Interactions between estradiol and ERK, but not mTOR, signaling is necessary for enhanced cocaine-induced conditioned place preference in female rats.

Women rapidly progress from recreational cocaine use to dependence, consume greater quantities of cocaine, experience more positive subjective effects of cocaine and have higher incidences of relapse during abstinence. These effects have been replicated in animal models of cocaine addiction and indicate an enhanced sensitivity and therefore, vulnerability of females to cocaine addiction. Furthermore, it has been demonstrated that estradiol (E2) is a key mediator of the aforementioned effects of cocaine in women and female animals. However, studies identifying the influence of E2 on cocaine-associated reward and its underlying neurobiological mechanisms are lacking. Here, we further explored the influence of E2 on cocaine conditioned place preference in female rats. We show that E2 mediates cocaine-conditioned reward by potentiating cocaine-context associations. In addition, the E2-mediated increases in cocaine-induced CPP are associated with increased activation of ERK1/2 and mTOR proteins in the nucleus accumbens, dorsal striatum, and ventral tegmental area. To assess the involvement of ERK1/2 and mTOR in E2-mediated enhanced cocaine-CPP, we inhibited ERK1/2 and/or mTOR activity during cocaine-conditioning and before CPP-test. Inhibition of ERK1/2 during conditioning blocked cocaine-CPP in females, inhibition mTOR was without effect, and inhibiting ERK1/2 and mTOR before CPP-test blocked cocaine-CPP. In conclusion, we have established that E2 enhances cocaine-conditioned reward by potentiating cocaine-context associations formed during conditioning. Additionally, activation of ERK1/2 during cocaine-conditioning is necessary for the potentiation of cocaine-conditioned reward by E2. SIGNIFICANCE STATEMENT: Studies characterizing the molecular substrates underlying the effects of E2 during the formation of cocaine-context associations are virtually unknown. In this study, we established the influence of E2 during the formation of cocaine-CPP and characterized the role of ERK1/2 and mTOR activity on this effect within significant nodes of the reward pathway. The elucidation of the role of E2 in cocaine-induced intracellular signaling fills a significant gap in our knowledge regarding the mechanisms by which E2 affects intracellular signaling pathways to indicate the motivational salience of a stimulus. These data are crucial to our understanding of how fluctuating hormone levels can render females increasing sensitive to the rewarding effects of cocaine.

Increased Excitability and Synaptic Plasticity of Drd1- and Drd2-Expressing Prelimbic Neurons Projecting to Nucleus Accumbens after Heroin Abstinence Are Reversed by Cue-Induced Relapse and Protein Kinase A Inhibition.

Dysregulation of the input from the prefrontal cortex (PFC) to the nucleus accumbens (NAc) contributes to cue-induced opioid seeking but the heterogeneity in, and regulation of, prelimbic (PL)-PFC to NAc (PL->NAc) neurons that are altered has not been comprehensively explored. Recently, baseline and opiate withdrawal-induced differences in intrinsic excitability of Drd1+ (D1+) versus Drd2+ (D2+) PFC neurons have been demonstrated. Thus, here we investigated physiological adaptations of PL->NAc D1+ versus D2+ neurons after heroin abstinence and cue-induced relapse. Drd1-Cre+ and Drd2-Cre+ transgenic male Long-Evans rats with virally labeled PL->NAc neurons were trained to self-administer heroin followed by 1 week of forced abstinence. Heroin abstinence significantly increased intrinsic excitability in D1+ and D2+ PL->NAc neurons and increased postsynaptic strength selectively in D1+ neurons. These changes were normalized by cue-induced relapse to heroin seeking. Based on protein kinase A (PKA)-dependent changes in the phosphorylation of plasticity-related proteins in the PL cortex during abstinence and cue-induced relapse to cocaine seeking, we assessed whether the electrophysiological changes in D1+ and D2+ PL->NAc neurons during heroin abstinence were regulated by PKA. In heroin-abstinent PL slices, application of the PKA antagonist (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium (RP-cAMPs) reversed intrinsic excitability in both D1+ and D2+ neurons and postsynaptic strength in only D1+ neurons. Additionally, in vivo bilateral intra-PL infusion of RP-cAMPs after abstinence from heroin inhibited cue-induced relapse to heroin seeking. These data reveal that PKA activity in D1+ and D2+ PL->NAc neurons is not only required for abstinence-induced physiological adaptations but is also required for cue-induced relapse to heroin seeking.SIGNIFICANCE STATEMENT Neuronal plasticity in the medial prefrontal cortex is thought to underlie relapse to drug seeking, yet the subpopulation of neurons that express this plasticity to functionally guide relapse is unclear. Here we show cell type-specific adaptations in Drd1-expressing versus Drd2-expressing prelimbic pyramidal neurons with efferent projections to nucleus accumbens. These adaptations are bidirectionally regulated during abstinence versus relapse and involve protein kinase A (PKA) activation. Furthermore, we show that disruption of the abstinence-associated adaptations via site-specific PKA inhibition abolishes relapse. These data reveal the promising therapeutic potential of PKA inhibition for preventing relapse to heroin seeking and suggest that cell type-specific pharmacologies that target subpopulations of prefrontal neurons would be ideal for future therapeutic developments.

Sex Differences and the Role of Estradiol in Mesolimbic Reward Circuits and Vulnerability to Cocaine and Opiate Addiction.

Although both men and women become addicted to drugs of abuse, women transition to addiction faster, experience greater difficulties remaining abstinent, and relapse more often than men. In both humans and rodents, hormonal cycles are associated with females' faster progression to addiction. Higher concentrations and fluctuating levels of ovarian hormones in females modulate the mesolimbic reward system and influence reward-directed behavior. For example, in female rodents, estradiol (E2) influences dopamine activity within the mesolimbic reward system such that drug-directed behaviors that are normally rewarding and reinforcing become enhanced when circulating levels of E2 are high. Therefore, neuroendocrine interactions, in part, explain sex differences in behaviors motivated by drug reward. Here, we review sex differences in the physiology and function of the mesolimbic reward system in order to explore the notion that sex differences in response to drugs of abuse, specifically cocaine and opiates, are the result of molecular neuroadaptations that differentially develop depending upon the hormonal state of the animal. We also reconsider the notion that ovarian hormones, specifically estrogen/estradiol, sensitize target neurons thereby increasing responsivity when under the influence of either cocaine or opiates or in response to exposure to drug-associated cues. These adaptations may ultimately serve to guide the motivational behaviors that underlie the factors that cause women to be more vulnerable to cocaine and opiate addiction than men.

Overlap in the neural circuitry and molecular mechanisms underlying ketamine abuse and its use as an antidepressant.

Ketamine, a dissociative anesthetic and psychedelic compound, has revolutionized the field of psychopharmacology by showing robust, and rapid-acting antidepressant activity in patients suffering from major depressive disorder (MDD), suicidal tendencies, and treatment-resistant depression (TRD). Ketamine's efficacy, however, is transient, and patients must return to the clinic for repeated treatment as they experience relapse. This is cause for concern because ketamine is known for its abuse liability, and repeated exposure to drugs of abuse often leads to drug abuse/dependence. Though the mechanism(s) underlying its antidepressant activity is an area of current intense research, both clinical and preclinical evidence shows that ketamine's effects are mediated, at least in part, by molecular adaptations resulting in long-lasting synaptic changes in mesolimbic brain regions known to regulate natural and drug reward. This review outlines our limited knowledge of ketamine's neurobiological and biochemical underpinnings mediating its antidepressant effects and correlates them to its abuse potential. Depression and addiction share overlapping neural circuitry and molecular mechanisms, and though speculative, repeated use of ketamine for the treatment of depression could lead to the development of substance use disorder/addiction, and thus should be tempered with caution. There is much that remains to be known about the long-term effects of ketamine, and our lack of understanding of neurobiological mechanisms underlying its antidepressant effects is a clear limiting factor that needs to be addressed systematically before using repeated ketamine in the treatment of depressed patients.

Sex differences in the expression of morphine withdrawal symptoms and associated activity in the tail of the ventral tegmental area.

Recent studies, in male rodents, have begun to elucidate a role for the GABAergic neurons in the tail of the ventral tegmental area (tVTA) in morphine withdrawal. To date, the mechanisms underlying morphine withdrawal have been studied almost exclusively in male animals. As a result, there is a considerable gap in our current understanding of the processes underlying sex differences in morphine withdrawal behaviors and its effects on cellular activity in the tVTA in females. The purpose of the present study was to investigate the influence of sex on the expression and duration of spontaneous somatic morphine withdrawal syndrome, and to characterize the relationship between spontaneous somatic withdrawal symptoms and cellular activation (measured as phosphorylated CREB; pCREB), in the GABAergic tVTA in male and female rats. Morphine-dependent adult male and female Long Evans rats underwent 72 h of spontaneous withdrawal, and somatic withdrawal symptoms were assessed every 12 h. Male morphine-dependent rats expressed more severe symptoms during the early phases of withdrawal compared to females. Although, females demonstrated lower overall symptom severity, their symptoms persisted for a longer period of time, thus demonstrating higher withdrawal-symptom severity than males during late withdrawal. pCREB activity in the tVTA was elevated in morphine-withdrawn rats and was positively correlated with the severity of withdrawal symptoms. These results demonstrate sex differences in the timing of the expression of somatic withdrawal. Our data add to the growing body of evidence demonstrating a role for the tVTA in morphine withdrawal and begin to establish a sex-dependent behavioral and molecular profile within this brain region.

Loss of Hepatic Oscillatory Fed microRNAs Abrogates Refed Transition and Causes Liver Dysfunctions.

Inability to mediate fed-fast transitions in the liver is known to cause metabolic dysfunctions and diseases. Intuitively, a failure to inhibit futile translation of state-specific transcripts during fed-fast cycles would abrogate dynamic physiological transitions. Here, we have discovered hepatic fed microRNAs that target fasting-induced genes and are essential for a refed transition. Our findings highlight the role of these fed microRNAs in orchestrating system-level control over liver physiology and whole-body energetics. By targeting SIRT1, PGC1α, and their downstream genes, fed microRNAs regulate metabolic and mitochondrial pathways. MicroRNA expression, processing, and RISC loading oscillate during these cycles and possibly constitute an anticipatory mechanism. Fed-microRNA oscillations are deregulated during aging. Scavenging of hepatic fed microRNAs causes uncontrolled gluconeogenesis and failure in the catabolic-to-anabolic switching upon feeding, which are hallmarks of metabolic diseases. Besides identifying mechanisms that enable efficient physiological toggling, our study highlights fed microRNAs as candidate therapeutic targets.

Prolonged ketamine exposure induces increased activity of the GluN2B-containing N-methyl-d-aspartate receptor in the anterior cingulate cortex of neonatal rats.

Ketamine is a commonly used anesthetic among pediatric patients due to its high efficacy. However, it has been demonstrated by several preclinical studies that, widespread accelerated programmed death of neurons (neuroapoptosis) occurs due to prolonged or repeated exposure to ketamine specifically in the neonatal brain. Therefore, an emphasis on understanding the molecular mechanisms underlying this selective vulnerability of the neonatal brain to ketamine-induced neuroapoptosis becomes important in order to identify potential therapeutic targets, which would help prevent or at least ameliorate this neuroapoptosis. In this study, we demonstrated that repeated ketamine administration (6 injections of 20mg/kg dose given over 12h time period) in neonatal (postnatal day 7; PND 7) Sprague-Dawley rats induced a progressive increase in N-methyl-d-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs) in the neurons of the anterior cingulate cortex (ACC) for up to 6h after the last ketamine dose. Specifically, we observed that the increased EPSCs were largely mediated by GluN2B-containing NMDARs in the neurons of the ACC. Along with increased synaptic transmission, there was also a significant increase in the expression of the GluN2B-containing NMDARs as well. Taken together, these results showed that after repeated exposure to ketamine, the synaptic transmission mediated by GluN2B-containing NMDARs was significantly increased in the neonatal brain. This was significant as it showed for the first time that ketamine had subunit-specific effects on GluN2B-containing NMDARs, potentially implicating the involvement of these subunits in the increased vulnerability of immature neurons of the neonatal brain to ketamine-induced neuroapoptosis.

Neonatal inhibition of Na+-K+-2Cl--cotransporter prevents ketamine induced spatial learning and memory impairments.

Prolonged ketamine exposure in neonates at anesthetic doses is known to cause long-term impairments of learning and memory. A current theoretical mechanism explains this phenomenon as being neuro-excitotoxicity mediated by compensatory upregulation of N-methyl-d-aspartate receptors (NMDARs), which then initiates widespread neuroapoptosis. Additionally, the excitatory behavior of GABAergic synaptic transmission mediated by GABAA receptors (GABAARs), occurring during the early neuronal development period, is proposed as contributing to the susceptibility of neonatal neurons to ketamine-induced injury. This is due to differential developmental expression patterns of Na+-K+-2Cl- co-transporter (NKCC1) and K+-Cl- co-transporter. Studies have shown that bumetanide, an NKCC1 inhibitor, allows neurons to become inhibitory rather than excitatory early in development. We thus hypothesized that bumetanide co-administration during ketamine treatment would reduce over excitation and protect the neurons from excitotoxicity. In this initial study, the Morris Water Maze test was used to assess the effects of co-administration of ketamine and bumetanide to neonatal Sprague-Dawley rats on long-term learning and memory changes seen later in life. It was revealed that bumetanide, when co-treated with ketamine neonatally, significantly impeded behavioral deficits typically seen in animals exposed to ketamine alone. Therefore, these findings suggest a new mechanism by which neonatal ketamine induced learning impairments can be prevented.