Role of System xc- in Addiction: Developing & Phenotyping a Slc7a11 knockout rat

描述（由申请人提供）：皮质纹状体通路中的异常谷氨酸信号与人类的渴望和大鼠的可卡因寻求有关。不幸的是，我们对谷氨酸的有限理解导致了许多中枢神经系统疾病（包括药物成瘾）缺乏有效、耐受性良好的治疗方法。虽然谷氨酸被描述为大脑中主要的兴奋性神经递质，但尚不清楚这个复杂的转运体网络的许多组成部分和释放机制如何以综合的方式调节兴奋性信号。由于缺乏选择性靶向这些新机制的可用工具，很难令人信服地证明这些新机制的重要性。其中一种成分是系统xc-，它是主要在星形胶质细胞上表达的非囊性谷氨酸释放的来源。它的功能是将细胞外半胱氨酸交换为细胞内谷氨酸。系统xc影响突触活动和可塑性通过

DESCRIPTION (provided by applicant): Abnormal glutamate signaling within corticostriatal pathways has been linked to craving in humans and cocaine seeking in rats. Unfortunately, our limited understanding of glutamate has contributed to the lack of effective, well-tolerated treatments for many CNS diseases, including drug addiction. While glutamate is described as the primary excitatory neurotransmitter in the brain, it is unclear how the many components of this complex network of transporters and release mechanisms function in an integrated manner to regulate excitatory signaling. Due to a lack of available tools that selectively target these novel mechanisms, it has been difficult to convincingly demonstrate the importance of these novel mechanisms. One such component is system xc-, a source of nonvesicular glutamate release that is primarily expressed on astrocytes. It functions by exchanging extracellular cysteine for intracellular glutamate. System xc influences synaptic activity and plasticity through the release of glutamate and dopamine in multiple brain regions. Repeated cocaine produces a persistent reduction in system xc- activity, which appears to be necessary for glutamate-induced compulsive drug seeking. In contrast, manipulations that prevent or reverse cocaine-induced changes in system xc- activity normalize glutamate levels and blunt cocaine-induced reinstatement. In humans, N-acetylcysteine has shown promise in the treatment of drug addiction and related compulsive disorders. Studies such as these indicate that system xc- function may have profound implications in revealing the cellular basis of addiction, as well as the role of astrocytes in central nervous system activity - especially if it is determined that system xc- is the primary mechanism of action for N-acetylcysteine. Efforts to manipulate system xc- in rats typically involve the use of pharmacological tools that are associated with predictable pharmacological concerns. Increasing system xc activity by direct infusion of cystine into the brain or systemic administration of a cysteine prodrug (e.g., N acetylcysteine) are both effective since the rate of cysteine-glutamate exchange is a function of the relative extracellular/intracellular concentration gradients of its substrates. Mutations in the gene giving rise to xCT, the active subunit for system xc, is present in multiple mouse strains. However, essentially every study linking system xc to glutamate homeostasis or addiction has been conducted in rats or primates. The goal of this proposal is to use the novel Zinc Finger Nucleases (ZFN) approach to mutate the Slc7a11 gene encoding xCT in rat. After creating an xCT deficient rat model (aim 1), we will verify and characterize the general phenotype (aim 2) as well as addiction-specific phenotypes (aim 3). The development and application of these technologies to generate transgenic rat strains may result in a major paradigm shift in studying the neural basis of addiction by enabling more sophisticated and highly specific manipulations in a species that better models critical aspects of human addiction.