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的发展提供基础。我们的假设是，成瘾的一个关键方面是异常的谷氨酸能神经元可塑性，涉及学习的共同机制，如LTP和LTD。本申请中提出的实验将通过检查跨膜AMPA受体调节蛋白（TARPs）在AMPA受体运输和伏隔核（NAc）中成瘾相关可塑性中的作用来验证这一假设，伏隔核是一个在成瘾的发展和持续中起关键作用的大脑区域。我建议在细胞水平上研究1)TARP亚型与AMPA受体亚基之间的相互作用，2)TARP磷酸化在NAc中AMPA受体运输中的作用，3)在成瘾动物模型中，与药物渴望增强相关的AMPA受体表面表达增加与TARP磷酸化和表面表达之间的关系。在此之前，美国国家航空公司还没有对tarp进行过研究。首先，我将使用免疫细胞化学和定量共免疫沉淀技术来确定TARP亚型的分布及其与NAc中AMPA受体亚基的关联。其次，我将在一个新的nac -前额皮质共培养系统中，利用免疫细胞化学方法确定AMPA受体突触结合对TARPs突触结合的影响，并利用Western blotting和等电聚焦确定TARP磷酸化的影响。最后，我将研究自我戒断可卡因后线索引发的药物渴求增强与TARP磷酸化和表面表达之间的关系。为此，将在停药后收集先前允许自我施用可卡因或生理盐水的大鼠的NAc组织，并使用几种生化技术来测定TARP磷酸化，激酶活性以及AMPA受体和TARP的表面表达。综上所述，来自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颖的知识，并将有助于我们理解与成瘾相关的谷氨酸能可塑性。多达300万美国人将在他们的一生中使用可卡因，可卡因成瘾（以及其他精神兴奋剂，如安非他明）仍然是一个全国性的健康问题。本应用程序中提出的实验将提供有关AMPA受体运输的细胞机制的知识，并将提供谷氨酸系统的改变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.