This proposal examines the role of neural activity in regulating two forms of retrograde signaling during the development and maturation of synapses. The proposal will investigate this form of signaling as a model for understanding how early synaptic critical periods operate. The proposal has two specific aims. Aim 1 examines how neural activity modulates a retrograde chemorepulsive signal from muscle (Sema-2a) that acts on the motoneuron during the early refinement of synaptic connections at the neuromuscular junction (NMJ). The responsiveness of the motoneuron to the retrograde Sema-2a is modulated by Ca2+, potentially through the activation of CaMKII. Using genetic and molecular approaches we will define the downstream effectors that mediate the activity-dependent repulsion. In addition we will elucidate how patterned rhythmic electrical activity regulates the withdrawal of processes from off-target muscles. Aim 2 addresses a second form of retrograde control by a muscle-derived TGF-beta ligand, Gbb, that acts through a canonical BMP signaling pathway. We have found two distinct roles for the retrograde control: an early critical period that defines the future growth of the NMJ, and is required for both the mature size of the NMJ, and for activity-dependent expansion. There is also an ongoing requirement for retrograde signaling to regulate synaptic function. We will examine the molecular mechanisms that govern these signals, and use new genetic methods to screen for the downstream effectors, using both inducible gene expression and RNAi knockdown. These tools will allow us to narrow the candidate genes to those involved in the critical period, to better resolve the underlying molecular mechanisms. We will use molecular tools we have developed, that include reengineered ion channels that either suppress or enhance membrane excitability, expressed at specific times in development on either side of the synapse. In addition, we have developed an inducible bipartite gene expression system to perform experiments testing temporal requirements for induced genes at specific synapses. The proposal is structured as a series of well- defined hypotheses that will help resolve the molecular and cellular mechanisms that govern synaptogenesis in a model genetic system. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: In this project we examine how muscle-derived molecular signals regulate the growth and maturation of the motoneuron. The cellular events we are studying are fundamental to the normal establishment of connections by nerve cells, and are relevant to events occurring both during normal development and in clinical conditions. As there are numerous neurological disorders that involve defects in synaptic growth, maintenance, and/or plasticity, these studies will contribute to a rational analysis of these disorders of the nervous system. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page