The general aims of this project are to study the factors controlling the numerical and qualitative composition of photoreceptor synapses formed in the first neuropile, or lamina, of the optic lobe of the flies Drosophila and Musca. 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. The modules of the lamina, called cartridges, comprise small, fixed numbers of identified neurons. We will sample these: 1) using quantitative single-section EM to estimate synaptic frequencies, measure synaptic contact sites and cell surface areas. 2) using serial EM to undertake computer 3-D reconstructions of immature cell morphologies, to relate dendritic growth to synapse formation. Under states of rapid change occurring after functional reversals of light or of temperature, we will plot the location of new synaptic sites relative to old ones, to examine the influence of an existing site on the formation of a new synaptic site nearby, and the presynaptic ribbon's mode of formation at the new site. We will also examine the recycling of membrane between synaptic organelles, using the Drosophila mutant shibire. The functional outcome of anatomical synaptic changes will be sought from ERG recordings, and the activity-dependence of synapse formation will be assessed in mutants with impaired pre- or postsynaptic lamina function. The control of circadian modulations in the lamina by two sets of widespread neurons, one thought to release 5-HT, the other the peptide PDF, will be analyzed as a further example of plasticity in the lamina. We will irradiate flies as pupae to kill the epithelial glial cells that envelop the lamina cartridges, and explore the synaptic consequences, and rapidly, of anticipated sprouting from the interneurons of intact cartridges to adjacent cartridges which have been acutely deafferented by retinal photo-ablation. 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 exhibit their ability to substitute synaptic partners. We will exploit the tissue-culture system we have developed to examine selective fasciculation amongst photoreceptor axons and their interactions with their lamina cell targets, and to procure synaptogenesis in vitro from co-cultures of photoreceptors and optic lobe cells. In collaboration with other labs, we will examine the expression of genes involved in either the formation or maintenance of synapses. We will examine GAL4 Drosophila lines, in which gene expression is marked by an exogenous reporter gene, to identify expression occurring under conditions known to promote synaptogenesis, as well as to secure specific molecular markers to identified optic lobe cells. We will also examine the possible synaptic expression of two promising candidate genes, irreC and demo, using immuno-EM. 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, will contribute to a general knowledge of the perturbations in disease states to which visual synapses are susceptible during their growth and development.
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