Morphologican and functional studies on the Drosophila neuromuscular system during postembryonic stages

Abstract: The nervous system of the fruit fly Drosophila melanogaster has emerged as an excellent model for fundamental neuroscience as well as for biomedical research of human neurological diseases. In this thesis, two aspects of the neuromuscular system have been investigated: the role of the IkB-protein Cactus in the larval neuromuscular junction and the morphology of motor terminals throughout adult life.We found that cactus mutant larvae have poor locomotion, morphological abnormalities at the presynaptic site of motor terminals and impaired mechanical and electrophysiological properties, demonstrating that Cactus is clearly involved in the normal functioning of Drosophila neuromuscular system. In the adult, we show that cactus, dorsal and dif are expressed in the brain but are not redistributed between cytoplasm and nucleus in a circadian manner as expected from a previous finding in larval brain. Both Cactus and Dif immunoreactivity was strong in mushroom bodies and antennal lobes, suggesting a putative role in olfactory memory. In the rat, proteins of the same family are involved in the regulation of sleep but we found no indication of such regulation in flies subjected to 6 hrs of sleep deprivation.We found that neuromuscular junctions continue to change throughout adult life. Two types of long-term changes in the morphology of neuromuscular junctions are demonstrated here: a daily change in the size of synaptic boutons and long-term changes in bouton size developing over several weeks. By careful morphological studies of flight neuromuscular terminals in clock-gene mutants and wild type flies of different ages we demonstrate that the daily changes depend on the biological clock and disappear in the old fly. Moreover, we show that light is necessary for the motor neurons studied to reach maximum size of synaptic boutons. Lastly, we found that the two clock genes period and timeless are also necessary to control axonal branching.Transmission electron microscopy revealed several ultrastructural features distinct of the aging fly and indicative of reduced plasticity. We used a temperature-sensitive allele of shibire that rapidly and reversibly blocks vesicle recycling to investigate whether the morphological phenotype found in neuromuscular junctions of aging flies could be explained by impairment of endocytotic mechanisms. Our results show a clear reduction of the time required for complete paralysis and an increased recovery time in old flies, indicating that aging correlates with impaired endocytosis and membrane dynamics.