Endocannabinoids mediate muscarine‐induced synaptic depression at the vertebrate neuromuscular junction

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Abstract
Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using theCeratomandibularismuscle from the lizardAnolis carolinensiswe asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB1cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M3muscarinic acetylcholine (ACh) receptors.N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide, a CB1antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB1receptor agonist, mimics M3activation and occludes the effect of muscarine. As for its mechanism of action, ACPA reduces the action‐potential‐evoked calcium transient in the nerve terminal and this decrease is more than sufficient to account for the observed inhibition of neurotransmitter release. Similar to muscarine, the inhibition of synaptic transmission by ACPA requires nitric oxide, acting via the synthesis of cGMP and the activation of cGMP‐dependent protein kinase. 2‐Arachidonoylglycerol (2‐AG) is responsible for the majority of the effects of eCB as inhibitors of phospholipase C and diacylglycerol lipase, two enzymes responsible for synthesis of 2‐AG, significantly limit muscarine‐induced inhibition of neurotransmitter release. Lastly, the injection of (5Z,8Z,11Z,14Z)‐N‐(4‐hydroxy‐2‐methylphenyl)‐5,8,11,14‐eicosatetraenamide (an inhibitor of eCB transport) into the muscle prevents muscarine, but not ACPA, from inhibiting ACh release. These results collectively lead to a model of the vertebrate neuromuscular junction whereby 2‐AG mediates the muscarine‐induced inhibition of ACh release. To demonstrate the physiological relevance of this model we show that the CB1antagonistN‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide prevents synaptic inhibition induced by 20 min of 1‐Hz stimulation.