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Endocytosis dependent critical periods mediated proper giant fiber escape circuit development in Drosophila melanogaster
Neurons extend processes that navigate to their synaptic target via a continuous sampling of guidance molecules and selective adhesion. In the Drosophila CNS, two large inter-neurons (the giant fibers) send their axons from the brain to the middle of the thorax, where each forms a large lateral synapse with a motor neuron (TTMn) that mediates the jump escape response. This mixed (electro-chemical) synapse is established during the first 30% of metamorphosis and mediates a fast (0.87 ms) and reliable response (100% at 100 Hz) to eye stimulation in adults. We examined the ability of the giant fiber (GF) to properly pathfind and form a synapse while selectively blocking endocytosis in, the GF, TTMn or both for 24 hours during a 100 hour metamorphosis. Through the directed expression of UAS-shibire (UAS-shi), under the control of a GAL4 driver, endocytosis is blocked when the fly is held at 30°C (considered a heat pulse (HP)). Blocking endocytosis in the GF during the first 60% of metamorphosis resulted in dramatic physiological and anatomical phenotypes. In these specimens, the probability of a GF to TTM or DLM connection was low and the connections made were significantly weaker then controls. In addition, the GF anatomy in these specimens was also aberrant; overgrowth resulted from an early HP (0–25%) and the GF was bendless in middle HP specimens (30–60%). Late HP (60% and afterwards) had no effect on GF anatomy or the probability of a connection to TTM or DLM, but weakened the strength of the synapses. When endocytosis was blocked in the postsynaptic TTMn, the critical period was only during the first 32% of metamorphosis. A HP 48% or afterwards had no effect on anatomy or physiology. Finally, a genetic interaction between a weak disruption in endocytosis by UAS- shi and weak over expression of UAS-semaphorin 1A (UAS-sema 1A) indicates a sema mediated repellent signaling aids in axon guidance and GF synaptogenesis. Based on the GF growth cone activity at the time of the HP and the resulting adult physiology and anatomy, three conclusions were drawn from these results. First, in late pupae (>60%) and adults endocytosis in the GF facilitates synaptic transmission and is not necessary to maintain the GF-TTMn synapse. From the beginning of metamorphosis to 60% was a presynaptic GF critical period where blocking endocytosis resulted in severe and permanent defects to TTM and DLM physiology and GF anatomy. In the postsynaptic TTMn, the endocytosis critical period was shorter, 0–32%. Second, once the GF reached the TTMn (30–60%), presynaptic endocytosis was critical for the removal of sema 1A to maintain synapse stability. In the postsynaptic TTMn, endocytosis was not critical for synapse stability. Third, maintaining sema 1A on the GF growth cone surface during pathfinding (0–25%) limited extra axonal branching.
Froggett, Stephan John, "Endocytosis dependent critical periods mediated proper giant fiber escape circuit development in Drosophila melanogaster" (2003). Doctoral Dissertations Available from Proquest. AAI3078683.