This study investigates mechanisms of activity-driven sharpening in the regenerating retinotectal projection of goldfish as a model system for development. It seeks to understand the role of activity and the mechanisms by which it causes the formation of precise connections, and it takes advantage of four features of this system: 1) the ease of surgical manipulation, 2) the ability of the optic fibers to regenerate a precise retinotopic projection, 3) the geometry of the eye which allows the containment of agents for long term manipulations, and 4) the known glutamatergic transmission of the projection through N-methyl-D-aspartate (NMDA) and quisqualate (QUIS) receptors. Correlated activity neighboring ganglion cells is used as a cue to concentrate the initially diffuse branches of retinal arbors into retinotopically correct areas in the overall map. At each tectal cell, the correlated activity of inputs causes summation of EPSP's that overcomes a voltage dependent block of NMDA receptors, and allow entry of calcium as a signal to stabilize these synapses. this proposal investigates mechanisms activated by calcium entry: whether kinase activation (either calmodulin-dependent kinase or C kinase) is necessary by infusing phorbols (which down-regulate C kinase), and kinase blockers. It also tests whether arachidonic acid (AA), known to be released by NMDA receptor activation, may serve as a retrograde messenger to the presynaptic terminal (to signal changes there), by infusing AA and inhibitors of AA release -- phospholipase A2 blockers. Sharpening of the projections is assessed by electrophysiological unit recording and horseradish peroxidase staining of retinal arbors. Synaptogenesis and effects on long-term potentiation are assessed by recording field potentials, and effects on outgrowth of retinal axons are assessed in culture assays. Finally, the culture system will be developed so that we can study growth cone interactions with tectal neurons during early phases of synaptogenesis with greater freedom to change the surrounding medium and assess effects at individual steps. 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. 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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