DCC Receptor is Required Downstream of PKA for CB₂R Induced Reorganization of the growth cone.

<p>Retinal explants were grown for 1 DIV and growth cones were immunolabeled for CB₂R (A) and DCC receptor (B). Merged image is presented in C. They were also immunostained for netrin-1, a ligand of DCC receptor (D). Dissociated neurons were cultured for 2 days <i>in vitro</i> and treated with pharmacological agents for 1 hour. (E) DCC function blocking (αDCC_fb) antibody 3.5 µg/ml was added 15 minutes prior to AM630 or JTE907 stimulation. αDCCfb abolished AM630 and JTE907 induced increases in growth cone surface area (F) and filopodia number (G) (mean ± SEM; n = from 134 to 159 per conditions, *P<0.05 vs control condition). (H) Photomicrographs of growth cone for <i>dcc</i>^+/+ or <i>dcc</i>^−/− mice. Histograms showing the size of growth cone area and the filopodia numbers in <i>dcc</i>^+/+ and <i>dcc</i>^−/− animals (I–L). Pharmacological modulation of the CB₂R did not induce any significant changes in growth cone surface area nor filopodia number in primary neuron cultures obtained from <i>dcc</i>^−/− mice embryos (I and K) whereas JWH133 and JWH015 induced a decrease in growth cone surface and filopodia number while AM630 and JTE907 augmented these endpoints in <i>dcc</i>^+/+ neuron cultures (J and L) (mean ± SEM; 125 to 219 per conditions, *P<0.05 vs control condition). (M) DIV2 neurons were treated with AM630, JTE907, AM630 and KT5720, AM630 and H89, or FSK for 15 minutes. Following biotinylation and western blot, expression of surface protein was assessed for DCC receptor, CB₂R and NCAM. Scale bar: 10 µm (A–C, E, H); 250 µm (D).