SUMMARY1. Ionic currents associated with the invasion of an action potential into the motor nerve ending of the lizard, Anolis carolinensis, were measured with a focal extracellular electrode at several locations along the nerve ending.2. These experimentally observed currents could be matched with computer simulations of action potential propagation into the nerve ending. They revealed that while Na+ channels are the major ionic current pathway in the heminode, K+ channels provide the major pathway in the terminal branches and boutons.3. Calcium current in the presynaptic ending was unmasked by the application of tetraethylammonium (TEA). This current was blocked by: (a) cadmium, (b) oconotoxin GVIA and (c) nifedipine, but was unaffected by nickel at concentrations less than or equal to 100 /tM. Nifedipine's action became more definitive when the duration of the action potential was greatly extended by pre-treatment with TEA. The effect of Bay K 8644 was inconsistent.4. Transmitter release, as measured by postsynaptic current, had a pharmacological response profile similar to that of the Ca2+ current, with the exception that transmitter release was increased reliably and reversibly by Bay K 8644. 5. This pharmacological response profile is identical to that of the L type Ca2+ channel identified by Fox, Nowycky & Tsien (1987a) in chick dorsal root ganglion neurones. We saw no evidence for more than a single type of Ca2+ channel in lizard motor nerve endings.6. A calcium-activated K+ current IK(Ca) was revealed by application of 3,4-diaminopyridine (DAP), a delayed-rectifier K+ channel blocker. This K(Ca) current was blocked by TEA, charybdotoxin and by substitution of cobalt for extracellular calcium.

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.

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