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David Thomas Stark, Joseph Caprioli; Differential subcellular recruitment of an endocannabinoid signaling cassette in developing retinal ganglion cell axons. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1785.
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© ARVO (1962-2015); The Authors (2016-present)
Endocannabinoids (eCBs) modulate axonal growth in developing glutamatergic neurons, but the role of these lipid messengers in retinal ganglion cells (RGCs) is not well understood. We investigated whether RGCs exhibit an enzyme topology consistent with formation of 2-arachidonoyl glycerol (2-AG)-enriched hotspots in an embryonic retinal explant model.
Retinal explants were stained for monoacylglycerol lipase (MGL), diacylglycerol lipase alpha (DGLα), beta-III-tubulin or F-actin and imaged by confocal microscopy. Thirty axons were analyzed per group. Z-stack projections were segmented into regions of interest (ROIs) that included growth cone central and peripheral domains (C- and P-domains) and consecutive 5 µm segments of stabilized axon. Mean gray value for each ROI was recorded and normalized to that of the C-domain. Data were analyzed with linear least squares regression and Student’s t-test.
The C-domain and most distal axon segment showed the maximal mean gray values for both MGL and DGLα, while P-domains stained lightly except in occasional areas invaded by pioneer microtubules. Normalized mean gray values were not significantly different in the P-domains of MGL versus DGLα-labeled cultures (48.9% +/- 4.1% versus 48.6% +/- 3.1%; P-value = 0.96, mean +/- SEM). Moving proximally from the growth cone along MGL-stained processes, mean gray values remained stable in the distal 35 µm of the axon shaft then subsequently tapered. In contrast, DGLα signal dropped precipitously beginning at the transition from the C-domain to the first segment of distal axon (comparison of regression coefficients, P-value = 0.05).
Previous studies of non-RGC glutamatergic neurons have demonstrated mutually exclusive distributions of DGLα and MGL in developing axons with DGLα enriched in growth cones and MGL restricted to axons. Putative 2-AG "hotspots" that result may establish spatially restricted signaling competence of 2-AG during axonal growth. Our model of RGC axon development shows intense staining of growth cone C-domains for both MGL and DGLα with differential expression in the distal axon. This topology is distinct from the previously reported pattern but consistent with the hotspot hypothesis, since a relative paucity of 2-AG-synthesizing DGLα in the proximal axon shaft should favor comparative enhancement of eCB tone in the distal axon and growth cone.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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