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

Abstract

Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using the Ceratomandibularis muscle from the lizard Anolis carolinensis we asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB 1 cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M 3 muscarinic acetylcholine (ACh) receptors. N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB 1 antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB 1 receptor agonist, mimics M 3 activation 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 CB 1 antagonist N-(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.

Expression of the endocannabinoid system in fibroblasts and myofascial tissues

“…Our hypothesis that MFTrPs were endowed with CB 1 receptors was supported by a GEO study (GDS1838) that showed greater CB 1 levels in skeletal muscle near the neuromuscular junction. Two new papers confirmed our results (Newman et al, 2007;Sánchez-Pastor et al, 2007), showing that CB 1 activation in motor endplates dampened acetylcholine release.…”

Muscarinic Modulation of Synaptic Transmission via Endocannabinoid Signalling in the Rat Midbrain Periaqueductal Gray

“…There is increasing evidence that 2-arachidonylglycerol mediates the M1/M3 mAChR and group I mGluR-induced inhibition of synaptic transmission because it is abolished by phospholipase C and DAG lipase inhibitors, and by knocking out phospholipase C␤ (Melis et al, 2004;Hashimotodani et al, 2005Hashimotodani et al, , 2007Maejima et al, 2005;Newman et al, 2007;Uchigashima et al, 2007). Although we did not examine the role of phospholipase C, it was found that the carbachol-induced inhibition of evoked IPSCs was reduced by the DAG lipase inhibitor tetrahydrolipstatin to a similar extent as that produced by AM251.…”

“…Studies in the hippocampus and striatum have shown that M1/M3-induced suppression of synaptic transmission is mediated by retrograde endocannabinoid signaling. Recent evidence suggests that the endocannabinoid involved is 2-arachidonoylglycerol, which is produced via the phospholipase C/DAG lipase pathway (Melis et al, 2004;Hashimotodani et al, 2005Hashimotodani et al, , 2007Maejima et al, 2005;Newman et al, 2007;. We therefore examined whether the M1/M3-induced effects on synaptic transmission in PAG are mediated by endocannabinoids.…”

Tetrahydrocannabinol (THC) for cramps in amyotrophic lateral sclerosis: a randomised, double-blind crossover trial

“…The current understanding of the pathophysiology of cramps suggests that peripheral nerve hyperexcitability and motor neuron bistability (anterior horn cell level) may play a role 14–17. The distribution of CB1 receptors in the peripheral nerves including the neuromuscular junction and spinal cord may pose an advantage of THC over medications acting solely at the peripheral nerve/neuromuscular junction (quinine sulfate) or centrally (neurontin) 18 19. Consequently, in view of the lack of randomised controlled trials, we sought to investigate the effectiveness of THC in the treatment of cramps by using a randomised, double-blind, placebo-controlled crossover design.…”