The cellular and molecular mechanisms that regulate the growth of axons in target regions are largely uncharacterized. Regulation of axonal growth in targets helps to specify which neurons can be accessed for synapse formation during development, and it may be important in recovery of function after injury. Fundamental questions about the control of CNS innervation include 1) how do axonal growth cones bypass most cells without forming stable connections and choose specific target neurons with which to synapse, 2) does inhibition of axonal growth play a role in target cell selection and regulation of arbor sizes among target cells, and 3) how do action potentials and the development of synaptic activity influence the extent of axons in their target regions? The proposed studies will identify regulatory mechanisms for axonal growth in the CNS using a recently developed in vitro system. One of the key findings from this system is that cerebellar mossy fiber extension is interrupted by target granule neurons. This """"""""stop-growing signal"""""""" is both target- and axon-specific, and requires action potentials and functional NMDA receptors. The in vitro axon-target system will be used to further characterize the stop-growing signal for appropriate axons. The involvement of plasma membrane components, and of secretion from target granule cells will be assessed. In addition, the requirement for NMDA receptors will be defined, by determining the time course of their involvement, and determining if they are located on mossy fibers as well as on granule cells. In addition to identifying mechanisms used by target neurons to regulate axonal growth, the relationship of the stop-growing signal to synapse formation will be defined. This is a key question in assessing the role of the stop-growing signal in the development of brain function, and in characterizing the requirements for development of functional synapses in the CNS.
