Research this past year focused on the molecular characterization of glutamate receptors and their function in the auditory system. Previously, we demonstrated that the delta 2 receptor is selectively expressed at parallel fiber synapses of cerebellar Purkinje neurons. This past year we established that this selective expression is developmentally regulated; in young animals both parallel and climbing fiber synapses express delta receptors, while in adults the receptor is found only at parallel fiber synapses. AMPA (amino-2-hydroxy-5-methyl-4- isoxazolepropionic acid) receptors, in contrast, are found at both synapses in adults and throughout development with no significant change in amount of receptor expression with development. In a study to investigate the molecular basis of silent synapses in the hippocampus, we determined the developmental expression of AMPA and NMDA (N-methyl-D-aspartate) receptors at pyramidal cell synapses. NMDA receptors are highly expressed as early as 2 days postnatal and maintain this level of expression into adulthood. However, AMPA receptors are rare in young animals, but significantly increase with development. In adult animals about 90% of the synapses contain AMPA receptors. These results support the idea that the addition of AMPA receptors to the postsynaptic membrane during development is related to the decrease in the number of silent synapses during this time period. We have previously shown in fusiform cells of the dorsal cochlear nucleus and in Purkinje cells of the cerebellum that different glutamate receptors are expressed at different synaptic populations. A fundamental question is how this selective targeting is achieved. We hypothesized two mechanisms: receptors are either selectively transported to the synapses or this transport is nonspecific, but specificity is obtained through a selective anchoring of the appropriate receptor at the postsynaptic site. By determining the distribution of intracellular receptors in apical and basal dendrites of fusiform cells, we find evidence supporting the idea of a selective transport of receptors. For receptors that are at synaptic sites in basal dendrites only, the intracellular pool of these receptors is greater in basal dendrites than in apical dendrites. Our investigation of the role of homer proteins in the expression of type I metabotropic receptors suggests that one member of this family, homer 1b, may play a role in the intracellular trafficking of the receptor. In transfected cells, homer 1b retains the receptor in the endoplasmic reticulum, and in the cerebellum, homer 1b is localized at the cytoplasmic side of the postsynaptic density, a site consistent with its control of receptor expression at the synapse. In an analysis of SNARE complex proteins expressed in the organ of Corti, we have found an absence of the expression of synaptotagmins I and II, which are generally considered essential components of neurotransmitter release. Other components are expressed. In an attempt to search for additional proteins involved in neurotransmitter release from hair cells, we have developed a cDNA library of guinea pig organ of Corti suitable for screening with the yeast two-hybrid system. Screening of the library with baits of SNAP25 and syntaxin 1 has identified several potential interacting proteins.
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