Figure 4.

NT-3 and GFP expression are regulated in vivo by doxycycline administration. A, Animals that were injected with tet-off-GFP lentivirus and were untreated (−Dox) show strong GFP immunolabeling, whereas (B) only very limited and in most cases no GFP expression can be found in the spinal cord of animals treated with doxycycline for 2 weeks (+Dox, 1 mg/ml in the drinking water). C, Regulated NT-3 expression 4 weeks after tet-off-NT-3 or tet-off-GFP lentivirus injection was measured by ELISA in dissected spinal cords. Tet-off NT-3-injected animals that were not treated with doxycycline (NT-3, −Dox) show significantly higher NT-3 levels in the spinal cord than animals that were treated with doxycycline (NT-3, +Dox), and animals that received GFP virus. NT-3 levels in doxycycline-treated animals with tet-off-NT-3 virus are not significantly different from GFP virus-injected control animals. D, Kinetics of NT-3 protein levels following doxycycline administration in animals that received tet-off-NT-3 lentivirus injections. NT-3 levels are highest in animals that were not treated with doxycycline for 2 weeks (−Dox, 2 weeks). NT-3 expression decreases significantly after treatment with doxycycline for just 3 d (−/+Dox, 2 weeks/3 d). There is an additional slight but insignificant decline in NT-3 levels in the subsequent 4–11 d of doxycycline treatment (−/+Dox, 2 weeks/1 wk and −/+Dox, 2 weeks/2 weeks). NT-3 levels were measured in 5 mm spinal cord segments rostral to the lesion site. Scale bar, 500 μm. ***p < 0.001, NS p > 0.05; ANOVA, followed by Fisher's post hoc test.