Netherlands Organization for Scientific Research(724.017.002)
Frontier Program(KKP129961)
NIH grant(P30DA056410)
Endocannabinoids (eCB) are lipid neurotransmitters that play a critical role in brain
function by activating the cannabinoid CB1 receptor. Unlike classical neurotransmitters,
their storage and release mechanisms have remained elusive, leading to major gaps
in our understanding of how these signals are regulated. In this study, we developed
an experimental system combining genetically encoded fluorescent sensors, electrophysiology,
and mathematical modeling to study endocannabinoid signaling with temporal precision.
Our findings led us to propose an ?on-demand release? model, where microvesicle formation
governs endocannabinoid release. This comprehensive model extends the ?on-demand production?
model and reconciliates the three previously proposed hypotheses for eCB trafficking.
The model reshapes our understanding of endocannabinoid signaling and addresses key
unanswered questions in the field. 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.
