The role of dopaminergic transmission in the development of manic-like behaviors

描述(申请人提供)：越来越多的证据表明，情绪障碍，如双相情感障碍，可能与昼夜节律紊乱有关。我们实验室之前的工作表明，时钟基因突变的小鼠，其时钟基因是核心昼夜节律机制的关键组成部分，表现出类似于双相情感障碍躁狂期患者的行为表型。重要的是，当功能时钟被引入腹侧被盖区(VTA)时，这种区域救援足以将一些行为恢复到野生型水平，这为时钟功能在哪里可能对调节情绪相关行为起重要作用提供了重要线索。VTA是边缘多巴胺能回路的一个重要脑区，向伏核等脑区提供投射。了解昼夜节律基因如何与多巴胺系统相互作用来调节情绪和奖励相关行为，对于设计新的情感障碍治疗方法至关重要。这项提议试图用时钟突变小鼠作为躁狂症的模型来描述这种相互作用。在具体目标1中，我们计划确定时钟基因功能中断对VTA和NAC中TH节律性表达模式的影响。我们有初步证据表明，时钟突变小鼠的多巴胺能系统可能处于失调状态。我们将通过分析在12小时明暗(LD)和恒定黑暗(DD)条件下的六个时间点的基因表达来检查昼夜节律。基因表达的变化可能会也可能不会被蛋白质水平的变化所反映，因此我们将通过蛋白质印迹来检测酪氨酸羟基酶(TH)和p-TH(SER31)蛋白表达的变化。我们还将用高效液相色谱法检测纹状体多巴胺的总水平。我们将在锂存在的情况下重复这些实验，以确定是否像我们在时钟突变中观察到的行为变化那样，通过治疗挽救分子变化。在特定的目标2中，我们将试图确定时钟突变是否改变了TH基因的调节，以及TH启动子是否是锂的作用目标。首先，我们将分析参与TH转录调控的转录因子CLOCK、BMAL1和CREB的节律性基因表达的变化。此外，我们将研究时钟、BMAL1、CREB和p-CREB(SER133)在六个时间点的蛋白水平的变化。我们还将通过使用Clock、BMAL1和CREB的特异性抗体进行染色质免疫沉淀分析，直接检查TH启动子的结合。这些实验将在锂治疗和不治疗的情况下进行，以确定该药物是否改变了时钟突变小鼠TH启动子的活性。在具体目标3中，我们计划确定多巴胺在时钟突变小鼠躁狂行为发展中的作用。我们将使用两种不同的方法来操纵多巴胺系统，以努力调节时钟突变体的行为。首先，我们将系统地应用α-甲基-p-酪氨酸来抑制TH活性和降低多巴胺水平，随后将进行一系列行为实验，包括运动活动、焦虑和抑郁相关行为。我们还将通过将K+通道病毒HSV Kir2.1直接送入VTA来操纵VTA神经元的放电频率。这一构造以前已经发布和验证过。这些动物将同样接受与情绪相关的行为测试。 公共卫生相关性：生物钟越来越多地与包括躁狂症在内的精神疾病的病因学有关。这项建议的目的是提高我们对生物钟在大脑中缘多巴胺能回路中的作用，以及昼夜节律基因的破坏在多巴胺能神经传递失调中所起的作用的理解。

DESCRIPTION (provided by applicant): There is a growing body of evidence to suggest that mood disorders, such as bipolar disorder, may be associated with disruptions in circadian rhythms. Previous work in our lab has shown that mice with a mutation in their Clock gene, a critical component of the core circadian machinery, display a behavioral phenotype similar to human patients in the manic phase of bipolar disorder. Importantly, when a functional Clock is introduced into the Ventral Tegmental Area (VTA), this regional rescue is sufficient to restore some of the behaviors back to wild type levels offering an important clue as to where Clock function may be important for modulating mood-related behaviors. The VTA is an important brain region of the limbic dopaminergic circuit providing projections to brain regions such as the Nucleus Accumbens (NAc). Understanding how circadian genes may interact with the dopamine system to regulate mood and reward related behaviors is critical in designing new treatments for affective disorders. This proposal seeks to characterize this interaction using the Clock mutant mice as a model of mania. In specific aim 1, we plan to determine the effects of disruption of Clock gene function on the rhythmic expression patterns of TH in the VTA and the NAc. We have preliminary evidence suggesting the dopaminergic system may be dysregulated in the Clock mutant mice. We will examine both the diurnal and circadian rhythms by assaying gene expression over six time points under both 12 hour light-dark (LD) and constant darkness (DD) conditions. Gene expression changes may or may not be mirrored by changes at the protein level, therefore we will assay for these alterations in the protein expression of Tyrosine Hydroxylase (TH) and p-TH (ser 31) by western blot. We will also examine the total levels of striatal dopamine by high performance liquid chromatography. We will repeat these experiments in the presence of lithium to determine whether molecular alterations are rescued by the treatment as we have observed with the behavioral changes in the Clock mutants. In specific aim 2, we will try to determine if the Clock mutation alters the regulation of the TH gene and whether the TH promoter is a target of lithium's action. First, we will assay for changes in rhythmic gene expression of transcription factors Clock, Bmal1, and Creb which are implicated in regulation of TH transcription. Additionally, we will investigate changes in the protein levels of CLOCK, BMAL1, CREB, and p-CREB (ser 133) over six time points. We will also directly examine binding at the TH promoter by performing Chromatin Immunoprecipitation assays using antibodies specific for CLOCK, BMAL1, and CREB. These experiments will be performed both with and without lithium treatment to determine if the drug alters activity at the TH promoter in the Clock mutant mice. In specific aim 3, we plan to establish a role for dopamine in the development of the manic-like behaviors of the Clock mutant mice. We will employ two different methods to manipulate the dopamine system in an effort to modulate the behavior of the Clock mutants. First, we will systemically administer Alpha-methyl-p-tyrosine to inhibit TH activity and decrease dopamine levels followed by a battery of behavioral experiments including locomotor activity, anxiety, and depression-related behaviors. We will also manipulate the firing rate of VTA neurons by delivering a K+ channel virus, HSV Kir2.1, directly into the VTA. This construct has been previously published and verified. These animals will similarly be tested for their mood-related behaviors. PUBLIC HEALTH RELEVANCE: The circadian clock is increasingly being implicated in the etiology of psychiatric diseases including bipolar mania. The goal of this proposal is to improve our understanding of the role of the circadian clock in the brain's mesolimbic dopaminergic circuit and the role that disruption of circadian genes plays in dysregulation of dopaminergic neurotransmission.