ELEMENTAL AND STRUCTURAL ORGANIZATION OF NEURONS AND GLIA

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This two-part project studies the organization and function of specialized membranes in neurons and glia. The first part aims to characterize calcium regulation during synaptic activity in the dendrites and dendritic spines of cerebellar Purkinje cells and CA3 hippocampal pyramidal cells. Previous electro-physiological and structural work had established that a new type of organotypic culture of hippocampus was an excellent preparation for studying free calcium transients in living slices, as well as for analyzing the cellular distribution of total calcium in directly frozen cultures by means of analytical electron microscopy. Further experiments have now revealed a dramatic increase in the calcium content of specific endoplasmic reticulum-like organelles within the dendrites of CA3 neurons under conditions of synaptic stimulation which produce a response similar to long-term potentiation (LTP). This provides the first direct evidence for and identification of calcium-sequestering organelles which are specifically involved in potentiating neural-inducing activity. Parallel experiments on cultures of isolated pyramidal cells have revealed a two-fold increase in the density of dendritic spines on neurons treated with estradiol during development of the dendritic arbor. This preparation appears promising for studying the development of the calcium-regulating apparatus in conjunction with hormonal effects on the development of spines and synapses. In Part Two, immunocytochemistry and confocal light microscopy have been used to study the expression, transport and assembly of myelin. Prior work had shown that myelinating Schwann cells have a characteristic organization of microtubules which is essential for the sorting, transport and targeting of myelin-specific proteins. We have now used microtubules assembly/disassembly experiments to show that an axon- specific signal mediates the appropriate organization of Schwann cell microtubules, i.e., the dispersion of microtubule minus ends and the induction of multiple microtubule organizing centers in perinuclear cytoplasm. This signal is absent during Wallerian degeneration, which therefore results in formation of disruptive microtubule organizing centers within the myelin internode.