The correct operation of the nervous system depends on the formation of highly specific patterns of axonal projections and synaptic connections among nerve cells. To understand how this occurs, the approach taken in this proposal is to study the development of one well characterized neuronal circuit in detail, the simple monosynaptic stretch reflex. Sensory neurons supplying stretch-sensitive spindle organs in muscles (Ia afferents) project into the spinal cord where they make highly specific synaptic connections with particular subsets of motoneurons. The experiments proposed here examine how each aspect of the development of this sensory pathway is controlled: the initial differentiation of Ia afferents and the support of their survival by peripheral or central target factors, the molecular cues responsible for Ia axons to begin growing into the spinal cord, the guidance of these axons towards the ventral horn, and finally their arborization and the formation of functional synapses with motoneurons. These issues are addressed using two different experimental preparations of the developing chicken embryo: Organotypic cultures of spinal cord and sensory ganglia will permit the detailed study of the development of Ia projection patterns at a higher resolution than is possible in vivo. A parallel set of experiment on intact, surgically manipulated embryos will check if the results from culture also apply to more normal development in vivo. Finally an analogous organotypic culture system will be developed using embryonic mice to test the generality of these findings in a mammalian system. The six specific aims of this proposal are: (1) to determine if NT-3 instructs the differentiation of Ia sensory neurons, (2) analyze the relative roles of motoneurons and peripheral target muscle in controlling the number of Ia afferents, (3) analyze the cues within the spinal cord that guide Ia axons from the dorsal roots through the dorsal horn to the ventral cord, (4) determine the nature and identity of signals within the spinal cord that promote arborization of Ia axons in the ventral horn, (5) analyze the role of peripheral targets on the formation of specific patterns of synaptic connections between Ia axons and motoneurons, and (6) develop an analogous preparation of cultured spinal explants from embryonic mice.
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