Ozburn Angela R, Falcon Edgardo, Twaddle Alan, Nugent Alexandria L, Gillman Andrea G, Spencer Sade M, Arey Rachel N, Mukherjee Shibani, Lyons-Weiler James, Self David W, McClung Colleen A
Department of Psychiatry and Translational Neuroscience Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219.
Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX 75390-9070.
Biol Psychiatry. 2015 Mar 1;77(5):425-433. doi: 10.1016/j.biopsych.2014.07.030. Epub 2014 Aug 13.
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.
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.
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.
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.
昼夜节律基因紊乱与包括成瘾在内的精神疾病的发生发展有关。然而，昼夜节律基因调节奖赏的机制仍知之甚少。
我们使用了在神经元 PAS 结构域蛋白 2（Npas2）中发生突变的小鼠，以及腺相关病毒短发夹 RNA 介导的伏隔核（NAc）中 Npas2 和 Clock 的敲低。我们进行了条件性位置偏爱试验。我们利用细胞分选定量实时聚合酶链反应，以及染色质免疫沉淀后进行深度测序。
Npas2 突变体对可卡因奖赏的敏感性降低，在 NAc 中特异性敲低神经元 PAS 结构域蛋白 2（NPAS2）可重现这一现象，表明 NPAS2 在该区域的重要性。有趣的是，在 NAc 中降低昼夜运动输出周期蛋白（CLOCK）（NPAS2 的同源物）没有效果，这表明 NPAS2 和 CLOCK 功能存在重要差异。此外，我们发现 NPAS2 的表达仅限于表达多巴胺 D1 受体（Drd1）的神经元，而 CLOCK 则普遍存在。而且，NPAS2 和 CLOCK 具有不同的 DNA 结合时间模式，并且我们确定了每种蛋白质的新的独特结合位点。我们确定多巴胺 D3 受体（Drd3）是 NPAS2 的直接转录靶点，并发现 NAc 中 NPAS2 的敲低会破坏其表达的昼夜节律。慢性可卡因治疗同样会破坏 NAc 中 Npas2 和 Drd3 表达的正常节律，这可能是对可卡因反应的行为可塑性的基础。
总之，这些发现确定了昼夜节律蛋白 NPAS2 在 NAc 中对多巴胺受体表达和药物奖赏调节中的重要作用。
