The general aims of this project are to study the factors controlling the numerical and qualitative composition of photoreceptor synapses that form in the first neuropile, or lamina, of the optic lobe of the flies Drosophila and Musca. The modules of this neuropile, called cartridges, comprise small, fixed numbers of identified neurons, which we will sample in two ways: 1) We will use quantitative single-section EM to estimate synaptic frequencies, measure synaptic contact sites and cell surface areas. 2) We will also use serial EM to trace out synaptic connections between elements and undertake computer 3-D reconstructions of cell morphologies. Based on our previous analysis of normal synaptogenesis, we especially want to understand the control of the developmental assembly of multiple-contact synapses (dyads, triads, etc.), for which the fly receptor tetrad synapses are a model. Cross-species transplant experiments designed to allow chimeric synaptogenesis will test the conservation of synaptogenetic signals between homologous neurons in different species. The triple mutant rol/sol/mnb will procure the loss of differing combinations of postsynaptic contributors to the tetrads, through spontaneous cell degeneration prior to synaptogenesis, and allow surviving cells the opportunity to replace those lost, and thereby exhibit their synaptogenetic preferences. We will irradiate flies as prepupae to kill the epithelial glial cells that envelop the lamina cartridges, and explore the synaptic consequences, and rapidity, of anticipated sprouting from the interneurons of intact cartridges to adjacent cartridges which have been acutely deafferented by retinal photo-ablation. We will extend our initial approaches to procure synaptogenesis in vitro through the co-culture of photoreceptors and optic lobe cells. We will continue a major computer 3-D reconstructions of a complete lamina cartridge EM series to quantify the spatial characteristics of synaptic populations distributed over neuronal surfaces, especially those which constitute reciprocal connections between neurons. We will examine dynamic changes in the lamina cartridge: 1) amongst synaptic populations during a circadian rhythm, and after cold stress, both of which cause short-term changes, and 2) amongst interneurons, which show daily size changes; as well as investigate these functionally from long-term ERG recordings. Lastly, we will employ enhancer trap methods: 1) to study gene expression in identified optic lobe interneurons, especially that occurring under conditions known to promote synaptogenesis, as well as 2) to generate specific molecular markers to these cells. The studies proposed here, because they aim to produce a basic model of synaptogenesis applicable to multiple-contact synapses, such as the dyads and triads found widely in visual systems, should contribute to a general understanding of the perturbations in disease states to which visual synapses are susceptible during their growth and development.
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