During the current funding cycle, we revealed trophic and tropic actions of neurotrophins on specific classes of developing trigeminal (V) primary afferent neurons, and showed that arborization and elongation effects of neurotrophins are mediated by the Rho family GTPases. Proposed experiments address the differentiation of V ganglion cells and their projections in mice with targeted deletion of specific genes known to be necessary for sensory neuron survival. Mice with deletion of a preapoptotic gene (bax) will be crossed with NGF-, NT3-,TrkA-, or TrkC-knockout mice to reveal axonal differentiation effects of neurotrophins on V ganglion cells, independent of their cell survival effects. To address chemotropic actions of NGF and NTS on embryonic V afferent projections, loss-of-function and gain-of-function studies will be performed. Synaptotrophic actions of NGF and NTS will be analyzed in these various preparations by ultrastructural and immunohistochemical studies. Pilot data prompt the following 3 hypotheses and experiments: 1. In Bax/NGF or Bax/NTS double knockout mice, all classes of primary sensory neurons in the V ganglion will survive, but they display deficits in axon differentiation, therein preventing whisker related brainstem pattern formation. Cell death markers, stereologic estimates of cell numbers (Core A), and cell class-specific markers will be used to assess embryonic V ganglion cell survival. Axonal labeling and quantification of projections will reveal changes in axonal differentiation relative to wildtype controls (Core B). Metabolic staining will reveal effects on CNS pattern formation (Core A). 2. Neurotrophins facilitate synaptogenesis in developing V primary afferents. Wholemount cultures of the V pathway subjected to exogenous neurotrophin augmentation regimens will be used to study development of synaptic protein expression and synaptic profiles in V brainstem nuclei (Core A). Complementary analyses will be performed in vivo in TrkA and TrkC knockout embryos to study synaptic development in the absence of high-affinity receptors for NGF and NTS. These 2 experiments will reveal NGF family actions in V system development. We further hypothesize: 3. The GDNF family ligands (GDNF, neurturin, artemin) contribute to perinatal V ganglion cell survival and axon development. V primary afferent survival, differentiation, phenotypes, morphology and patterning will be assessed in comparable gene targeting and factor augmentation wholemount paradigms as they pertain to the GDNF family ligands and receptors (Core A). Chemotropic effects will also be tested by using isolated V ganglia embedded with a localized source of GDNF family ligand. V axon growth towards or away from this GDNF source will be analyzed (Core B).
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