Subcellular Localization of a 2-Arachidonoyl Glycerol Signaling Cassette in Retinal Ganglion Cell Axonal Growth In Vitro.

Purpose To investigate whether the subcellular distribution of endocannabinoid (eCB) system (ECS) components in growing RGC axons is consistent with the formation of eCB-enriched "hotspots" and the role of the ECS in RGC axonal growth. Methods We used immunocytochemistry and image analysis to quantify axonal expression of the ECS components diacylglycerol lipase alpha (DGLα), monoacylglycerol lipase (MGL), and cannabinoid receptor type 1 (CB1R) in a mouse retinal explant model. We tested whether pharmacologic antagonists of CB1R and inhibitors of eCB degradation modulate ECS component expression and axonal growth. Results DGLα expression was higher in the distal RGC axon than in the growth cone central domain (GCCD) (95% confidence interval [CI], 106.5%-122.4% at 15 μm proximal to the GCCD), whereas MGL expression in the same region was not significantly different (95% CI, 88.8%-102.1%). In more proximal axon segments, DGLα and MGL expression were both lower than in the GCCD, whereas CB1R expression was 2.5-fold higher in this region (95% CI, 220.3%-278.4% at 50 μm proximal to the GCCD). The presence of CB1R antagonist O-2050 disrupted profiles of ECS component expression and increased axonal growth (95% CI for the difference of median axon lengths 26.6-55.6 μM). Conclusions Our results demonstrate an ECS topology in RGC axons that is consistent with formation of eCB-enriched hotspots and suggest that the ECS has a role in CB1R-dependent inhibition of RGC axonal growth in vitro.