The research outlined in this portion of the program project is designed to define the putative excitatory amino acid transmitters and receptors associated with cerebellar afferent systems in normal rats. Although neurotransmitters in the majority of cerebellar cortical neurons have been identified, information on the transmitters of cerebellar climbing fibers remains controversial and the major transmitters of the mossy fibers have yet to be defined. The experiments proposed will examine several acidic amino acid compounds, nitric oxide and polyamines as possible transmitter candidates for the olivocerebellar pathway and selected mossy fiber pathways. This research will provide basic information relevant to our understanding of the biochemical and pharmacological characteristics of cerebellar afferent systems. Our proposed investigation is described by the following three specific aims: 1. To determine whether glutamate, aspartate, NAAG, nitric oxide synthase or spermidine and putresine immunoreactivity is associated with identified climbing fiber or mossy fiber terminals by employing anterograde tracing with PHA-L and biotinylated dextran amine (BDA) in combination with immunocytochemistry at both light and electron microscopic levels. 2. Based on the results of 1, we will ascertain whether aspartate, glutamate, NAAG, citrulline, spermine or putresine are released in the cerebellar cortex following olivary, vestibular, LRN or middle cerebellar peduncle stimulation by combining electrical or chemical stimulation with in vivo microdialysis and electrophysiological recording or C-fos immunostaining. 3. The types of excitatory amino acid receptors associated with cerebellar afferents will be defined using: A) direct ultrastructural examination of PHA-L or BDA labeled terminals in combination with immunocytochemical localization of glutamate receptor subtypes and B) lesions of selected afferent pathways will be employed in combination with immunoprecipitation techniques and in situ hybridization.
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