This study investigates activity-driven sharpening in the regenerating retinotectal projection of goldfish as a model system for development. It seeks to understand the roles of activity and calcium- activated mechanisms that drive the formation of precise connections, and takes advantage of 1) the ease of surgery, 2) the ability of the optic fibers to regenerate, 3) the geometry of the eye and the cranium both of which allow the containment of agents for long-term potentiation (LTP). LTP occurs during the sensitive period and LTP may be a first stage in sharpening. Correlated activity of neighboring ganglion cells is the cue to concentrate the initially diffuse branches of retinal arbors into retinotopically correct areas in the visual map. Correlated activity of inputs causes summation of EPSPs that allows calcium entry through NMDA receptors as an intracellular signal to stabilize these synapses. Beyond calcium entry, C-kinase activation and arachidonic acid release were both implicated in sharpening by infusing blockers and activators. Arachidonic acid may serve as a retrograde messenger to the presynaptic terminal to signal changes there. The current experiments 1) demonstrate the expression of C-kinase in developing retinal fibers and a reexpression during regeneration using immunostaining and phosphorylation assays, 2) test whether NMDA receptors stimulate the activation of phospholipase A2 to release arachidonic acid, 3) test whether blocking metabotropic glutamate receptors' prevents both LTP and retinotopic sharpening, 4) test with whole cell patch recording in cultured ganglion cells whether C-kinase activation uncouples the AP4 glutamate receptors inhibition of calcium channels, a proposed presynaptic mechanism for LTP, and 5) test with DiI staining in live zebrafish larvae the effects of these agents on the growth of retinal arbors. The culture system will also be developed to study how growth cones form synapses with tectal neurons in the presence of the above agents. There are two health related aspects. First, the phenomenon of activity-driven synaptic stabilization and competition in neural development is germane to developmental disorders such as amblyopia where one eye's connections become ineffective. Secondly, the failure to regenerate severed projections in mammalian central nervous system makes most neurological damage irreversible. Better understanding of successful regeneration in lower vertebrates may lead to strategies for therapeutic restoration of this ability in humans.
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