Our broad goal is to understand how precise patterns of neuronal connections are established during development of the central nervous system. This proposal focuses on the role of presynaptic signaling in organizing in organizing the structure of the postsynaptic substrate, the dendritic arbor, during synapse formation and refinement of the retina. The dendrites of immature retinal ganglion cells (RGCs) undergo structural and functional remodeling as they contact and receive input from presynaptic cells. The role of activity-dependent and activity- independent signaling from two classes of retinal interneurons (cholinergic amacrine cells and bipolar cells) in the remodeling of RGC arbors will be investigated.
Aim 1 will characterize the structural and potential functional relationship between the terminal processes of the retinal interneurons and the dendrites of RGCs before and during RGC dendritic remodeling. RGC arbors will be labeled with multicolors by a novel method of delivering carbocyanine dyes to retinal explants (collaboration with Jeff Lichtman). Cholinergic amacrines and a subset of bipolar cells will be labeled by introducing yellow fluorescent protein downstream of promoters specific to these cells, using knock-in technology.
Aims 2 and 3 will assess the in vivo roles of presynaptic signaling by ablating these interneurons or blocking their release of neurotransmitter. Transmission will be blocked by knocking out the choline acetyltransferase gene, or by introducing the tetanus toxoid (TeTx) gene downstream of the cell-specific promoters. Ablation of cells will be carried out by replacing the TeTx gene with the diptheria toxin gene. Cre-lox technology will e used to ensure that gene or cell ablation and neurotransmission blockade occurs only when induced at an appropriate developmental stage. RGC arbors from wildtype and mutant mice will be reconstructed and compared. All transgenic mice will be generated in collaboration with Josh Sanes.
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