The goal of this project is to understand the mechanisms through which glutarnatergic synapses shape the development of visual circuitry. This knowledge should be useful in preventing or treating disabilities arising from early visual dysfunction. Our planned studies focus on the NMDA subtype (NR) of glutamate receptor (GR), because the MR is involved in intraocular synaptic competition, retinotopic map refinement and the early differentiation of glutamatergic synapses. We propose to use Xenopus tectal neurons in vitro and the superficial visual layers of the superior colliculus (sSC) in mice and rats. This research will: 1) Define the relationships among axon and dendritic sprouting, glutamate receptor function, and the maturation of synapses at the single neuron level using gene gun transfections to label neurons with fluorescent proteins and alter signaling proteins. Time-lapse imaging of such neurons during pharmacological blockade of different GRs, followed by post-imaging immunocytochemistry will be employed, and the role of NR-dependent protein synthesis and cadherin/B-catenin signaling will be explored. 2) Define the regulation of post-synaptic density proteins and of proteins involved in neuronal adhesion and process growth relative to developmental changes in normal visual function in the rat sSC. This work will use quantitative immunoblotting of rat sSC synaptoneurosome proteins to examine PSD proteins that anchor GR' s and proteins implicated in both synaptogenesis and sprouting (N-CAM, PSA-NCAM, Li, TrkB, BDNF, B-catenin and glycogen synthetase kinase 3b). After establishing normal profiles for these proteins, we will examine the effects of early NR blockade using sSC tissue exposed from birth to Elvax containing an NR antagonist. 3) Define structural changes in sSC afferents during later disruption of NR function and during termination in zones of low activity correlation. One set of studies will use blockade of the NR with antagonist in Elvax during the 2nd postnatal week to examine late effects on intraocular competition as monitored by sprouting of fluorescein-tagged ipsilateral retinocollicular afferents. A second Set of studies will construct a mouse carrying at the Rosa 26 locus a VAMP-GFP fusion protein cDNA behind a """"""""floxed"""""""" stopper fragment. This construct will label synaptic vesicles with GFP when the Cre recombinase is actived in neuronal somata by an adenovirus carrying the Cre gene. After characterization, living sSC slices from this mouse will be used to study the stability of presynaptic puncta in Dil-labeled retinocollicular terminals at topographic and non-topographic sSC loci during different stages of retinocollicular map refinement.
