Fig. 3. Reduction of Crk and CrkL prevents reelin-enhanced dendrite extension in hippocampal neurons. Primary hippocampal neurons harvested from E17 mouse brains were infected with lentiviruses. Two days later, neurons were replated in neurobasal/B-27 growth media (A,D,G), control conditioned media (CM) (B,E,H) or reelin CM (C,F,I). Map2-positive dendrites of neurons infected with GFP control virus, measured after 5 days of growth, were longer when grown in reelin CM (C) than control CM (B) or neurobasal media (A). Reelin CM treatment also enhanced dendritogenesis of neurons infected with CrkL shRNA virus (F) as compared with control CM (E) or neurobasal media (D) treatment of the same population of neurons. By contrast, reelin CM treatment did not promote extension of neurons infected with Crk&CrkL shRNA virus (I) relative to control CM (H) or neurobasal (G) treatments. Bar, 50 µm. (J) Quantification of these and similar experiments demonstrated that reelin CM promoted an twofold increase in dendrite length that was blocked by Crk&CrkL shRNA virus infection, but not by Crk&CrkL mutant (mt) shRNA, or GFP-expressing control viruses. SFK inhibitors PP1 and PP2 also prevented reelin CM-enhanced dendritogenesis. The combined effect of SFK inhibitors and Crk&CrkL shRNA viruses did not cause further reductions in process lengths over that induced by either agent alone. The CrkL shRNA virus did not significantly inhibit reelin CM-enhanced dendritogenesis. The number of neurons measured is indicated at the base of each bar. ^*P<0.001. Error bars indicate s.e.m.