Previous studies from our lab showed that terminal Schwann cells (TSCs) are actively involved in the restoration of functional synapses during reinnervation at mammalian neuromuscular junctions (NMJs). However, it is unclear what induces TSCs to extend processes that guide the nerve growth (Son and Thompson, 1995a,b). Is it the loss of the axon or instead, some signal arising from denervated muscle fibers? The main objective of my dissertation was to examine whether muscle fibers can be the source of signals affecting TSC growth. In Chapter 2, I report that both TSCs and nerve terminals are maintained at the former junction even after their underlying muscle fiber degenerates. Some of the original AChRs are surprisingly sustained at the synaptic sites for a long time with the preserved pretzel pattern. These results show that the postsynaptic target is not necessary for the maintenance of presynaptic structures. In Chapter 3, I report that following fiber regeneration, newly formed AChRs are clearly separated from the persisting receptors at most locations and they are apposed by the nerves. Moreover, as the fiber regenerates, TSCs begin to grow processes. Nerve sprouts then follow these processes to form new synaptic sites beyond the old receptor territory. My observations therefore show that signals for nerve growth arise from regenerating fibers and they appear to act by first affecting TSCs. Such signals seem diffusible because I saw that TSCs on surrounding fibers also began to grow during regeneration. In Chapter 4, I report that new junctions on the regenerated fibers are very dynamic. They undergo continual remodeling and eventually take on an 'en grappe' pattern. Since the synapses on undamaged fibers are normally very stable, these observations suggest that regeneration has set in place a process whereby the synapses are unable to stabilize. Interestingly, this appears to be the case in muscles that degenerate as a consequence of muscular dystrophy. My findings are important because they suggest an active role of the postsynaptic muscle fiber not in synapse maintenance but rather in generating signals that attract innervation after injury. / text

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

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Myosin VI, encoded by jaguar (jar) in Drosophila melanogaster, is the only member of the myosin superfamily of actin-based motor proteins known to move towards the minus ends of actin filaments. In vitro studies demonstrate that Myosin VI has the ability to perform distinct functions as a cargo transporter and anchor in the cell, however which of these roles Myosin VI plays in the nervous system has yet to be determined. A locomotor defect, observed as sluggish movement in severe jar mutant larvae, was confirmed by behavioural assays. As this can indicate problems at the neuromuscular synapse, microscopy and electrophysiology were used to investigate neuromuscular junction (NMJ) structure and function in jar loss of function mutants of varying severity. Confocal imaging studies revealed a decrease in NMJ length, a reduction in bouton number per NMJ, alterations to the microtubule cytoskeleton and mislocalization of the synaptic vesicle protein Synaptotagmin in jar mutant boutons. FM dye labeling was consistent with the immunostaining data revealing vesicles endocytosed following electrical stimulation occupy the bouton centre in jar mutants. The data is indicative of a function for Myosin VI in maintaining proper peripheral vesicle localization. Electrophysiological experiments revealed a role for Myosin VI in basal synaptic transmission, with a reduction in low frequency nerve-evoked responses and spontaneous release in severe jar mutants. Changes in short-term synaptic plasticity were also observed in Myosin VI mutants by using both paired-pulse experiments to examine release probability and high-frequency stimulation paradigms to recruit vesicles from different functional pools. Taken together, the data suggest that Myosin VI functions as an anchor to peripherally localize vesicles within the bouton enabling their efficient release during nerve stimulation. Synaptic vesicles are mobile at the Drosophila NMJ; thus if Myosin VI is acting as a vesicle tether, it would normally be expected to restrain vesicle mobility at the synapse. FRAP analysis revealed a significant increase in synaptic vesicle mobility in jar mutant boutons. This study elucidates novel roles for Myosin VI function in the nervous system via regulation of the synaptic microtubule architecture and localization of synaptic vesicles within the nerve terminal.

Myosin VI, encoded by jaguar (jar) in Drosophila melanogaster, is the only member of the myosin superfamily of actin-based motor proteins known to move towards the minus ends of actin filaments. In vitro studies demonstrate that Myosin VI has the ability to perform distinct functions as a cargo transporter and anchor in the cell, however which of these roles Myosin VI plays in the nervous system has yet to be determined. A locomotor defect, observed as sluggish movement in severe jar mutant larvae, was confirmed by behavioural assays. As this can indicate problems at the neuromuscular synapse, microscopy and electrophysiology were used to investigate neuromuscular junction (NMJ) structure and function in jar loss of function mutants of varying severity. Confocal imaging studies revealed a decrease in NMJ length, a reduction in bouton number per NMJ, alterations to the microtubule cytoskeleton and mislocalization of the synaptic vesicle protein Synaptotagmin in jar mutant boutons. FM dye labeling was consistent with the immunostaining data revealing vesicles endocytosed following electrical stimulation occupy the bouton centre in jar mutants. The data is indicative of a function for Myosin VI in maintaining proper peripheral vesicle localization. Electrophysiological experiments revealed a role for Myosin VI in basal synaptic transmission, with a reduction in low frequency nerve-evoked responses and spontaneous release in severe jar mutants. Changes in short-term synaptic plasticity were also observed in Myosin VI mutants by using both paired-pulse experiments to examine release probability and high-frequency stimulation paradigms to recruit vesicles from different functional pools. Taken together, the data suggest that Myosin VI functions as an anchor to peripherally localize vesicles within the bouton enabling their efficient release during nerve stimulation. Synaptic vesicles are mobile at the Drosophila NMJ; thus if Myosin VI is acting as a vesicle tether, it would normally be expected to restrain vesicle mobility at the synapse. FRAP analysis revealed a significant increase in synaptic vesicle mobility in jar mutant boutons. This study elucidates novel roles for Myosin VI function in the nervous system via regulation of the synaptic microtubule architecture and localization of synaptic vesicles within the nerve terminal.

No description available.