MORPHOCHEMISTRY OF CEREBELLAR BRAINSTEM NEURONS AND CIRCUITS

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The overall goal of this project is to define the morphological basis for the spatial and temporal properties of calcium signaling in cerebellar neurons. The proposed experiments will focus on P-type calcium channels, plasmalemmal calcium pumps, and calcium concentration management molecules. The hypotheses to be tested and specific aims fall into two categories: 1) For the P-type calcium channel signaling and management system we propose that: i) P-type calcium channels are localized at synaptic junctions on the dendritic tree and somata of Purkinje, granule, and cerebellar nuclear neurons and that this localization relates to neuronal integration and second messenger signaling. ii) In order to restrict calcium concentration levels, calcium pumps (ATPases) are distributed at punctate sites over the dendritic arbor of Purkinje cells with the highest concentrations occurring distally on dendrites and spines in close association with P channels. iii) P-type calcium channels and plasmalemmal calcium pumps are co-localized and associated with mobile and bound calcium-buffering molecules, such calmodulin and calcineurin, which further increase the [Ca2+]i regulation abilities of these neurons. 2) For the IP3 intracellular calcium signaling and management system we propose that the endoplasmic reticulum, in association with IP3 receptor-related calcium channels and smooth ER calcium pumps, form a framework which establishes functional microdomains of intracellular calcium signaling and management. The studies will be carried out in the cerebellum of the rat using immunocytochemical techniques at both the light and electron microscopic levels. Ultrastructural studies will take advantage of recent advances in the high resolution localization of protein macromolecules, improved methods we have developed to pre-treat tissue sections, and the recent generation of specific antibodies against the P channel. An understanding of the morphological basis of both the plasmalemmal and the endomembrane calcium concentration management systems, and the relationship between these systems in controlling the cytosolic calcium concentration and in providing a mechanism for extruding calcium from neurons are critical elements in understanding neuronal function.