Incubation of cue-induced reinstatement of cocaine, but not sucrose, seeking in C57BL/6J mice.

Prior studies have shown that drug-seeking behaviors increase, rather than dissipate, over weeks to months after withdrawal from drug self-administration. This phenomenon - termed incubation - suggests that drug-craving responses elicited by conditioned environmental or discrete cues may intensify over pronged abstinence. While most of this work is conducted in rats with intravenous drug self-administration models, there is less evidence for incubation in mice that have greater utility for molecular genetic analysis and perturbation. We tested whether incubation of cocaine-seeking behavior is evident in C57BL/6J mice following 3weeks (5days/week) of cocaine self-administration in 2h self-administration sessions. We compared cocaine-seeking (drug-paired lever) responses 1, 7, or 28days after withdrawal from cocaine self-administration, and over similar times following sucrose pellet self-administration. We found that the initial re-exposure to the self-administration test chambers elicited increased reward-seeking behavior in both sucrose and cocaine self-administering mice, with maximal responses found at 7days compared to 1 or 28days after self-administration with either reinforcer. However, following extinction training, reinstatement of cocaine seeking reinforced by response-contingent presentation of reward-associated cues (tone/light) was significantly higher after 28days compared to 1 or 7days following cocaine self-administration. In contrast, cue-induced reinstatement of sucrose-paired lever pressing did not increase over this time frame, demonstrating a drug-specific incubation effect not seen with a natural reward. Thus, C57BL/6J mice display incubation of cue-induced reinstatement of cocaine seeking similar to findings with rats, but only show a transient incubation of context-induced cocaine seeking.

Direct regulation of diurnal Drd3 expression and cocaine reward by NPAS2.

BACKGROUND: Circadian gene disruptions are associated with the development of psychiatric disorders, including addiction. However, the mechanisms by which circadian genes regulate reward remain poorly understood. METHODS: We used mice with a mutation in Npas2 and adeno-associated virus-short hairpin RNA mediated knockdown of Npas2 and Clock in the nucleus accumbens (NAc). We performed conditioned place preference assays. We utilized cell sorting quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation followed by deep sequencing. RESULTS: Npas2 mutants exhibit decreased sensitivity to cocaine reward, which is recapitulated with a knockdown of neuronal PAS domain protein 2 (NPAS2) specifically in the NAc, demonstrating the importance of NPAS2 in this region. Interestingly, reducing circadian locomotor output cycles kaput (CLOCK) (a homologue of NPAS2) in the NAc had no effect, suggesting an important distinction in NPAS2 and CLOCK function. Furthermore, we found that NPAS2 expression is restricted to Drd1 expressing neurons while CLOCK is ubiquitous. Moreover, NPAS2 and CLOCK have distinct temporal patterns of DNA binding, and we identified novel and unique binding sites for each protein. We identified the Drd3 dopamine receptor as a direct transcriptional target of NPAS2 and found that NPAS2 knockdown in the NAc disrupts its diurnal rhythm in expression. Chronic cocaine treatment likewise disrupts the normal rhythm in Npas2 and Drd3 expression in the NAc, which may underlie behavioral plasticity in response to cocaine. CONCLUSIONS: Together, these findings identify an important role for the circadian protein, NPAS2, in the NAc in the regulation of dopamine receptor expression and drug reward.

ΔFosB induction in striatal medium spiny neuron subtypes in response to chronic pharmacological, emotional, and optogenetic stimuli.

The transcription factor, ΔFosB, is robustly and persistently induced in striatum by several chronic stimuli, such as drugs of abuse, antipsychotic drugs, natural rewards, and stress. However, very few studies have examined the degree of ΔFosB induction in the two striatal medium spiny neuron (MSN) subtypes. We make use of fluorescent reporter BAC transgenic mice to evaluate induction of ΔFosB in dopamine receptor 1 (D1) enriched and dopamine receptor 2 (D2) enriched MSNs in ventral striatum, nucleus accumbens (NAc) shell and core, and in dorsal striatum (dStr) after chronic exposure to several drugs of abuse including cocaine, ethanol, Δ(9)-tetrahydrocannabinol, and opiates; the antipsychotic drug, haloperidol; juvenile enrichment; sucrose drinking; calorie restriction; the serotonin selective reuptake inhibitor antidepressant, fluoxetine; and social defeat stress. Our findings demonstrate that chronic exposure to many stimuli induces ΔFosB in an MSN-subtype selective pattern across all three striatal regions. To explore the circuit-mediated induction of ΔFosB in striatum, we use optogenetics to enhance activity in limbic brain regions that send synaptic inputs to NAc; these regions include the ventral tegmental area and several glutamatergic afferent regions: medial prefrontal cortex, amygdala, and ventral hippocampus. These optogenetic conditions lead to highly distinct patterns of ΔFosB induction in MSN subtypes in NAc core and shell. Together, these findings establish selective patterns of ΔFosB induction in striatal MSN subtypes in response to chronic stimuli and provide novel insight into the circuit-level mechanisms of ΔFosB induction in striatum.

Morphine suppresses MHC-II expression on circulating B lymphocytes via activation of the HPA.

Morphine has been shown to alter gene expression of the major histocompatibility complex, class II (MHC-II) in circulating rat immunocytes. Here, we demonstrate that a single morphine injection (10 mg/kg) reduces basal MHC-II protein expression on circulating B lymphocytes by 33%, while also impairing the ability of B lymphocytes to increase MHC-II upon interleukin-4 induction. As these data implicate opioids in the regulation of antigen presentation, studies were undertaken to examine the potential mechanisms through which morphine exerts this suppressive effect. Central injection studies utilized Tyr-D-Ala-Gly-(me) Phe-Gly-ol (DAMGO), an opioid receptor agonist, which mimicked morphine's effect on MHC-II, while D-Phe-Cys_Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) pretreatment, prior to morphine, blocked the suppression of MHC-II. As central opioid receptor activation results in the activation of the hypothalamic-pituitary-adrenal axis, thereby, signaling increased circulating corticosterone levels, we examined whether MHC-II expression was suppressed after incubation with corticosterone at concentrations similar to those observed after morphine. Interestingly, corticosterone dramatically decreased basal MHC-II (88%) expression while completely preventing the induction of MHC-II. Additionally, MHC-II suppression was absent in morphine-treated adrenalectomized animals. Since prolonged morphine exposure has previously been shown to result in tolerance to both the steroidogenic and immunosuppressive effects of morphine, the effect of prolonged morphine exposure on MHC-II was also examined. Interestingly, MHC-II expression is no longer suppressed after chronic morphine, while morphine withdrawal results in both a renewed increase in circulating corticosterone levels and a renewed suppression of MHC-II in previously tolerant animals. Taken together, these data strongly implicate corticosterone in mediating the suppressive effects of morphine on circulating B-lymphocyte MHC-II expression.

Enhanced immune sensitivity to stress following chronic morphine exposure.

Chronic administration of escalating doses ofmorphine leads to neuroadaptive changes precipitating development of tolerance to many of the acute effects of morphine, such as analgesia, activation of the hypothalamic-pituitary-adrenal (HPA) axis and suppression of immune cell activities. Interestingly, morphine tolerance has also been shown to be accompanied by heightened immunosuppressive effects of restraint stress using a rodent model. These observations have led to the hypothesis that the altered neuronal state accompanying opioid tolerance may contribute to this enhanced immune sensitivity to stress. To further test this hypothesis using different stressors, Sprague-Dawley rats were treated chronically with morphine for at least 8 days and then challenged with either psychological (water stress) or systemic stressors [morphine withdrawal, lipopolysaccharide (10 mug/kg i.p. challenge)]. It was found that, independent of the type of stress employed, morphine-tolerant animals displayed significantly lower mitogen-stimulated blood lymphocyte responses when compared to the responses of similarly treated saline controls. To determine whether direct activation of central stress pathways may also lead to enhanced immune sensitivity, morphine-tolerant animals were centrally injected with IL-1beta (1 ng/mul i.c.v.), a cytokine that activates the HPA axis by central mechanisms. Similar to the other types of stress, this direct central challenge was also found to be more immunosuppressive in morphine-tolerant animals compared to controls. Collectively, these studies demonstrate that morphine-tolerant animals have an enhanced susceptibility to the debilitating effects of a variety of stressors on immune cell function, an effect that is likely due to the neuroadaptive changes that develop during chronic morphine exposure.