Date of Award
Doctor of Philosophy
Zoology and Genetics
Philip G. Haydon
This dissertation presents work in two parts. First, a presentation of effects of astrocytes on synapse formation of hippocampal neurons. The second describes a new near-field microscope for biological samples.;The formation of chemical synapses is a poorly understood process that is likely to critically rely on the presence of molecular cues that arise from presynaptic and postsynaptic neurons as well as associated glial cells. Rat hippocarnpal cultures were established in astrocyte-deplete and enriched conditions to ask whether the presence of astrocytes stimulated formation of chemical synapses. Co-culture with astrocytes selectively augmented formation of excitatory synapses. Immunostaining demonstrated that astrocytes stimulate export of synaptic proteins to the neurites, and the nerve terminal, which on ultrastructural examination were shown to contain increased number of synaptic vesicles within the presynaptic terminal. Also, local contact with astrocytes led to a selective augmentation of the magnitude of the N-type calcium current. The N-type calcium current is known to be associated with newly formed synapses. We propose that local interactions between the process of an astrocyte and developing pre- and postsynaptic terminals will augment calcium influx and thus synaptic transmission to place this tripartite structure at a competitive advantage over neighboring developing synapses that are devoid of interactions with astrocytes.;Sub-diffraction optical resolution achieved using near-field optical microscopy has the potential for new approaches and insights into sub-cellular function and molecular dynamics. Despite this potential it has been difficult to apply near-field microscopy to biology. Sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell. Increased fluorescence intensity during z-translation contains two components; far-field fluorescence and combined near- and far-field fluorescence. Fitting a regression curve to the far-field intensity as the illumination aperture approaches the cell; it is possible to isolate near-field fluorescent signals. We demonstrate ability to resolve actin filaments in fixed glial cells. Comparison of composite signals with extracted near-field fluorescence demonstrates this approach significantly increases ability to detect sub-cellular structures at sub-diffraction resolution.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Robert Thomas Doyle
Doyle, Robert Thomas, "Astrocyte effects on hippocampal synaptogenesis in culture and near-field microscopy " (2001). Retrospective Theses and Dissertations. 423.