At the cellular level, learning and memory are represented by changes in the efficacy of the neuronal communication. It is now understood that growth of new synaptic connections underlies rewiring of neuronal networks. Although tremendous progress has been made recently in understanding postsynaptic mechanisms of this process, little is known about activity-dependent modifications at the presynaptic level. Although advanced imaging approaches allow monitoring the growth of dendritic spines in the brain in a real time, this is not yet the case for the growth and proliferation of presynaptic terminals. However, this process can be reliably monitored in the Drosophila larval neuromuscular synapse, a model system that is readily amendable to genetic and molecular biology approaches. In the present application we build upon our preliminary results in the Drosophila neuromuscular junction to investigate the activity-dependent budding and outgrowth of synaptic buttons at the functional and molecular levels. We have adopted a synaptic growth assay in Drosophila that uses patterned depolarization's to induce robust budding of new presynaptic buttons. Our preliminary results suggest that the activity-dependent outgrowth of synaptic buttons depends on the presence of synapsing, a neuronal phosphoprotein that is thought to regulate synaptic vesicle clustering, synaptic plasticity, and neuronal development. The central question that we propose to investigate is what are the molecular mechanisms underlying maturation of early synapses, more specifically, what is the role of synapsin in the activity-dependent synaptic growth and differentiation. We propose to address this issue by taking advantage of the molecular tools available in Drosophila to investigate the initial stages o bouton formation, differentiation, and activation in real time. Specifically, we will employ live confocal imaging to monitor formation and differentiation of new synaptic terminals and to investigate how this process depends on synapsing levels. To complement these experiments, we propose to employ optical methods to monitor the activity-dependent redistribution of synapsing within synaptic terminals. Since synapsis had been implicated in mediating formation and maturation of new varicosities in both vertebrates and invertebrates, the results of these studies are likely to have wide applicability beyond Drosophila.
New synapses can be formed in response to intense activity, and impairments in this process are associated with a broad range of neurological disorders including neurodegeneration, mental retardation, and Huntington's disease. However, the presynaptic mechanisms that govern synaptic restructuring remain obscure. This application aims to investigate the activity-dependent formation, differentiation, and maturation of new synaptic terminals.
