Fig. 6.
NT3 does not regulate the number of CGRP-IR neurons. A, Antibody dose–response. To determine the amount of antibody required to neutralize NT3 activity, a short-term neuronal survival assay was used. E14.5 DRG neurons were supported for 2 d by NT3 (10 ng/ml) and serum, and that survival activity was neutralized by the addition of 50, 100, or 200 μg/ml anti-NT3. Twenty-five hundred cells, of which about half are neurons, were plated/96 well, with duplicates for each condition, and the experiments were performed twice. The mean number of neurons and SEM are shown. The lowest effective antibody concentration, 50 μg/ml of anti-NT3, was used for subsequent neutralization experiments in co-cultures.B, Anti-NT3 had no effect on CGRP expression. Dissociated E14.5 DRG neurons were grown for 10 d in the growth medium containing RS and 25 ng/ml NGF in the presence or absence of 50 μg/ml anti-NT3. Cells were processed for CGRP and neuron-specific tubulin immunocytochemistry. The percentage of CGRP-IR neurons and overall neuronal survival were equivalent in control and anti-NT3 conditions (unpaired t test; p > 0.1).