This proposal examines a novel mechanism for the activity-dependent regulation of synaptic targeting and error correction. At the developing Drosophila neuromuscular junction a low frequency (0.01-0.03Hz) Ca oscillation modulates or gates the cell?s response to retrograde transsynaptic chemotropic signals from target cells. The dependence on neural activity is presynaptic and heterosynaptic. This system provides a powerful avenue for studying an alternate mechanism for early synaptic refinement in the establishment of neural circuits, distinct from a Hebbian matching that involves spike timing and competition. A similar mechanism involving low frequency oscillations has been observed in the development of connectivity at the mammalian superior colliculus. In our system, the low frequency Ca oscillation regulates the response of the growth cone to a postsynaptically derived chemorepellant, Semaphorin 2a (Sema2a), acting through a Plexin B receptor in the neuron. Experimental manipulation of the frequency and duration of the Ca oscillator disrupts the synaptic wiring process. Our previous works shows that the responsiveness of the neuron to Sema-2a involves at least three Ca-dependent signaling systems: principally a Ca-activated adenylyl cyclase, as well as the Ca-activated serine/threonine kinase CaMKII, and the Ca-dependent PP2B phosphatase Calcineurin. This proposal will examine in detail the signaling events that are involved in early synaptic targeting and refinement, in the context of a time-dependent Ca-wave environment. This includes live imaging of growth cones and their contacts, molecular genetic manipulation of the second messenger systems, and genetic tests to examine the roles of candidate molecules, including kinases and phosphatases, in mediating this time-dependent system of second messenger activity. We use several molecular tools we have developed, that include reengineered ion channels that either suppress or enhance membrane excitability, and an inducible bipartite gene expression system to perform experiments testing the temporal requirements for induced genes at specific synapses.
A major goal of neuroscience is to identify how synapses form and refine their initial contacts, removing those made onto the wrong cells. We have found that there is a low frequency oscillation in neural activity that controls how the cell responds to both repulsive and attractive signals. The results are of significance in understanding how nervous systems acquire their precise patterns of connections.
