We propose to analyze the neural functions of two genes that we previously cloned and have found to be highly expressed in the striatum. CalDAG-GEFI has a highly striatum-enriched expression pattern. CalDAGGEFII is also highly expressed in the striatum but less selectively so. These genes are particularly important to study because they have coding regions for calcium and diacylglycerol binding and also a coding region for a guanine nucleotide exchange factor that targets the Ras superfamily proteins (Rap for CalDAG-GEFI and Ras for CalDAG-GEFII). Ras superfamily proteins have recently been implicated in learning and memory functions at the behavioral level and in neuroplasticity and synaptic functions at the cellular level. Ras superfamily proteins are activated by proteins called GEFs and our proposal thus focuses on potential activators of Rap and Ras in the striatum. We have generated CalDAG-GEFI-/- and CalDAG-GEFI/II-/- mice. Based on our preliminary work on CalDAG-GEFI, we hypothesize that this striatum-enriched gene participates in cellular functions related to learning. To test this overall hypothesis, we have developed four Specific Aims. First, we will assess CalDAG-GEFI-/- and +/+ mice in a series of learning paradigms to test our hypothesis that the knockouts will have a striatum-dependent learning deficit. Second, we will record neural activity in the striatum of the mice both under baseline conditions and during procedural learning to test the hypothesis that the knockout mice have a deficit in learning-related neuroplasticity. Third, we will assess the responses of the CalDAG-GEFI-/- and +/+ mice in experiments designed to test our hypothesis that CalDAG-GEFI-/- mice will exhibit abnormal behavioral and cellular responsively to dopamine-receptor agonist and antagonist drugs. Finally, we will carry out Golgi-like in vivo labeling to test for altered morphology of striatal neurons in the knockout mice, perform experiments with immortalized striatal cell lines and primary striatal cultures, and carry out collaborative electron microscopy to test our hypothesis that CalDAG-GEFI participates in the regulation of aspects of striatal neuron morphology, including synaptic structure and process output. These detailed studies of CalDAG-GEFI will be accompanied by initial characterization of CalDAG-GEFII-/- and CalDAG-GEFI/II-/- mice. The work proposed is highly significant for understanding signaling in key basal ganglia neurons whose function is essential for normal behavior and whose dysfunction leads to major extrapyramidal disorders.
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