Abstract

The endocannabinoid system is a critical modulator of synaptic transmission in the central nervous system, but its role at the peripheral neuromuscular junction (NMJ) remains less understood. This study investigated the participation of endocannabinoid receptors, specifically the cannabinoid receptor type 1 (CB1), in the regulation of spontaneous synaptic activity at the mouse NMJ. Using intracellular microelectrode recordings of miniature endplate potentials (MEPPs) in mouse diaphragm preparations, we examined the effects of the CB1 receptor antagonist AM-251 and the non-selective cannabinoid receptor agonist WIN 55,212-2 (WIN). Our results indicate that acute application of AM-251 (1 µM) had no significant effect on MEPP frequency or amplitude, suggesting a low basal tone of endocannabinoid signaling. However, activation of cannabinoid receptors with WIN (20 µM) significantly increased the frequency of spontaneous acetylcholine (ACh) release by over 50% after 30–60 minutes of exposure, without altering MEPP amplitude. This effect was completely blocked by pre-treatment with AM-251, confirming the involvement of CB1 receptors. Further pharmacological analysis revealed that the WIN-induced potentiation of spontaneous ACh secretion was dependent on a signaling cascade involving phospholipase C (PLC) and protein kinase C (PKC), and the subsequent release of calcium from internal stores via ryanodine receptors (RyRs). These findings demonstrate, for the first time, a presynaptic CB1 receptor-mediated signaling pathway at the mouse NMJ that potentiates spontaneous quantal ACh release, suggesting a novel mechanism for the short-term regulation of synaptic efficacy at the peripheral synapse.