long-term impact on the individual's phenotype. Such effects are particularly robust when they occur during sensitive periods of brain development (e.g., first postnatal weeks in rodents). We review this emerging literature as it relates to the known neurobiology of AUDs and SUDs and suggest new avenues of research. Such findings will have implications for the treatment and prevention of AUDs and SUDs and could provide insight into factors that support resiliency.

Preclinical efficacy of an anti-methamphetamine vaccine using E6020 adjuvant.

BACKGROUND AND OBJECTIVE: Methamphetamine (MA) substance use disorder (SUD) does not have an efficacious pharmacotherapy. We developed a MA vaccine and investigated its potential to attenuate MA induced responses. METHODS: We examined a novel adjuvant, E6020, a Toll-like receptor-4 (TLR-4) agonist combined with tetanus-toxoid conjugated to succinyl-methamphetamine (TT-SMA) adsorbed on aluminum hydroxide (alum). Adult BALB/c female mice received the vaccine and booster injections at weeks 0, 3, and 6. The efficacy of the vaccine was assessed by the level and affinity of anti-MA antibodies elicited, its ability to attenuate MA induced locomotor activation and its reduction in the amount of MA entering the brains of vaccinated mice. RESULTS: The TT-SMA vaccine containing alum and E6020 adjuvant produced anti-MA antibodies with nanomolar affinities and showed threefold greater peak titer levels than without E6020 (700 vs 250 µg/ml). These antibodies significantly decreased MA-induced locomotor activation (p < .05), and reduced the brain (p < .005) MA levels following MA administration in actively immunized mice. CONCLUSIONS: Thus, this anti-MA vaccine formulated with E6020 demonstrated effective functional protection against behavioral disruptions induced by MA. SCIENTIFIC SIGNIFICANCE: Together, anti-MA vaccine showing a promising improvement in the efficacy of the vaccine that could be an effective candidate vaccine for methamphetamine use disorder (MUD). Furthermore, combinations of adjuvants may be a tool to design vaccines for MA dependence in humans. (Am J Addict 2019;XX:1-8).

Naltrexone alters alcohol self-administration behaviors and hypothalamic-pituitary-adrenal axis activity in a sex-dependent manner in rats.

BACKGROUND: The mu-opioid antagonist, naltrexone (NTX), is a FDA-approved treatment for alcohol use disorder (AUD); however, the data on whether it differentially affects males vs. females are mixed. NTX increases hypothalamic-pituitary-adrenal (HPA) axis activity that associates with subjective responses to alcohol and craving in individuals with AUD. The present study tested for sex differences in the ability of NTX to decrease appetitive and consummatory behaviors in rats in operant alcohol self-administration. Because the opioid system and HPA axis are sexually dimorphic, we examined NTX's effect on adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels. METHODS: Male and female Sprague-Dawley rats (n's = 6-8) were trained to lever press for alcohol (10% v/v) under a fixed-ratio 2 schedule of reinforcement. NTX doses (0, 0.1-10 mg/kg) were assessed in tests conducted under a progressive ratio schedule of reinforcement. Separate groups of alcohol and water drinking rats (n's = 8) were used to assess NTX's (10 mg/kg) effects on HPA axis hormones. RESULTS: NTX decreased consummatory behaviors for alcohol in a dose-related manner, but not appetitive behaviors in males. In females, NTX decreased appetitive behaviors for alcohol in a dose-dependent manner, but only decreased consummatory behaviors at the highest (10 mg/kg) NTX dose. NTX increased ACTH levels in alcohol drinking females in diestrus, but not in other groups. However, NTX increased CORT levels for longer durations in alcohol drinking males relative to alcohol drinking females in diestrus. CONCLUSIONS: Our findings suggest that NTX selectively reduces consummatory behaviors for alcohol in males and appetitive behaviors in females, while also showing differential sex effects on HPA hormones.

Enhanced Hypothalamic NMDA Receptor Activity Contributes to Hyperactivity of HPA Axis in Chronic Stress in Male Rats.

Chronic stress stimulates corticotrophin-releasing hormone (CRH)-expressing neurons in the paraventricular nucleus (PVN) of the hypothalamus and leads to hypothalamic-pituitary-adrenal (HPA) axis hyperactivity, but the mechanisms underlying this action are unknown. Because chronic stress enhances N-methyl-d-aspartate receptor (NMDAR) activity in various brain regions, we hypothesized that augmented NMDAR activity contributes to the hyperactivity of PVN-CRH neurons and the HPA axis in chronic stress. We performed whole-cell patch-clamp recordings on PVN-CRH neurons expressing CRH promoter-driven enhanced green fluorescent protein in brain slices from rats exposed to chronic unpredictable mild stress (CUMS) and unstressed rats. CUMS rats had significantly higher expression levels of the NMDAR subunits GluN1 in the PVN than unstressed rats. Furthermore, puff NMDA-elicited currents, evoked NMDAR currents, and the baseline frequency of the miniature excitatory postsynaptic currents (mEPSCs) in PVN-CRH neurons were significantly larger in CUMS rats than in unstressed rats. The NMDAR-specific antagonist 2-amino-5-phosphonopentanoic acid (AP5) significantly decreased the frequency of mEPSCs of PVN-CRH neurons in CUMS rats but did not change the frequency or amplitude of mEPSCs in unstressed rats. Bath application of AP5 normalized the elevated firing activity of PVN-CRH neurons in CUMS rats but not in unstressed rats. In addition, microinjection of the NMDAR antagonist memantine into the PVN normalized the elevated corticosterone (CORT) levels in CUMS rats to the levels in unstressed rats, but did not alter CORT levels in unstressed rats. Our findings suggest that synaptic NMDAR activity is enhanced in CUMS rats and contributes to the hyperactivity of PVN-CRN neurons and the HPA axis.

Neuroadaptations of presynaptic and postsynaptic GABAB receptor function in the paraventricular nucleus in response to chronic unpredictable stress.

BACKGROUND AND PURPOSE: Chronic stress impairs GABAA (GABA type A) receptor-mediated inhibition in the hypothalamic paraventricular nucleus (PVN). It is not clear whether GABAB receptor function is also altered. We hypothesize that chronic stress alters GABAB receptor function in PVN corticotrophin-releasing hormone (CRH) neurons to control hypothalamus-pituitary-adrenal axis activity. EXPERIMENTAL APPROACH: Whole-cell patch clamp recordings were made of PVN-CRH neurons expressing eGFP driven by CRH promoter in brain slices from unstressed rats and rats exposed to chronic unpredictable mild stress (CUMS). KEY RESULTS: CUMS elevated the basal circulating corticosterone levels and increased the basal firing activity of PVN-CRH neurons. Microinjection of GABAB receptor agonist baclofen into the PVN suppressed the increased corticosterone levels in CUMS rats compared with unstressed rats. CUMS blunted the baclofen-induced inhibition on PVN-CRH neurons and outward currents in these neurons. Furthermore, CUMS reduced expression of GABAB1 (GABAB R1) protein in the PVN. Blocking NMDA receptors with AP5 restored the reduced baclofen-induced currents in CUMS rats but had no effect on GABAB1 expression. Furthermore, CUMS treatment augmented the baclofen-induced decrease in the frequency of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitor postsynaptic currents in PVN-CRH neurons. The GABAB receptor antagonist CGP55845 increased the firing activity of PVN-CRH neurons only in CUMS-treated rats and not in unstressed rats. CONCLUSIONS AND IMPLICATIONS: These findings suggest that chronic stress impairs postsynaptic GABAB receptor function but augments presynaptic GABAB receptor function in controlling glutamatergic and GABAergic synaptic inputs in PVN-CRH neurons.