The aim of this program is to study those aspects of the physiology, molecular biology and morphology of calcium-triggered Purkinje cell death that relate to normal adult and aged rats as well as to the accelerated aging syndrome (AAS) in the same animal. Our basic hypothesis is that the increased tendency for neuronal death which is prevalent in aged and AAS animals, may represent an exaggerated tendency to express a self-destruct mechanism normally present in all neurons. The study will be composed of four approaches which bring together several disciplines in neuroscience. Project Ia will use in vitro electrophysiological techniques to study intracellular calcium regulation in normal adult, aged and AAS in rat Purkinje cells. The study will correlate intracellular voltage recordings with changes in [Ca2+]i using the calcium-sensitive dye Fura 2 under conditions which induce cell death. In addition, a study of the biophysical properties of single channels under the same conditions will be carried out using patch clamp techniques. Project Ib will address the role of IP3 in the regulation of [Ca2+]i in Purkinje cells of normal, aged and AAS rats. It will attempt to determine the biochemistry of IP3 modulation and the role of IP3 in calcium release from intracellular stores. The experiments will examine the intracellular effects of IP3 and IP3 blockers on [Ca2=]i, as well as the localization of phosphoinositide hydrolysis and IP3 generation following specific pharmacological challenges as well as during cell death. Project II will study the molecular biology of the so-called """"""""P"""""""" channel from aged and AAS animals. The project entails the isolation of calcium channels from rat cerebellum, their molecular characterization and the generation of polyclonal and monoclonal antibodies against the channels. Channels isolated from adult, aged and AAS Purkinje cells will be incorporated into lipid bilayers having different compositions, and their biophysical and biochemical properties will be determined. Project III will generate a quantitative model of the major structural parameters of Purkinje cells in aged and AAS animals compared to young adults. Antibodies developed as part of Project II will be used to determine immunocytochemically the distribution of both voltage-dependent """"""""P"""""""" and intracellular calcium channels in aged and AAS cells. Finally, the in vitro recordings made as part of Project I will be correlated with this morphological information to integrate the functional and structural aspects of the role of calcium in the process of aging.
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