Mild traumatic brain injury (mTBI) can often impair prefrontal cortex (PFC)-mediated higher-order decision making processes that lead to increased risk-taking behaviors, and repetitive mTBIs (rmTBIs) increase susceptibility to more severe and lasting symptoms. The catecholamine transmitters, dopamine and norepinephrine, modulate neural activity within the PFC and catecholamine dysregulation associated with TBI may underly aberrant decision making. The psychostimulant, methylphenidate (MPH), elevates catecholamine levels by blocking their reuptake transporters and is often used off-label to treat post-injury cognitive symptoms. However, to establish a treatment regimen, it is necessary to characterize how chronic therapeutic doses of MPH affect rmTBI-induced disruptions of decision making. Here, male and female rodents were trained on the probabilistic discounting task (PDT) prior to receiving rmTBI induced by the closed head-controlled cortical impact model. Rats then received daily administration of saline or low-dose MPH (0.5 or 2 mg/kg, i.p.) upon return to the PDT to assess their risk/reward decision-making behavior for 4 weeks post-surgery. The combination of rmTBI and MPH treatment significantly increased risky choice preference in males for 2 weeks. In contrast, MPH (2 mg/kg) disrupted choice preference only in uninjured females within the first week post-surgery. Upon completion of chronic MPH treatment and PDT testing, no changes in catecholamine transporter protein levels within subregions of the PFC were observed. These results indicate MPH treatment transiently exacerbates risky behavior in males, but not females, following rmTBI. These sex-specific responses advise caution for males exposed to making decisions that involve uncertain risk/reward outcomes when considering MPH to treat post-rmTBI cognitive symptoms.

Head trauma often impairs cognitive processes mediated within the prefrontal cortex (PFC), leading to impaired decision making and risk-taking behavior. Mild traumatic brain injury (mTBI) accounts for approximately 80% of reported head injury cases. Most neurological symptoms of a single mTBI are transient; however, growing evidence suggests that repeated mTBI (rmTBI) results in more severe impairments that worsen with each subsequent injury. Although mTBI-induced disruption of risk/reward decision making has been characterized, the potential for rmTBI to exacerbate these effects and the neural mechanisms involved are unknown. Catecholamine neurotransmitters, dopamine (DA) and norepinephrine (NE), modulate PFC-mediated functions. Imbalances in catecholamine function have been associated with TBI and may underlie aberrant decision making. We used a closed head-controlled cortical impact (CH-CCI) model in rats to evaluate the effects of rmTBI on performance of a probabilistic discounting task of risk/reward decision making behavior and expression levels of catecholamine regulatory proteins within the PFC. RmTBI produced transient increases in risky choice preference in both male and female rats, with these effects persisting longer in females. Additionally, rmTBI increased expression of the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), within the orbitofrontal (OFC) region of the PFC in females only. These results suggest females are more susceptible to rmTBI-induced disruption of risk/reward decision making behavior and dysregulation of catecholamine synthesis within the OFC. Together, using the CH-CCI model of rodent rmTBI to evaluate the effects of multiple insults on risk-taking behavior and PFC catecholamine regulation begins to differentiate how mTBI occurrences affect neuropathological outcomes across different sexes.

Irregular transmitter activity is theorized to underly impaired prefrontal cortex (PFC)-mediated executive functions following repetitive mild traumatic brain injury (rmTBI). The psychostimulant, methylphenidate (MPH), enhances catecholamine neurotransmission by blocking reuptake transporters and is used off-label to treat post-TBI executive dysfunction. Both rmTBI and MPH are known to independently alter catecholamine transporter levels. The present report evaluated the interactive effects of rmTBI and a sub-chronic therapeutic dose of MPH on expression levels of vesicular monoamine transporter-2 (VMAT2) and norepinephrine reuptake transporter (NET) within the medial, anterior cingulate, and orbitofrontal subregions of the PFC in both male and female rats. MPH failed to rescue, and in some cases exacerbated, rmTBI-induced reductions in VMAT2 and NET expression in males, whereas transporter expression was largely unaltered in females. These results suggest MPH treatment produces further protein-level perturbations of catecholaminergic activity that are proposed to underlie executive dysfunction in males, but negligible effects in females, following rmTBI.

It is theorized that low concentrations of dopamine (DA) and norepinephrine (NE) within in the prefrontal cortex (PFC) following traumatic brain injury (TBI) leads to increased risky behavior. Our lab has shown that repeated mild TBI (rmTBI) sex-differentially increases risky behavior in a rodent model. Methylphenidate (MPH) is a psychostimulant drug used to treat symptoms of Attention-Deficit Hyperactivity Disorder (ADHD), also driven by a hypo-catecholaminergic PFC. MPH elevates catecholamine levels by blocking DA and NE transporters, DAT and NET. While the potential of psychostimulants to treat post-TBI symptoms have been explored, the effects of sub-chronic MPH on transporter levels following rmTBI has not. To investigate this gap, we used the closed head-controlled cortical impact model to induce 3 mild injuries in Long Evans rats of both sexes. Rats received either saline or MPH (2mg/kg) daily for 7 days (4 groups; sham/saline, sham/MPH, rmTBI/saline, rmTBI/MPH). Brain tissue from the medial (mPFC) and orbitofrontal (OFC) regions of the PFC were collected and standard western blotting protocols were used to measure protein levels of NET, tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), catechol-O-methyltransferase (COMT) and monoamine oxidase (MOA). Within the mPFC, female NET and VMAT levels were decreased in the rmTBI/saline group, while the rmTBI/MPH group’s protein levels did not differ from controls. In males, mPFC VMAT levels were decreased in both rmTBI groups. Within the OFC, NET and VMAT levels were decreased in the male rmTBI/MPH group only. These results suggest that rmTBI reduces transporter levels within regions of the PFC and that sub-chronic MPH treatment may produce restorative benefits on these protein levels in female, but not male rodents following rmTBI. We conclude that interactions between rmTBI and MPH on levels of catecholamine regulatory proteins may begin to elucidate sex differential changes in risk-taking behavior following injury and treatment.

IntroductionIncreasing evidence indicates that sex is a factor that impacts the abuse liability and relapse vulnerability of prescription opioids like oxycodone. However, while women are more likely than men to be prescribed and to use these drugs, the impact of sex and ovarian hormones on prescription opioid use and relapse vulnerability remains unclear. Accurately assessing these measures is complicated by the fact that chronic opioid exposure can lower ovarian hormone levels and cause cycle irregularities.MethodsAdult male and female Sprague–Dawley rats self-administered oxycodone (0.1 mg/kg/infusion) under extended-access conditions (6 h/day, 10 days) followed by forced abstinence. Separate groups of animals received cue-induced seeking tests in a drug-free state during early (1–2 days) or later periods of abstinence (43–45 days). To track estrous cycle stage, animals were regularly vaginally swabbed throughout the study.ResultsWe observed oxycodone-induced estrous cycle dysregulation in the majority (~60%) of the animals during both self-administration and the first month of abstinence. In animals whose cycles were not dysregulated, we found a reduction in oxycodone intake during estrus compared to all other cycle stages (non-estrus). We also found that males but not females showed a time-dependent intensification or incubation of cue-induced oxycodone craving over the first 6 weeks of abstinence. This sex difference was estrous cycle-dependent, driven by a selective reduction in drug seeking during estrus.DiscussionThese findings highlight the importance of tracking drug-induced estrous cyclicity and identify a clear impact of ovarian hormones on oxycodone taking and seeking behavior.

Mild traumatic brain injury (mTBI) disrupts cognitive processes that influence risk taking behavior. Little is known regarding the effects of repetitive mild injury (rmTBI) or whether these outcomes are sex specific. Risk/reward decision making is mediated by the prefrontal cortex (PFC), which is densely innervated by catecholaminergic fibers. Aberrant PFC catecholamine activity has been documented following TBI and may underlie TBI-induced risky behavior. The present study characterized the effects of rmTBI on risk/reward decision making behavior and catecholamine transmitter regulatory proteins within the PFC. Rats were exposed to sham, single (smTBI), or three closed-head controlled cortical impact (CH-CCI) injuries and assessed for injury-induced effects on risk/reward decision making using a probabilistic discounting task (PDT). In the first week post-final surgery, mTBI increased risky choice preference. By the fourth week, males exhibited increased latencies to make risky choices following rmTBI, demonstrating a delayed effect on processing speed. When levels of tyrosine hydroxylase (TH) and the norepinephrine reuptake transporter (NET) were measured within subregions of the PFC, females exhibited dramatic increases of TH levels within the orbitofrontal cortex (OFC) following smTBI. However, both males and females demonstrated reduced levels of OFC NET following rmTBI. These results indicate the OFC is susceptible to catecholamine instability after rmTBI and suggests that not all areas of the PFC contribute equally to TBI-induced imbalances. Overall, the CH-CCI model of rmTBI has revealed time-dependent and sex-specific changes in risk/reward decision making and catecholamine regulation following repetitive mild head injuries.

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.

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.

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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.

Many studies have demonstrated that negative allosteric modulators (NAM) of metabotropic glutamate receptor 5 (mGlu5) reduce cocaine and methamphetamine seeking in extinction-reinstatement animal models of addiction. Less is known about effects of mGlu5 NAMs in abstinence models, particularly for methamphetamine. We used the incubation of drug craving model, in which cue-induced craving progressively intensifies after withdrawal from drug self-administration, to conduct the first studies of the following aspects of mGlu5 function in the rat nucleus accumbens (NAc) core during abstinence from methamphetamine self-administration: 1) functionality of the major form of synaptic depression in NAc medium spiny neurons, which is induced postsynaptically via mGlu5 and expressed presynaptically via cannabinoid type 1 receptors (CB1Rs), 2) mGlu5 surface expression and physical associations between mGlu5, Homer proteins, and diacylglycerol lipase-α, and 3) the effect of systemic and intra-NAc core administration of the mGlu5 NAM 3-((2-methyl-4-)ethynyl)pyridine (MTEP) on expression of incubated methamphetamine craving. We found that mGlu5/CB1R-dependent synaptic depression was lost during the rising phase of methamphetamine incubation but then recovered, in contrast to its persistent impairment during the plateau phase of incubation of cocaine craving. Furthermore, whereas the cocaine-induced impairment was accompanied by reduced mGlu5 levels and mGlu5-Homer associations, this was not the case for methamphetamine. Systemic MTEP reduced incubated methamphetamine seeking, but also reduced inactive hole nose-pokes and locomotion, while intra-NAc core MTEP had no significant effects. These findings provide the first insight into the role of mGlu5 in the incubation of methamphetamine craving and reveal differences from incubation of cocaine craving.

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca2+-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg2+ block and Ca2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain.SIGNIFICANCE STATEMENT "Incubation of craving" is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction.

Abstract 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.

Cue-induced drug craving progressively intensifies after withdrawal from self-administration of cocaine, methamphetamine, and other drugs of abuse, a phenomenon termed incubation of craving. For cocaine and methamphetamine, expression of incubated craving ultimately depends on strengthening of nucleus accumbens (NAc) synapses through an accumulation of high conductance Ca2+-permeable AMPA receptors (CP-AMPARs) that is detectable with electrophysiological approaches. This study sought to further characterize glutamate receptor adaptations in NAc core during methamphetamine incubation. Previous biochemical studies revealed that the CP-AMPARs accumulating after cocaine incubation are mainly homomeric GluA1 receptors and that their accumulation is reflected by increased cell surface GluA1. Here, for methamphetamine, we observed no significant change in surface or total GluA1 (GluA2 and GluA3 were also unchanged). Nonetheless, GluA1 translation was elevated after incubation of methamphetamine craving, as recently found for cocaine. Additionally, for cocaine, we previously observed a withdrawal-dependent decrease in mGlu1 surface expression that precedes and enables CP-AMPAR accumulation and incubation of craving, reflecting weakening of mGlu1-dependent mechanisms that normally limit synaptic CP-AMPAR levels in the NAc core. Here, we observed no change in surface or total mGlu1 protein or its coupling to Homer scaffolding proteins after methamphetamine withdrawal, nor did elevation of mGlu1 tone through repeated injections of an mGlu1-positive allosteric modulator delay incubation of craving. These findings suggest a common role for increased GluA1 translation, but not decreased mGlu1 function, in the incubation of methamphetamine and cocaine craving. We speculate that increased GluA1 translation near synapses may drive formation and synaptic insertion of homomeric GluA1 receptors in the absence of detectable changes in GluA1 protein levels.

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.