Figure 4
Extracellular 2-AG promotes spherical exclusion of cholinergic growth cones (GCs).
(a) Cholinergic neurons from C56Bl6 mice co-cultured with COS-7-DAGLα cells allowed measurement of GC exclusion triggered by extracellular 2-AG. (a1) Schema of the in vitro method and the parameters determined and plotted in (b2–f). (b,b1) Representative images of cholinergic GCs apposing control (“parent”) or DAGLα-overexpressing COS-7 cells (COS-7-DAGLα). Arrows point to filopodia. Dotted lines indicate the membrane surface of COS-7(-DAGLα) cells with arrowheads pinpointing their proximal segment facing the GCs. (b2) Percentage of GCs that were unable to make contact upon DAGLα overexpression. (b3) The distance (in μm) of cholinergic GCs from COS-7(-DAGLα) cells. GCs gradually approached COS-7 cells in control. DAGLα overexpression occluded this response, suggesting GC repulsion by extracellular 2-AG. DAGLα overexpression did not affect the angle at which the GC faced the proximal surface of the COS-7 cell (c), neurite outgrowth per se (d) or the distance of cholinergic somata from COS-7 cells (e). Upon making contact, cholinergic neurites were no longer affected by DAGLα overexpression (f). Data were averaged from 3 (1,2DIV) or 4 (3DIV) separate experiments; n = 25–77 cells/group. Abbreviations: DIV, days in vitro; N, neuron; n, nucleus. Data were expressed as means ± s.e.m. except for (b3) showing a combination of individual data points and mean values (horizontal lines); **p < 0.01, *p < 0.05, +p < 0.1. Scale bars = 10 μm (a), 3 μm (b).
