The overall objective of the proposed research is to understand mechanisms of 1) neural reorganization after insults such as stroke, and 2) host-graft interactions after transplantation, a potential future therapeutic approach to stroke. Specifically, the focus of this project is on the role of neural activity in the reorganizational changes following injury and transplantation. Cerebellar cultures will be chronically exposed during and after treatment with cytosine arabinoside to tetrodotoxin, to block all electrical activity, and to picrotoxin, to enhance the normally present cortical spontaneous activity. The cultures will be monitored by light and electron microscopy for possible alterations of the reorganizational changes consequent to cytosine arabinoside induced granule cell depletion and glial inhibition. These include 1) Purkinje cell recurrent axon collateral sprouting, 2) enhanced Purkinje cell survival, 3) hyperinnervation of Purkinje cell somata by sprouted collaterals, and formation of heterotypical recurrent axon collateral-Purkinje cell dendritic spine synapses. Cytosine arabinoside treated cerebellar cultures will similarly be exposed to tetrodotoxin and picrotoxin immediately after transplantation with granule cells and glia. These transplantation, including reduction of sprouted collaterals and of excess Purkinje cells, astrocytic stripping of excess axosomatic synapses, and the formation of parallel fiber-Purkinje cell dendritic spine synapses. After definition of the ultrastructural characteristics of catecholaminergic terminals in cerebellar cultures with incorporated locus coeruleus, as well as cytosine arabinoside treated coeruleocerebellar cultures and transplanted cytosine arabinoside treated coeruleocerebellar cultures, such cultures will be similarly exposed to tetrodotoxin and picrotoxin, and similarly monitored to determine if the effects of neural activity are different with regard to catecholaminergic axons as compared with other axons.
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