Direct Regulation of Diurnal Drd3 Expression and Cocaine Reward by NPAS2

Abstract

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.

The intertwined roles of circadian rhythmsand neuronal metabolism fueling drug reward and addiction

“…For example, while CLOCK is largely expressed across many brain regions, expression of the paralog NPAS2, a circadian transcription factor structurally and functionally similar to CLOCK, is primarily limited to the mammalian forebrain and striatum [19,35,37,38]. Significantly, striatal expression of NPAS2 has been implicated as a modulator of drug reward and addiction [19,39]. Recent work also suggests NPAS2 may drive circadian transcription of genes with important roles in neuronal metabolism and energy homeostasis [40–45].…”

“…Similar impairments of glycolysis either via direct blockade or by preventing lactate transport through MCT1 or MCT2 knockdown reduce the acquisition and maintenance of cocaine conditioned place preference and the number of infusions during cocaine self-administration [23,24]. Intriguingly, NPAS2 knockdown in the NAc also attenuates the acquisition of cocaine conditioned place preference, further suggesting the induction of NPAS2 is important for cocaine conditioned reward[19]. Given these data, we speculate that NPAS2 may be a transcriptional regulator of energy production in the NAc via the induction of Ldha and lactate production necessary for drug-paired contextual conditioning.…”

Clock genes × stress × reward interactions in alcohol and substance use disorders

“…First, they demonstrated that Clock Δ 19 -mutant mice, carrying a single point mutation inducing the protein CLOCK, are devoid of any transcriptional activity in all CLOCK-expressing cells, and display increased cocaine-induced conditioned place preference and self-administration when compared to wild-type mice (McClung, Nestler, & Zachariou, 2005; Ozburn, Larson, Self, & McClung, 2012). Most recently, they showed that, when applied specifically within the nucleus accumbens, adeno-associated virus-short hairpin RNA mediating knockdown of the gene Clock does not seem to affect cocaine-induced behaviors, whereas such expression knockdown of the gene Npas2 decreases cocaine-induced conditioned place preference and self-administration (Ozburn et al, 2015). …”

Chromatin immunoprecipitation and gene expression analysis of neuronal subtypes after fluorescence activated cell sorting

“…Using FACS, ~30,000 tdTomato+ cells were collected from NAc with minimal tissue processing time (~45 min), providing sufficient RNA and chromatin for downstream analysis. This one day ChIP protocol is an improvement over previous protocols that describe pooling multiple animals (16, 22, 33) and is expected to facilitate epigenetic analysis of neuronal subtypes in a variety of experimental settings.…”

“…A recent study utilized BAC transgenic strains along with antibody-mediated tagging of histone modifications to examine how cocaine alters histone modifications in D1R and D2R MSNs (14). Other groups have used antibody-mediated FACS to study gene regulation induced by cocaine (15, 16), opioids (17, 18), and methamphetamine (19) in cellular subtypes. However, while antibody-based approaches allow for selection of a wider array of cell types compared to BAC transgenic strains, they also require fixing cells and have issues with off-target binding by antibodies that may reduce their specificity.…”