A Genetic Analysis of Structural and Functional Homeostatic Plasticity at the Drosophila Neuromuscular Junction

The structure and function of the nervous system is continuously changing due to genetic programs and environmental interactions. Regardless, despite such a degree of plasticity, the nervous system can remain functional under normal conditions. It is widely accepted that homeostatic regulatory mechanisms, some operating at the synapse, are responsible for stabilizing neural activity in the face of perturbations. From studies of the Drosophila neuromuscular junction (NMJ), a model glutamatergic synapse, mechanisms of homeostatic synaptic plasticity are being unraveled. These mechanisms include, but are not limited to, enhancement of neurotransmitter release from nerve terminals and a restructuring of synaptic morphology to maintain a set point of muscle depolarization. A reduction in the postsynaptic sensitivity to the neurotransmitter, glutamate, is countered by a homeostatic increase in the amount of glutamate release at Drosophila NMJs. This phenomenon, termed homeostatic potentiation, can be rapidly induced by an antagonist of glutamate receptors or chronically expressed following genetic ablation of a subset of glutamate receptors. Conversely, undergrowth of fly NMJs can be offset by homeostatic increases in the number of active zones, the sites of neurotransmitter release. Likewise, overgrowth of fly NMJs can be mollified by the redistribution of neurotransmitter release machinery throughout synapses. Although several synaptic proteins have been linked to these homeostatic regulatory mechanisms, it is not currently known if these phenomena are distinct at a molecular level. Furthermore, it is unclear if homeostatic plasticity at the fly NMJ is separable from overtly similar forms of synaptic plasticity, including long-term potentiation and presynaptic homeostatic scaling, in the mammalian brain. In this thesis, I tried to address these outstanding issues via genetics, electrophysiology, and imaging.In Chapter 2, I describe mutagenesis and characterization of the gene encoding Synaptotagmin 12 (Syt12) in Drosophila. This study indicates that Syt12 is required for the normal growth and elaboration of the fly NMJ. Yet, counter to expectations, synaptic undergrowth in Syt12 loss-of-function mutants was concomitant with a decreased active zone number and an enlargement of the readily-releasable pool of synaptic vesicles, as measured by two-electrode voltage-clamp recordings. These data implicate Syt12 in the morphogenesis of synapses and in the limiting of neurotransmitter release. Additionally, these findings are consistent with a function for Syt12 in presynaptic structural plasticity due to synaptic undergrowth. We suggest that the mechanisms of presynaptic structural plasticity are separable from those of homeostatic potentiation, which was expressed in the absence of Syt12, but may be related to those of presynaptic long-term potentiation.In Chapter 3, I describe the characterization of Cdk5 and its activator, p35, in the fly. This study indicates that Cdk5 signaling is not essential for homeostatic potentiation at the fly NMJ, despite its earlier association with a similar form of homeostatic synaptic plasticity in the hippocampus. Moreover, we validated a previous report by showing that NMJ overgrowth is compensated by a decrease in active zone density in the absence of Cdk5 signaling. These results suggest that homeostatic potentiation and another form of homeostatic structural plasticity at Drosophila NMJs are also molecularly separable. In a series of voltage-clamp experiments, we have also gathered evidence that Cdk5 activity regulates the composition of synaptic vesicle pools, largely consistent with its role in the hippocampus. Yet, microscopical techniques revealed only weak effects, if any, of Cdk5 signaling on the distribution of synaptic vesicle markers and glutamate receptors. These findings should add to our understanding of Cdk5 in brain evolution and human disease