Cocaine Exposure Increases Excitatory Synaptic Transmission and Intrinsic Excitability in the Basolateral Amygdala in Male and Female Rats and across the Estrous Cycle.

INTRODUCTION: Sex and ovarian hormones influence cocaine seeking and relapse vulnerability, but less is known regarding the cellular and synaptic mechanisms contributing to these behavioral sex differences. One factor thought to influence cue-induced seeking behavior following withdrawal is cocaine-induced changes in the spontaneous activity of pyramidal neurons in the basolateral amygdala (BLA). However, the mechanisms underlying these changes, including potential sex or estrous cycle effects, are unknown. METHODS: Ex vivo whole-cell patch clamp electrophysiology was conducted to investigate the effects of cocaine exposure, sex, and estrous cycle fluctuations on two properties that can influence spontaneous activity of BLA pyramidal neurons: (1) frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and (2) intrinsic excitability. Recordings of BLA pyramidal neurons were conducted in adult male and female rats and across the estrous cycle following 2-4 weeks of withdrawal from extended-access cocaine self-administration (6 h/day for 10 days) or drug-naïve conditions. RESULTS: In both sexes, cocaine exposure increased the frequency, but not amplitude, of sEPSCs and neuronal intrinsic excitability. Across the estrous cycle, sEPSC frequency and intrinsic excitability were significantly elevated only in cocaine-exposed females in the estrus stage of the cycle, a stage when cocaine-seeking behavior is known to be enhanced. CONCLUSIONS: Here, we identify potential mechanisms underlying cocaine-induced alterations in the spontaneous activity of BLA pyramidal neurons in both sexes along with changes in these properties across the estrous cycle.

Cocaine and chronic stress exposure produce an additive increase in neuronal activity in the basolateral amygdala.

Cocaine addiction is a chronic, relapsing disorder. Stress and cues related to cocaine are two common relapse triggers. We have recently shown that exposure to repeated restraint stress during early withdrawal accelerates the time-dependent intensification or "incubation" of cue-induced cocaine craving that occurs during the first month of withdrawal, although craving ultimately plateaus at the same level observed in controls. These data indicate that chronic stress exposure during early withdrawal may result in increased vulnerability to cue-induced relapse during this period. Previous studies have shown that chronic stress exposure in drug-naïve rats increases neuronal activity in the basolateral amygdala (BLA), a region critical for behavioral responses to stress. Given that glutamatergic projections from the BLA to the nucleus accumbens are critical for the incubation of cue-induced cocaine craving, we hypothesized that cocaine withdrawal and chronic stress exposure produce separate increases that additively increase BLA neuronal activity. To assess this, we conducted in vivo extracellular single-unit recordings from the BLA of anesthetized adult male rats following cocaine or saline self-administration (6 h/day for 10 days) and repeated restraint stress or control conditions on withdrawal days (WD) 6-14. Recordings were conducted from WD15 to WD20. Interestingly, cocaine exposure alone increased the spontaneous firing rate in the BLA to levels observed following chronic stress exposure in drug-naïve rats. Chronic stress exposure during cocaine withdrawal further increased firing rate. These studies may identify a potential mechanism by which both cocaine and chronic stress exposure drive cue-induced relapse vulnerability during abstinence.

mGlu5 inhibition in the basolateral amygdala prevents estrous cycle-dependent changes in cue-induced cocaine seeking.

Drug associated cues are a common relapse trigger for individuals recovering from cocaine use disorder. Sex and ovarian hormones influence patterns of cocaine use and relapse vulnerability, with studies indicating that females show increased cue-induced craving and relapse vulnerability compared to males. In a rodent model of cocaine craving and relapse vulnerability, cue-induced cocaine seeking behavior following weeks of withdrawal from extended-access cocaine self-administration is higher in females in the estrus stage of the reproductive (estrous) cycle (Estrus Females) compared to both Males and females in all other stages (Non-Estrus Females). However, the neuronal substrates and cellular mechanisms underlying these sex differences is not fully understood. One region that contributes to both sex differences in behavioral responding and cue-induced cocaine seeking is the basolateral amygdala (BLA), while one receptor known to play a critical role in mediating cocaine seeking behavior is metabotropic glutamate receptor 5 (mGlu5). Here we assessed the effects of BLA mGlu5 inhibition following prolonged withdrawal from cocaine self-administration on observed estrous cycle-dependent changes in cue-induced cocaine seeking behavior. We found that BLA microinjections of the mGlu5 antagonist MTEP selectively reduced the enhanced cue-induced cocaine seeking normally observed in Estrus Females while having no effect on cocaine seeking in Males and Non-Estrus Females. These findings identify a unique interaction between cocaine-exposure, estrous cycle fluctuations and BLA mGlu5-dependent transmission on cue-induced cocaine seeking behavior.

Effects of sex and estrous cycle on intravenous oxycodone self-administration and the reinstatement of oxycodone-seeking behavior in rats.

Introduction: The increasing misuse of both prescription and illicit opioids has culminated in a national healthcare crisis in the United States. Oxycodone is among the most widely prescribed and misused opioid pain relievers and has been associated with a high risk for transition to compulsive opioid use. Here, we sought to examine potential sex differences and estrous cycle-dependent effects on the reinforcing efficacy of oxycodone, as well as on stress-induced or cue-induced oxycodone-seeking behavior, using intravenous (IV) oxycodone self-administration and reinstatement procedures. Methods: In experiment 1, adult male and female Long-Evans rats were trained to self-administer 0.03 mg/kg/inf oxycodone according to a fixed-ratio 1 schedule of reinforcement in daily 2-h sessions, and a dose-response function was subsequently determined (0.003-0.03 mg/kg/inf). In experiment 2, a separate group of adult male and female Long-Evans rats were trained to self-administer 0.03 mg/kg/inf oxycodone for 8 sessions, followed by 0.01 mg/kg/inf oxycodone for 10 sessions. Responding was then extinguished, followed by sequential footshock-induced and cue-induced reinstatement tests. Results: In the dose-response experiment, oxycodone produced a typical inverted U-shape function with 0.01 mg/kg/inf representing the maximally effective dose in both sexes. No sex differences were detected in the reinforcing efficacy of oxycodone. In the second experiment, the reinforcing effects of 0.01-0.03 mg//kg/inf oxycodone were significantly attenuated in females during proestrus/estrus as compared to metestrus/diestrus phases of the estrous cycle. Neither males nor females displayed significant footshock-induced reinstatement of oxycodone seeking, but both sexes exhibited significant cue-induced reinstatement of oxycodone seeking at magnitudes that did not differ either by sex or by estrous cycle phase. Discussion: These results confirm and extend previous work suggesting that sex does not robustly influence the primary reinforcing effects of oxycodone nor the reinstatement of oxycodone-seeking behavior. However, our findings reveal for the first time that the reinforcing efficacy of IV oxycodone varies across the estrous cycle in female rats.

Effects of sex and estrous cycle on intravenous oxycodone self-administration and the reinstatement of oxycodone-seeking behavior in rats.

The increasing misuse of both prescription and illicit opioids has culminated in a national healthcare crisis in the United States. Oxycodone is among the most widely prescribed and misused opioid pain relievers and has been associated with a high risk for transition to compulsive opioid use. Here, we sought to examine potential sex differences and estrous cycle-dependent effects on the reinforcing efficacy of oxycodone, as well as on stress-induced or cue-induced oxycodone-seeking behavior, using intravenous (IV) oxycodone self-administration and reinstatement procedures. In experiment 1, adult male and female Long-Evans rats were trained to self-administer 0.03 mg/kg/inf oxycodone according to a fixed-ratio 1 schedule of reinforcement in daily 2-hr sessions, and a dose-response function was subsequently determined (0.003-0.03 mg/kg/inf). In experiment 2, a separate group of adult male and female Long-Evans rats were trained to self-administer 0.03 mg/kg/inf oxycodone for 8 sessions, followed by 0.01 mg/kg/inf oxycodone for 10 sessions. Responding was then extinguished, followed by sequential footshock-induced and cue-induced reinstatement tests. In the dose-response experiment, oxycodone produced a typical inverted U-shape function with 0.01 mg/kg/inf representing the maximally effective dose in both sexes. No sex differences were detected in the reinforcing efficacy of oxycodone. In the second experiment, the reinforcing effects of 0.01-0.03 mg//kg/inf oxycodone were significantly attenuated in females during proestrus/estrus as compared to metestrus/diestrus phases of the estrous cycle. Neither males nor females displayed significant footshock-induced reinstatement of oxycodone seeking, but both sexes exhibited significant cue-induced reinstatement of oxycodone seeking at magnitudes that did not differ either by sex or by estrous cycle phase. These results confirm and extend previous work suggesting that sex does not robustly influence the primary reinforcing effects of oxycodone nor the reinstatement of oxycodone-seeking behavior. However, our findings reveal for the first time that the reinforcing efficacy of IV oxycodone varies across the estrous cycle in female rats.

Age- and Sex-Dependent Changes in Locus Coeruleus Physiology and Anxiety-Like Behavior Following Acute Stressor Exposure.

Adolescence is a critical period of development with increased sensitivity toward psychological stressors. Many psychiatric conditions emerge during adolescence and animal studies have shown that that acute stress has long-term effects on hypothalamic-pituitary-adrenal axis function and behavior. We recently demonstrated that acute stress produces long-term electrophysiological changes in locus coeruleus and long-lasting anxiety-like behavior in adolescent male rats. Based on prior reports of increased stress sensitivity during adolescence and increased sensitivity of female locus coeruleus toward corticotropin releasing factor, we hypothesized that the same acute stressor would cause different behavioral and physiological responses in adolescent female and adult male and female rats one week after stressor exposure. In this study, we assessed age and sex differences in how an acute psychological stressor affects corticosterone release, anxiety-like behavior, and locus coeruleus physiology at short- and long-term intervals. All groups of animals except adult female responded to stress with elevated corticosterone levels at the acute time point. One week after stressor exposure, adolescent females showed decreased firing of locus coeruleus neurons upon current injection and increased exploratory behavior compared to controls. The results were in direct contrast to changes observed in adolescent males, which showed increased anxiety-like behavior and increased spontaneous and induced firing in locus coeruleus neurons a week after stressor exposure. Adult males and females were both behaviorally and electrophysiologically resilient to the long-term effects of acute stress. Therefore, there may be a normal developmental trajectory for locus coeruleus neurons which promotes stress resilience in adults, but stressor exposure during adolescence perturbs their function. Furthermore, while locus coeruleus neurons are more sensitive to stressor exposure during adolescence, the effect varies between adolescent males and females. These findings suggest that endocrine, behavioral, and physiological responses to stress vary among animals of different age and sex, and therefore these variables should be taken into account when selecting models and designing experiments to investigate the effects of stress. These differences in animals may also allude to age and sex differences in the prevalence of various psychiatric illnesses within the human population.

Effects of Sex and Estrous Cycle on the Time Course of Incubation of Cue-Induced Craving following Extended-Access Cocaine Self-Administration.

Cocaine addiction is a devastating public health epidemic that continues to grow. Studies focused on identifying biological factors influencing cocaine craving and relapse vulnerability are necessary to promote abstinence in recovering drug users. Sex and ovarian hormones are known to influence cocaine addiction liability and relapse vulnerability in both humans and rodents. Previous studies have investigated sex differences in the time-dependent intensification or "incubation" of cue-induced cocaine craving that occurs during withdrawal from extended-access cocaine self-administration and have identified changes across the rat reproductive cycle (estrous cycle). Female rats in the estrus stage of the cycle (Estrus Females), the phase during which ovulation occurs, show an increase in the magnitude of incubated cue-induced cocaine craving compared with females in all other phases of the estrous cycle (Non-Estrus Females). Here we extend these findings by assessing incubated craving across the estrous cycle during earlier withdrawal periods (withdrawal day 1 and 15) and later withdrawal periods (withdrawal day 48). We found that this increase in the magnitude of incubated craving during estrus (Estrus Females) is present on withdrawal day 15, but not on withdrawal day 1, and further increases by withdrawal day 48. No difference in the magnitude of incubated craving was observed between Males and Non-Estrus Females. Our data indicate that the effects of hormonal fluctuations on cue-induced cocaine craving intensify during the first month and a half of withdrawal, showing an interaction among abstinence length, estrous cycle fluctuations, and cocaine craving.

Association of Bystander and First-Responder Efforts and Outcomes According to Sex: Results From the North Carolina HeartRescue Statewide Quality Improvement Initiative.

Background The Institute of Medicine has called for actions to understand and target sex-related differences in care and outcomes for out-of-hospital cardiac arrest patients. We assessed changes in bystander and first-responder interventions and outcomes for males versus females after statewide efforts to improve cardiac arrest care. Methods and Results We identified out-of-hospital cardiac arrests from North Carolina (2010-2014) through the CARES (Cardiac Arrest Registry to Enhance Survival) registry. Outcomes for men versus women were examined through multivariable logistic regression analyses adjusted for (1) nonmodifiable factors (age, witnessed status, and initial heart rhythm) and (2) nonmodifiable plus modifiable factors (bystander cardiopulmonary resuscitation and defibrillation before emergency medical services), including interactions between sex and time (ie, year and year2). Of 8100 patients, 38.1% were women. From 2010 to 2014, there was an increase in bystander cardiopulmonary resuscitation (men, 40.5%-50.6%; women, 35.3%-51.8%; P for each <0.0001) and in the combination of bystander cardiopulmonary resuscitation and first-responder defibrillation (men, 15.8%-23.0%, P=0.007; women, 8.5%-23.7%, P=0.004). From 2010 to 2014, the unadjusted predicted probability of favorable neurologic outcome was higher and increased more for men (men, from 6.5% [95% confidence interval (CI), 5.1-8.0] to 9.7% [95% CI, 8.1-11.3]; women, from 6.3% [95% CI, 4.4-8.3] to 7.4% [95% CI, 5.5-9.3%]); while adjusted for nonmodifiable factors, it was slightly higher but with a nonsignificant increase for women (from 9.2% [95% CI, 6.8-11.8] to 10.2% [95% CI, 8.0-12.5]; men, from 5.8% [95% CI, 4.6-7.0] to 8.4% [95% CI, 7.1-9.7]). Adding bystander cardiopulmonary resuscitation and defibrillation before EMS (modifiable factors) did not substantially change the results. Conclusions Bystander and first-responder interventions increased for men and women, but outcomes improved significantly only for men. Additional strategies may be necessary to improve survival among female cardiac arrest patients.