Prior studies have revealed the trophic actions of neurotrophins in sensory neuron development. Proposed studies will address the trophic actions of neurotrophins in trigeminal (V) primary afferent development. Our working hypothesis is that neurotrophins regulatory sensory axon growth rate and mode during a critical period in development. Preliminary studies in expand co-cultures of the rat V periphery and brainstem show that NGF and NTF3 influence over axon elongation and brainstem arbor formation, respectively. Moreover, co-cultured V axons develop synaptic interactions with V brainstem cells that are similar to those seen in vivo. These data prompt the following 3 hypotheses; 1. Ion co-cultures of the V periphery and brainstem from embryonic mice, NGF produces axon elongation and NT3 produces axon arborization. TO test this, we will grow embryonic V explant co-cultures and wholemounts in the presence of exogenously added NGF or NT3. Axon growth rates in arborization responses will be quantified under these conditions. Immunohistochemical markers will be used to determine whether NGF and NT3 affect growth of all or specific V ganglion cell axons. Axon growth patterns will also be studied in dissociated trigeminal cell cultures under conditions of NGF or NT3 augmentation (or appropriate blocking agents). Explant co-cultures will also be used to assess whether NGF and NT3 promote synaptic interactions between V primary afferent and brainstem neurons. 2.In transgenic mice that over-express NGF or NT3 in the brainstem under a controllable promoter, V ganglion cells will develop aberrant axon elongation and arborization properties, respectively. Conversely, transgenic mice that over-express NGF or NT3 in the mysacial whiskerpad will develop normal axon morphologies in the brain stem. To test this, transgenic mice will be generated with controlled over- expression of NGF or NT3 in the brainstem or whiskerpad, by this, transgenic mice will be generated with controlled over-expression of NGF or NT3 in the brainstem or whiskerpad, by using the tetracycline- regulated gene system in CORE B. In the presence of tetracycline administered by diet or injection, NGF or NT3 will be over-expressed in particular target tissues during specified times in development. The morphology of identified V primary afferents will be revealed by intra- axonal recording and staining in these transgenic mice in adulthood. Axonal growth patterns will be quantified in CORE C. 3. Topographic aggregation patterns of brainstem cell (barrelettes) will be absent in transgenic mice that display aberrant primary afferent elongation and arborization properties induced by controlled over- expression of NGF or NT3 in the brainstem. Such patterning abnormalities will be transmitted to the thalamic barreloids and cortical barrels. To test this, above-described NGF or NT3 over-expressing mice that display altered primary afferent structure in the brainstem will be histochemically processed to reveal potential patterning alterations in the brainstem, thalamus and cerebral cortex. Image analysis of whisker- related patterns will be carried out in CORE C.
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