Figure 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-B27 growth media (A, D, G), control conditioned media (CM) (B, E, H) or Reelin CM (C, F, I). MAP2 positive dendrites of GFP control virus infected neurons, 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 neurons infected with CrkL shRNA virus (F) as compared to control CM (E) or neurobasal media (D) media treatment of the same population of neurons. In contrast Reelin CM treatment did not promote extension of Crk&CrkL shRNA virus infected neurons (I) relative to Control CM (H) or neurobasal media(G) treatments. The scale bar is 50 μm. Quantification of these and similar experiments demonstrated Reelin CM promoted an approximately 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 (J). 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 (J). The CrkL shRNA virus did not significantly inhibit Reelin CM enhanced dendritogenesis (J). The number of neurons measured is indicated at the base of each bar. * P<0.001. Error bars indicate s.e.m.