Excitatory amino acid (EAA)-mediated neurotransmission is widespread throughout the mammalian CNS and is involved in many important functions during neurodevelopment, synaptic plasticity, and neuropathology. Two EAAs, L-glutamate (Glu) and L-aspartate (Asp), fulfill most of the criteria for endogenous neurotransmitters in various regions of the CNS, including the hippocampus and the cerebellum. However, the mechanisms responsible for Asp-mediated neuronal responses, unlike those mediated by Glu, are largely unexplored. Although Asp is a highly selective NMDA receptor agonist in hippocampal neurons, Asp was found to activate a distinct response in Purkinje cells from genetically modified mice that lack functional NMDA receptors. Thus, Purkinje cells may express a novel receptor that is specifically activated by Asp. The goal of this application is to characterize this unique Asp response and to identify its functional role in Purkinje cells. In the first specific aim, the Asp response will be characterized using electrophysiological and pharmacological approaches in cultured Purkinje cells. The sensitivity of the Asp response to agonists and antagonists of known EAA receptors will be determined, and efforts will be made to define antagonists specific for the Asp response. Single channel properties of Asp responses will also be investigated. In the second aim, these properties will be used to investigate the role of the putative Asp receptor in synaptic transmission at climbing fiber- and parallel fiber-Purkinje cell synapses in cerebellar slices. The role of the Asp receptor in synaptic plasticity will also be analyzed. In the third aim, as a first step to identify the putative Asp receptor, glutamate receptor subtypes expressed in Purkinje cells will first be identified. Those subtypes, including several splice variants and RNA editing variants, will be co-expressed in heterologous cells to test whether they can reconstitute the Asp response. Further, two complementary approaches will be taken. A sequence similarity strategy will be pursued that takes advantage of possible functional similarities between the Asp response and glutamate receptor-mediated responses. In addition, expression cloning approaches will be taken that utilize the pharmacological profile of the Asp response to identify potential Asp receptors. The results of these studies will clarify the nature of the unique Asp response in Purkinie cells. Moreover, since Asp may function as a neurotransmitter in other regions of the CNS, the results will provide a better understanding of the general role of Asp in neurotransmission and in the pathogenesis of certain brain disorders.
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