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Koper, Ewa (2007): Differentiation of dendrites and the analysis of spine- like structures on Lobula Plate Tangential Cells in Drosophila melanogaster. Dissertation, LMU München: Faculty of Biology
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Abstract

The development of dendrites leads to the establishment of cell-type specific morphology of dendritic trees that eventually determines the way in which synaptic information is processed within the nervous system. The aim of this study was to investigate dendritogenesis of Drosophila motion-sensitive Lobula Plate Tangential Cells (LPTCs) and to understand the role of cytoskeletal molecules in these developmental processes. I employed genetic techniques to obtain fluorescent labeling exclusively in the neurons of interest. In order to visualize the LPTCs confocal imaging was applied. Time point analysis allowed me to follow and describe the phases of LPTC differentiation in the intact Drosophila brain starting from the third instar larva throughout the pupal stages until adulthood. I determined the time when the initial growth of LPTC dendrites starts and showed it to be directional from the beginning. Additionally, I demonstrated that the phase of extensive dendritic growth and branching precedes reorganization processes that lead to establishment of the final architecture of LPTC dendritic trees. In parallel, I attempted to analyze the contribution of actin and tubulin in the shaping of the neurons. In these experiments actin-GFP localized to dendritic termini whereas tubulin-GFP was mainly observed in the primary dendritic branches. These data showed clear similarities between the cytoskeletal organization of LPTCs dendrites and vertebrate neurons. The discovery of the actin enrichment in dendritic termini made me conduct a set of experiments to test if these protrusions are the counterparts of vertebrate spines. I performed a thorough quantitative analysis of spine- like protrusions present on LPTC dendrites. Morphological features like the density and shape of the LPTC spine- like protrusions appeared to be comparable to hippocampal spines. Using immunohistochemical methods I demonstrated that LPTC spine-like protrusions are sites of synaptic contacts. The ultrastructural analysis supported the immunohistochemical data and showed that synaptic transmission takes place at the LPTC spine-like protrusions. Next, I tried to genetically modify these structures by generating LPTC mutant for genes which have vertebrate homologues known to alter spine morphology. I showed that dRac1 can modulate significantly the LPTC spine-like structure density. Finally, I tried to check if Drosophila LPTC spine-like structures are motile. To conclude, I showed an initial description of LPTC dendritogenesis and the subcellular localization of actin and tubulin in these neurons. The actin enriched spine-like structures detected on the LPTC dendrites are sites of synaptic contacts, thus resemble vertebrate spines.