Role of TARPs in Addiction-Related Plasticity

描述(由申请人提供)：了解成瘾物质改变大脑的细胞机制是研究的关键目标，旨在为成瘾患者的治疗策略提供基础。我们的假设是，成瘾的一个关键方面是疏忽的谷氨酸能神经元可塑性，涉及学习中常见的机制，如LTP和LTD。在这项申请中提出的实验将通过检测跨膜AMPA受体调节蛋白(TARP)在AMPA受体运输和伏核(NAC)与成瘾相关的可塑性中的作用来检验这一假说，NAC是大脑中在成瘾发展和持续中发挥关键作用的区域。我建议在细胞水平上研究TARP亚型和AMPA受体亚单位之间的相互作用，2)TARP磷酸化在NAc中AMPA受体转运中的作用，3)在成瘾动物模型中AMPA受体表面表达增加与药物渴求增强和TARP磷酸化与表面表达之间的关系。TARP以前没有在NAC进行过检查。首先，我将使用免疫细胞化学和定量免疫共沉淀技术来确定TARP亚型在NAC中的分布及其与AMPA受体亚单位的关系。其次，我将用免疫细胞化学的方法来确定AMPA受体突触掺入对TARP突触掺入的影响，并用Western blotting和等电聚焦来确定在一个新的NAC-前额叶皮质共培养系统中AMPA受体突触掺入对TARP磷酸化的影响。最后，我将考察戒断可卡因后增强的线索诱发的药物渴求与TARP磷酸化和表面表达的关系。为此，以前被允许自我给药可卡因或生理盐水的大鼠的NAC组织将在戒断后被收集，并将使用几种生化技术来测定TARP的磷酸化、激酶活性以及AMPA受体和Tarp的表面表达。综上所述，这些实验的数据将为NAC中AMPA受体运输的机制提供必要的和新的知识，并将有助于我们理解与成瘾相关的谷氨酸可塑性。多达300万美国人将在一生中使用可卡因，可卡因成瘾(以及安非他明等其他精神刺激剂)在国家一级仍然是一个健康问题。这项申请中提出的实验将提供关于AMPA受体运输的细胞机制的知识，还将提供谷氨酸能系统的变化与成瘾行为之间的联系，例如增强的药物渴望。

DESCRIPTION (provided by applicant): Understanding the cellular mechanisms by which addictive substances modify the brain is a key target of research that is designed to provide a basis for development of treatment strategies for addicted patients. Our hypothesis is that a critical aspect of addiction is abberant glutamatergic neuronal plasticity involving mechanisms common to learning, such as LTP and LTD. The experiments proposed in this application will test this hypothesis by examining the role of transmembrane AMPA receptor regulatory proteins (TARPs) in both AMPA receptor trafficking and addiction-related plasticity in the nucleus accumbens (NAc), a brain region that plays a critical role in the development and persistence of addiction. I propose to investigate 1) the interactions between TARP isoforms and AMPA receptor subunits at the cellular level, 2) the role of TARP phosphorylation in AMPA receptor trafficking in the NAc, 3) the relationship between increases in AMPA receptor surface expression associated with enhanced drug-craving and TARP phosphorylation and surface expression in an animal model of addiction. TARPs have not previously been examine in the NAc. First, I will use immunocytochemical and quantitative co-immunoprecipitation techniques to determine the distribution of TARP isoforms and their association with AMPA receptor subunits in the NAc. Second, I will determine the effect of AMPA receptor synaptic incorporation on the synaptic incorporation of TARPs using immunocytochemistry and on TARP phosphorylation using Western blotting and isoelecrtric focusing in a novel NAc-prefrontal cortex co-culture system. Finally, I will examine the relationship between enhanced cue elicited drug-craving after withdrawal from cocaine self-administration and TARP phosphorylation and surface expression. To this end, NAc tissue from rats previously allowed to self-administer cocaine or saline will be collected after withdrawal and several biochemical techniques will be used to determine TARP phosphorylation, kinase activity, and surface expression of AMPA receptors and TARPs. Taken together, data from these experiments will provide essential and novel knowledge about the mechanisms underlying AMPA receptor trafficking in the NAc and will contribute to our understanding of glutamatergic plasticity associated with addiction. As many as 3 million Americans will use cocaine within their lifetimes, and addiction to cocaine (as well as other psychostimulants such as amphetamines) continues to be a health concern at the national level. The experiments proposed in this application will provide knowledge about the cellular mechanisms of AMPA receptor trafficking, and will also provide a link between alterations in the glutamatergic system and behaviors indicative of addiction, such as enhanced drug craving.