Growth cones are the motile tips of growing axons. During neural development axons are guided to their targets by responses of their growth cones to molecular cues in the environment. In the mammalian central nervous system (CNS) axon branches innervate targets. Thus branching is an important form of axon guidance. The long term goal of this work is to understand the signaling mechanisms that regulate axon outgrowth and branching. This work will be carried out on developing rodent cortical neurons and the findings will thus be important for promoting axon collateral sprouting after CNS injury. Intracellular calcium activity is known to regulate cortical axon outgrowth. The first specific aim will address mechanisms by which calcium signaling can independently regulate axon outgrowth and branching. In dissociated cortical cultures calcium indicator dyes will be used with time lapse fluorescence imaging to correlate expression, frequencies and amplitudes of calcium transients with outgrowth of a primary axon and its collateral branches. Netrin-1 and semaphorin 3A are guidance cues that respectively attract or repel cortical axons. These guidance molecules also promote or inhibit cortical axon branching. To determine whether the axon and its branches can respond differentially to these cues with different calcium activity in the axon and its branches, these cues will be locally applied to different regions of cortical axons in dissociated cultures. To determine whether calcium transients regulate axon outgrowth in pathways of the CNS in vivo, calcium imaging will be carried out with two photon microscopy on living cortical slices. CaMKII (calcium-calmodulin dependent kinase II) is known to be a detector of calcium spike frequency in neurons during synaptic plasticity. In the second specific aim the role of CaMKII in axon branching will be addressed. CaMKII DNA fused to the fluorescent marker EGFP (enhanced green fluorescent protein) will be used to transfect cultured cortical neurons to determine whether overexpression of CaMKII increases axon branching and whether this occurs independent of axon outgrowth. Mutant forms of the enzyme will also be used to test the importance of CaMKII in axon branching. In the third specific aim similar techniques will be used to determine the role in cortical axon outgrowth and branching of another kinase related to calcium signaling, MAPK (mitogen-activated protein kinase). Taken together, these experiments will elucidate some of the signaling components whereby calcium activity in concert with guidance cues can regulate axon outgrowth and branching in the CNS.
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