The purpose of this work is to assess the influence of neuronal growth factors on axon branching in the mammalian spinal cord. The dorsal root afferent projection has been chosen as a model system because four members of the """"""""neurotrophin"""""""" family of growth factors are known to promote neurite outgrowth of dorsal root ganglion (DRG) cells in vitro. Furthermore, each of the physiologically specified classes of DRG cells has a characteristic axonal arborization in the spinal cord that can be fully and conveniently stained with lipid-soluble tracers. A first step is to determine which classes of DRG cells express which members of the trk family of high-affinity neurotrophin receptors. Rat DRG cells expressing different trk's will be identified by retrograde tracing from characteristic peripheral and central target fields, followed by in situ hybridization with probes for trkA, trkB, and trkC. Second, we will determine the developmental time course of synthesis of the different neurotrophins in the central target fields of DRG cells. In situ hybridization in developing rat spinal cord will be performed with probes for NGF, BDNF, NT3, and NT5. Expression of neurotrophins by spinal cord neurons will be correlated with the development of axonal arborizations of the different classes of DRG cells as revealed by staining with lipid-soluble tracers. Finally, we will determine whether neurotrophins can influence dorsal root axon branching in the spinal cord. NGF, BDNF, NT3, and NT5 will in injected into embryos in utero and the dorsal root afferent projections in control and experimental animals will be stained with Dil. The important characteristics of axonal arbors will be quantitated and compared in control and growth factor-treated animals. These experiments will determine which high-affinity neurotrophin receptors are expressed by identified classes of DRG cells, the relationship of neurotrophin expression in spinal target fields to the development of arborizations of different classes of DRG neurons, and whether neurotrophins can influence the spinal arbors of DRG cells in vivo. Determining the effects of neurotrophins on the growth and branching of dorsal root axons in vivo is crucial to an assessment of their potential as agents to promote regeneration of sensory axons in the spinal cord.
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