Student Theses and Dissertations

Date of Award

1991

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Previous study indicates that in the cerebellum. Nerve Growth Factor (NGF) and NGF receptors are highly expressed during late embryonic and early postnatal development, suggesting that NGF may play an important role in cerebellar ontogeny. However, while receptor had been localized to some cerebellar populations, delineation of receptor subtypes, and potential physiologic function remained to the defined. The objective of this thesis was to define the role that NGF plays in cerebellar ontogeny. Initial studies were devoted to the localization of NGF binding sites to cell types in the developing cerebellum, and to the definition of receptor subtypes. 125I-.NGF binding was employed to delineate low- and high-affinity receptor sites in the postnatal rat cerebellum. Both high-affinity and low-affinity receptor sites were localized to cell bodies, dendrites and axonal processes of Purkinje cells. The expression of the biologically active, high-affinity receptors was found to correlate with cerebellar development, and suggested that N G F may regulate the normal ontogeny of Purkinje cells. The development of cerebellar Purkinje cells is subject to regulation by multiple epigenetic signals. To define mechanisms by which trophic and presynaptic stimulation may potentially regulate Purkinje cell ontogeny, I studied the effects of N G F and excitatory transmitters on Purkinje cell survival and morphological maturation in dissociated cell culture. Purkinje cells were identified by expression of Vitamin D-dependent Calcium Binding Protein (CaBP), and by their characteristic morphology. N G F receptors were selectively localized to Purkinje cells both by ligand and monoclonal antibody binding, suggesting responsivity to the trophic agent. Simultaneous exposure to the pharmacologic depolarizing agents, high-potassium or veratridine, and N G F specifically enhanced Purkinje cell survival in culture. Furthermore, N G F together with the excitatory neurotransmitters, aspartate or glutamate, promoted a 2-fold increase in survival. In addition, N G F increased Purkinje cell size and promoted neurite elaboration. These effects required simultaneous exposure to N G F and either aspartate, glutamate or pharmacologic depolarizing agents. Effects on survival or neurite elaboration were not evoked by exposure to trophic factor or transmitters alone. These results suggested a novel mechanism for regulation of development, in which trophic factors and afferent stimulation interact. To begin to investigate potential mechanisms by which excitatory signals and NGF interact in the regulation of Purkinje cell development I studied regulation of NGF receptor expression by excitatory influences in dissociated cerebellar cell cultures. Sensitive receptor-protein cross-linking, and ribonuclease protection assays were employed to evaluate NGF receptor protein, and message levels respectively. Exposure of neurons in culture to either pharmacologic depolarizing agents, or to the normal afferent neurotransmitter aspartate, resulted in a 2 to 3 fold increase in N G F receptor expression. In addition to the N G F receptor, expression of the NGF gene was examined. Cerebellar glia were identified as the source of N G F expression, and the synthesis of the trophic factor was also subject to regulation by depolarizing signals. Together, the results presented in this thesis suggest that afferent innervation and N G F interact to promote survival and morphogenesis of developing Purkinje cells.

Comments

A thesis submitted to the faculty of Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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