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 CB1 cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M3 muscarinic acetylcholine (ACh) receptors. N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB1 antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB1 receptor agonist, mimics M3 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 CB1 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.
Highlights
Cannabinoids, the active ingredients found in the marijuana plant Cannabis sativa (Ameri, 1999; Martin et al, 1999), produce their biological effects through binding to specific G-protein-coupled receptors (Howlett et al, 2002)
We used immunofluorescence to determine whether the CB1 receptor is present at the lizard neuromuscular junction (NMJ)
To determine where the receptors are located at the NMJ, we back-filled the nerve terminals with Texas red dextran and processed the tissue for immunofluorescence, using fluorescein-labelled secondary antibodies to reveal the CB1 receptors
Summary
Cannabinoids, the active ingredients found in the marijuana plant Cannabis sativa (Ameri, 1999; Martin et al, 1999), produce their biological effects through binding to specific G-protein-coupled receptors (Howlett et al, 2002). ECBs have been shown to act as retrograde signalling molecules in several areas of the central nervous system (for reviews see Kreitzer & Regehr, 2002; Wilson & Nicoll, 2002). Depolarization of the postsynaptic neurone and the resulting elevation of intracellular Ca2+ triggers eCB release (Ohno-Shosaku et al, 2001; Wilson & Nicoll, 2001; Brenowitz & Regehr, 2003). The activation of muscarinic acetylcholine (ACh) receptors (mAChRs) (Kim et al, 2002; Ohno-Shosaku et al, 2003; Fukudome et al, 2004) can trigger the release of eCBs. The eCBs released in neural tissue usually bind to the CB1 receptor subtype and inhibit the release of neurotransmitter from the presynaptic terminal
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