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

多巴胺能传递在躁狂样行为发展中的作用

• 批准号: 8111205
• 负责人: SADE MONIQUE SPENCER
• 金额: $ 2.35万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8111205
• 关键词: Acids; Affect; Animal Experimentation; Animal Housing; Animals; Antibodies; Anxiety; BHLH Protein; Back; Behavior; Behavioral; Binding; Binding Sites; Biological Assay; Biological Rhythm; Bipolar Disorder; Body Temperature; Boxing; Brain; Brain region; CREB1 gene; Cells; Circadian Rhythms; Corpus striatum structure; Cyclic AMP; Darkness; Data; Development; Dominant-Negative Mutation; Dopamine; Dopamine Receptor; Elements; Enzyme Activation; Enzymes; Etiology; Gene Expression; Gene Expression Profile; Genes; Genetic Polymorphism; Genetic Transcription; Goals; High Pressure Liquid Chromatography; Homovanillic Acid; Hour; Human; Human Genetics; Immunoprecipitation; Kir2.1 channel; Lead; Light; Lithium; Lithium Chloride; Manic; Manic Disorder; Measures; Mediating; Mental Depression; Mental disorders; Messenger RNA; Methods; Modeling; Molecular; Molecular Profiling; Monoamine Oxidase A; Mood Disorders; Moods; Motor Activity; Mus; Mutant Strains Mice; Mutation; Neurons; Nucleus Accumbens; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Physiological; Play; Potassium Channel; Process; Proteins; Publishing; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Rewards; Role; Simplexvirus; Sleep Wake Cycle; System; Testing; Time; Tyrosine 3-Monooxygenase; Ventral Tegmental Area; Virus; Western Blotting; Wild Type Mouse; Work; alpha-Methyltyrosine; behavior measurement; chromatin immunoprecipitation; circadian pacemaker; design; dopamine system; dopamine transporter; gene function; improved; mood regulation; mutant; neurotransmission; overexpression; promoter; protein expression; research study; reward circuitry; transcription factor; transmission process

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.

越来越多的证据表明，情绪障碍，如双相情感障碍，可能与昼夜节律的破坏有关。我们实验室以前的工作已经表明，Clock基因突变的小鼠，核心昼夜节律机制的关键组成部分，表现出与双相情感障碍躁狂期人类患者相似的行为表型。重要的是，当一个功能性时钟被引入到大脑被盖区（VTA）时，这种区域性拯救足以将一些行为恢复到野生型水平，这为时钟功能在调节情绪相关行为方面可能很重要提供了重要线索。腹侧被盖区是边缘多巴胺能回路的一个重要脑区，为伏隔核（NAc）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，对于设计情感障碍的新疗法至关重要。该提案试图使用Clock突变小鼠作为躁狂症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的中断对VTA和NAc中TH的节律性表达模式的影响。我们有初步证据表明，多巴胺能系统可能是失调的时钟突变小鼠。我们将通过在12小时光暗（LD）和恒定黑暗（DD）条件下测定六个时间点的基因表达来检查昼夜节律和昼夜节律。基因表达的变化可能会或可能不会反映在蛋白质水平的变化，因此，我们将分析这些变化的酪氨酸羟化酶（TH）和p-TH（ser 31）的蛋白质表达的蛋白质印迹。我们还将通过高效液相色谱法检测纹状体多巴胺的总水平。我们将在锂的存在下重复这些实验，以确定分子改变是否被治疗所拯救，正如我们在Clock突变体中观察到的行为变化一样。在具体目标2中，我们将尝试确定Clock突变是否改变TH基因的调控，以及TH启动子是否是锂作用的靶点。首先，我们将检测转录因子Clock、Bmal 1和Creb的节律性基因表达的变化，这些转录因子与TH转录的调节有关。此外，我们还将研究CLOCK、BMAL 1、CREB和p-CREB（ser 133）蛋白水平在6个时间点的变化。我们还将通过使用CLOCK、BMAL 1和CREB特异性抗体进行染色质免疫沉淀试验，直接检查TH启动子的结合。这些实验将在有和没有锂处理的情况下进行，以确定药物是否改变Clock突变小鼠中TH启动子的活性。在具体目标3中，我们计划确定多巴胺在Clock突变小鼠的躁狂样行为发展中的作用。我们将采用两种不同的方法来操纵多巴胺系统，以调节时钟突变体的行为。首先，我们将全身给予α-甲基-p-酪氨酸以抑制TH活性并降低多巴胺水平，然后进行一系列行为实验，包括自发活动、焦虑和抑郁相关行为。我们还将通过将K+通道病毒HSV Kir2.1直接递送到腹侧被盖区来操纵腹侧被盖区神经元的放电率。该结构先前已发布并验证。这些动物也将接受类似的情绪相关行为测试。