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.
Genetically silencing the xCT protein in the rat will advance our understanding of the function of system xc-, which may be a contributing mechanism to central nervous system disease states such as drug addiction. Moreover, generating a rat knockout, in place of a mouse knockout, will have profound effects on the field of neuroscience by enabling the ability to study more complex behavioral models and central nervous system diseases.
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