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

基本信息

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
项目类别：
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

项目摘要

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，躁郁症等情绪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（核心昼夜节律机制的关键组成部分）发生突变的小鼠表现出与躁郁症躁狂期人类患者相似的行为表型。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，这提供了重要线索，说明时钟功能在调节情绪相关方面可能发挥重要作用。行为。 VTA 是边缘多巴胺能回路的重要大脑区域，提供对伏核 (NAc) 等大脑区域的投射。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。该提案旨在使用时钟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破坏对 VTA 和 NAc 中 TH 节律表达模式的影响。我们有初步证据表明时钟突变小鼠的多巴胺能系统可能失调。我们将通过在 12 小时明暗 (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。基因表达变化可能会也可能不会反映在蛋白质水平的变化上，因此我们将通过蛋白质印迹分析酪氨酸羟化酶 (TH) 和 p-TH (ser 31) 蛋白质表达的这些变化。我们还将通过高效液相色谱法检查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，以确定治疗是否可以挽救分子改变，正如我们在时钟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 启动子是否是锂作用的目标。首先，我们将检测与 TH 转录调节有关的转录因子 Clock、Bmal1 和 Creb 的节律基因表达的变化。此外，我们将研究六个时间点上 CLOCK、BMAL1、CREB 和 p-CREB (ser 133) 的蛋白质水平的变化。我们还将使用 CLOCK、BMAL1 和 CREB 特异性抗体进行染色质免疫沉淀测定，直接检查 TH 启动子的结合。这些实验将在有或没有锂治疗的情况下进行，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，以努力调节时钟突变体的行为。首先，我们将系统地施用α-甲基-p-酪氨酸来抑制 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 直接递送至 VTA 来操纵 VTA 神经元的放电率。该构造先前已发布并经过验证。这些动物也将接受类似的情绪相关行为测试。