Abstract

The endocannabinoid system plays a critical role in modulating synaptic transmission, primarily through the retrograde suppression of neurotransmitter release. This study investigates the effects of the endogenous cannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) on the evoked quantal release of acetylcholine (ACh) at the mouse neuromuscular junction, a model cholinergic synapse. Contrary to the canonical inhibitory mechanism, our objective was to explore a potential **noncanonical potentiation** of ACh release. Using electrophysiological techniques, specifically intracellular recordings of endplate potentials (EPPs) and miniature endplate potentials (MEPPs) in mouse motor synapses, we assessed the impact of AEA and 2-AG on presynaptic function. We found that both AEA and 2-AG significantly increased the quantal content of evoked ACh release without altering the amplitude or frequency of spontaneous MEPPs, suggesting a selective effect on the machinery for evoked release. This potentiation was observed even in the presence of CB1 receptor antagonists, indicating a mechanism independent of the classical cannabinoid receptor pathway. The key finding is the identification of a novel, noncanonical pathway by which endocannabinoids can enhance, rather than suppress, neurotransmitter release. This discovery is significant for understanding the complex regulatory roles of lipid signaling molecules in synaptic plasticity and provides new insights into the diverse mechanisms controlling membrane fusion and exocytosis at the presynaptic terminal.