Date of Award
Doctor of Philosophy
Zoology and Genetics
Philip G. Haydon
This dissertation deals with the regulation of synapse formation between cultured hippocampal neurons, and the volume dynamics of astrocytes during hyposmotic-induced swelling;The developmental time course of synapse formation in cultured hippocampal neurons was examined. After 12 days in culture (DIC) inhibitory and excitatory synapses formed which were sensitive to the N-type calcium channel antagonist [omega]-conotoxin GVIA ([omega]-CgTx) while, at 4 DIC, immature connections were present in which spontaneous, but rarely evoked, synaptic currents were detected. A comparison of 4 and 12 DIC neurons revealed the presence of the synaptic proteins rab3a, synapsin I, and synaptotagmin, but the subcellular distribution changed from one in which immunoreactivity was initially distributed within the soma and neurites to a punctate varicose appearance. Correlated with these changes from immature to mature synaptic states was the development of [omega]-CgTx-sensitive calcium influx. These data suggest that the expression of functional [omega]-CgTx-sensitive calcium influx is temporally coincident with synapse formation, and that during the maturation of the synapse there is a redistribution of synaptic proteins;While astrocytes are known to actively regulate cell volume, the role of the F-actin cytoskeleton and membrane cycling in this process are ill-defined. A combination of scanning probe microscopy and a fluorescent dye dilution assay were utilized to dynamically examine hyposmotic-induced volume changes. Treatment of astrocytes with cytochalasin B to disrupt the F-actin cytoskeleton enhanced the hyposmotic-induced volume increase, but the rate of the compensatory regulatory volume decrease (RVD) was unaffected. Hyposmotic saline neither enhanced cell surface area nor caused altered membrane trafficking. These data are consistent with the hypothesis that astrocytes unfold plasma membrane upon treatment with hyposmotic saline, and reconvolute membrane during RVD. Furthermore, the F-actin based cytoskeleton may function to retard peak volume changes, presumably by restricting membrane unfolding, but does not function in controlling RVD. In addition to hyposmotic saline, glutamate release from astrocytes can also be stimulated by [alpha]-latrotoxin. Application of [alpha]-latrotoxin in the presence or absence of external calcium does not cause a significant increase in astrocyte volume, indicating that [alpha]-latrotoxin is not exerting its secretagogue effect through a swelling-activated pathway.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Trent Allan Basarsky
Basarsky, Trent Allan, "Astrocyte volume regulation " (1996). Retrospective Theses and Dissertations. 11106.