Straub_2025_Proc.Natl.Acad.Sci.U.S.A_122_e2421717122

While it is known that endocannabinoids (eCB) modulate multiple neuronal functions, the molecular mechanism governing their release and transport remains elusive. Here, we propose an "on-demand release" model, wherein the formation of microvesicles, a specific group of extracellular vesicles (EVs) containing the eCB, 2-arachidonoylglycerol (2-AG), is an important step. A coculture model system that combines a reporter cell line expressing the fluorescent eCB sensor, G protein-coupled receptor-based (GRAB)(eCB2.0), and neuronal cells revealed that neurons release EVs containing 2-AG, but not anandamide, in a stimulus-dependent process regulated by protein kinase C, Diacylglycerol lipase, Adenosinediphosphate (ADP) ribosylation factor 6 (Arf6), and which was sensitive to inhibitors of eCB facilitated diffusion. A vesicle contained approximately 2,000 2-AG molecules. Accordingly, hippocampal eCB-mediated synaptic plasticity was modulated by Arf6 and transport inhibitors. The "on-demand release" model, supported by mathematical analysis, offers a cohesive framework for understanding eCB trafficking at the molecular level and suggests that microvesicles carrying signaling lipids in their membrane regulate neuronal functions in parallel to canonical synaptic vesicles.