Background Circadian gene disruptions are associated with the development of psychiatric disorders, including addiction. However, the mechanisms by which circadian genes regulate drug reward remain poorly understood. Methods We used mice with a mutation in Npas2, and AAV-shRNA mediated knock-down of Npas2 and Clock in the nucleus accumbens (NAc). We performed conditioned place preference (CPP) assays for cocaine. We utilized cell sorting techniques, qPCR and chromatin immunoprecipitation (ChIP) assays followed by deep sequencing (ChIP-seq). Results Npas2 mutants exhibit decreased sensitivity to cocaine reward which can be recapitulated with a knock-down of NPAS2 specifically in the NAc, demonstrating the functional importance of NPAS2 in this region. Interestingly, reducing CLOCK (a homologue of NPAS2) expression in the NAc had no effect, suggesting an important distinction in NPAS2 and CLOCK function. Furthermore, we find that NPAS2 expression is restricted to Drd1 expressing neurons, (i.e. “direct” pathway circuitry) 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 find that NPAS2 knock-down 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 and novel role for the circadian protein, NPAS2, in the NAc in the regulation of dopamine receptor expression and drug reward.
Lithium has been used extensively for mood stabilization, and it is particularly efficacious in the treatment of bipolar mania. Like other drugs used in the treatment of psychiatric diseases, it has little effect on the mood of healthy individuals. Our previous studies found that mice with a mutation in the Clock gene (ClockD19) have a complete behavioral profile that is very similar to human mania, which can be reversed with chronic lithium treatment. However, the cellular and physiological effects that underlie its targeted therapeutic efficacy remain unknown. Here we find that ClockD19 mice have an increase in dopaminergic activity in the ventral tegmental area (VTA), and that lithium treatment selectively reduces the firing rate in the mutant mice with no effect on activity in wild-type mice. Furthermore, lithium treatment reduces nucleus accumbens (NAc) dopamine levels selectively in the mutant mice. The increased dopaminergic activity in the Clock mutants is associated with cell volume changes in dopamine neurons, which are also rescued by lithium treatment. To determine the role of dopaminergic activity and morphological changes in dopamine neurons in manic-like behavior, we manipulated the excitability of these neurons by overexpressing an inwardly rectifying potassium channel subunit (Kir2.1) selectively in the VTA of ClockD19 mice and wild-type mice using viral-mediated gene transfer. Introduction of this channel mimics the effects of lithium treatment on the firing rate of dopamine neurons in ClockD19 mice and leads to a similar change in dopamine cell volume. Furthermore, reduction of dopaminergic firing rates in ClockD19 animals results in a normalization of locomotor-and anxiety-related behavior that is very similar to lithium treatment; however, it is not sufficient to reverse depression-related behavior. These results suggest that abnormalities in dopamine cell firing and associated morphology underlie alterations in anxiety-related behavior in bipolar mania, and that the therapeutic effects of lithium come from a reversal of these abnormal phenotypes.
Chronic cocaine use reduces glutamate levels in the nucleus accumbens (NAc), and is associated with experience-dependent changes in (±)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptor membrane expression in NAc neurons. These changes accompany behavioral sensitization to cocaine and increased susceptibility to cocaine relapse. The functional relationship between neuroplasticity in AMPA receptors and the behavioral manifestation of cocaine addiction remains unclear. Thus, we examined the behavioral effects of up-and downregulating basal AMPA receptor function in the NAc core and shell using viral-mediated gene transfer of wild-type glutamate receptor 1 (wt-GluR1) or a dominant-negative pore-dead GluR1 (pd-GluR1), respectively. Transient increases in wt-GluR1 during or after cocaine treatments diminished the development of cocaine sensitization, while pd-GluR1 expression exacerbated cocaine sensitization. Parallel changes were found in D2, but not D1, receptormediated behavioral responses. As a correlate of the sensitization experiments, we overexpressed wt-or pd-GluR1 in the NAc core during cocaine self-administration, and tested the effects on subsequent drug-seeking behavior 3 weeks after overexpression declined. wt-GluR1 overexpression during self-administration had no effect on cocaine intake, but subsequently reduced cocaine seeking in extinction and cocaine-induced reinstatement, whereas pd-GluR1 facilitated cocaine-induced reinstatement. When overexpressed during reinstatement tests, wt-GluR1 directly attenuated cocaine-and D2 agonist-induced reinstatement, while pd-GluR1 enhanced reinstatement. In both experimental procedures, neither wt-nor pd-GluR1 expression affected cue-induced reinstatement. Together, these results suggest that degrading basal AMPA receptor function in NAc neurons is sufficient to facilitate relapse via sensitization in D2 receptor responses, whereas elevating basal AMPA receptor function attenuates these behaviors.
SummaryDiurnal and circadian rhythms are prominent in nearly all bodily functions. Chronic disruptions in normal sleep wake and social schedules can lead to serious health problems such as those seen in shift worker's syndrome. Moreover, genetic disruptions in normal circadian gene functions have recently been linked to a variety of psychiatric conditions including depression, bipolar disorder, seasonal affective disorder and alcoholism. Recent studies are beginning to determine how these circadian genes and rhythms are involved in the development of drug addiction. Several of these studies suggest an important role for these genes in limbic regions of the brain, outside of the central circadian pacemaker in the suprachiasmatic nucleus (SCN). This review summarizes some of the basic research into the importance of circadian genes in drug addiction.
Rationale Buprenorphine (BPN) has been shown to rapidly improve mood in treatment-resistant depressed patients in small clinical studies. However, BPN’s effects in preclinical tests for mood and antidepressant efficacy are largely unexplored. Objective The current study examined the effects of BPN in the forced swim test (FST) and novelty-induced hypophagia (NIH) test as measures of antidepressant and anxiolytic-like effects in C57BL/6J mice. Microdialysis was used to measure whether BPN engaged KORs in the nucleus accumbens shell (NAcSh) at a behaviorally active dose (0.25 mg/kg). Methods BPN was tested in the FST at both 30 min and 24 h post administration. Also measured in the FST at 24 h post administration were the KOR antagonist norbinaltorphimine (nor-BNI), the MOR agonist morphine and the reference antidepressant desipramine. The anxiolytic effects of BPN were examined in the NIH test 24 h after treatment. The effects of acute injection of BPN and the KOR agonist U50,488 were measured on extracellular DA levels in the NAcSh. Results BPN produced significant reductions in FST immobility without changing locomotor activity and reduced approach latencies in the novel environment of the NIH test when tested 24 h after treatment. Repeated daily BPN injections for 6 d did not produce tolerance to these behavioral effects. nor-BNI produced a similar antidepressant-like response in the FST 24 h postinjection but morphine and desipramine were ineffective. BPN (0.25 mg/kg) did not alter DA levels when given alone but prevented the KOR agonist U50,488 from reducing DA levels. Conclusions Acute and subchronic treatment with BPN produced antidepressant and anxiolytic-like responses in mice at doses that engage KORs. These studies support the clinical evidence that BPN may be a novel rapid-acting antidepressant medication and provides rodent models for investigating associated neurochemical mechanisms.
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