Activation of cannabinoid receptors inhibits voltage-gated Ca2+ channels and activates K+ channels, reminiscent of other G-protein-coupled signaling pathways that produce presynaptic inhibition. We tested cannabinoid receptor agonists for effects on excitatory neurotransmission between cultured rat hippocampal neurons. Reducing the extracellular Mg2+ concentration to 0.1 mM elicited repetitive, transient increases in intracellular Ca2+ concentration ([Ca2+]i spikes) that resulted from bursts of action potentials, as measured by combined whole-cell current clamp and indo-1-based microfluorimetry. Pharmacological characterization indicated that the [Ca2+]i spikes required glutamatergic synaptic transmission. Cannabinoid receptor ligands inhibited stereoselectively the frequency of [Ca2+]i spiking in the rank order of potency: CP 54,939 > CP 55,940 > Win 55,212-2 > anandamide, with EC50 values of 0.36, 1.2, 2.7, and 71 nM, respectively. CP 55,940 was potent, but not efficacious, and reversed the inhibition produced by Win 55,212-2, indicating that it is a partial agonist. Inhibition of [Ca2+]i spiking by Win 55,212-2 was prevented by treatment of cultures with active, but not heat-treated, pertussis toxin. Win 55,212-2 (100 nM) inhibited stereoselectively CNQX-sensitive excitatory postsynaptic currents (EPSCs) elicited by presynaptic stimulation with an extracellular electrode, but did not affect the presynaptic action potential or currents elicited by direct application of kainate. Consistent with a presynaptic site of action, Win 55,212-2 increased both the number of response failures and the coefficient of variation of the evoked EPSCs. In contrast, cannabimimetics did not affect bicuculline-sensitive inhibitory postsynaptic currents. Thus, activation of cannabinoid receptors inhibits the presynaptic release of glutamate via an inhibitory G-protein.

Cannabinoid receptor agonists act presynaptically to inhibit the release of glutamate. Because other drugs with this action are known to reduce excitotoxicity, we tested several cannabimimetics in a model of synaptically mediated neuronal death. Reduction of the extracellular Mg2+ concentration to 0.1 mM evoked a repetitive pattern of intracellular Ca2+ concentration ([Ca2+]i) spiking that, when maintained for 24 hr, resulted in significant neuronal death. The [Ca2+]i spiking and cell death in this model result from excessive activation of N-methyl-D-aspartate receptors, as indicated by the inhibition of both [Ca2+]i spiking and neuronal death by the N-methyl-D-aspartate receptor antagonist CGS19755 (10 microM). The cannabimimetic drug Win55212-2 (100 nM) completely blocked [Ca2+]i spiking and prevented neuronal death induced by low extracellular Mg2+ concentrations. These effects on [Ca2+]i spiking and viability were stereoselective and were prevented by the CB1 receptor antagonist SR141716 (100 nM). The partial agonist CP55940 (100 nM) also afforded significant protection from excitotoxicity. Cannabimimetic drugs did not protect cells from the direct application of glutamate (30 microM). These data suggest that cannabimimetic drugs may slow the progression of neurodegenerative diseases.

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Delta9-tetrahydrocannabinol (Delta9-THC) is the principal psychoactive ingredient in marijuana. We examined the effects of Delta9-THC on glutamatergic synaptic transmission. Reducing the extracellular Mg++ concentration bathing rat hippocampal neurons in culture to 0.1 mM elicited a repetitive pattern of glutamatergic synaptic activity that produced intracellular Ca++ concentration spikes that were measured by indo-1-based microfluorimetry. Delta9-THC produced a concentration-dependent inhibition of spike frequency with an EC50 of 20 +/- 4 nM and a maximal inhibition of 41 +/- 3%. Thus, Delta9-THC was potent, but had low intrinsic activity. Delta9-THC (100 nM) inhibition of spiking was reversed by 300 nM N-piperidino-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716), indicating that the inhibition was mediated by CB1 cannabinoid receptors. Delta9-THC attenuated the inhibition produced by a full cannabinoid receptor agonist, (+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1, 4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate (Win 55212-2), indicating that Delta9-THC is a partial agonist. The effect of Delta9-THC on synaptic currents was also studied. 6-Cyano-2,3-dihydroxy-7-niroquiinoxaline (CNQX)-sensitive excitatory postsynaptic currents were recorded from cells held at -70 mV in the whole-cell configuration of the patch-clamp and elicited by presynaptic stimulation with an extracellular electrode. Win 55212-2 and Delta9-THC inhibited excitatory postsynaptic current (EPSC) amplitude by 96 +/- 2% and 57 +/- 4%, respectively. Excitatory postsynaptic current amplitude was reduced to 75 +/- 5% in the presence of both drugs, demonstrating that Delta9-THC is a partial agonist. The psychotropic effects of Delta9-THC may result from inhibition of glutamatergic synaptic transmission. The modest physical dependence produced by Delta9-THC as well as its lack of acute toxicity may be due to the ability of the drug to reduce, but not block, excitatory neurotransmission.

Prolonged exposure to cannabinoids results in tolerance in vivo and desensitization of cannabinoid receptors in vitro. We show here that cannabinoid-induced presynaptic inhibition of glutamatergic neurotransmission desensitized after prolonged exposure to the cannabinoid receptor agonist ( Win55,. Synaptic activity between hippocampal neurons in culture was determined from network-driven increases in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i spikes) and excitatory postsynaptic currents. Win55,212-2-induced (100 nM) inhibition partially desensitized after 2 h and completely desensitized after 18-to 24-h exposure. The desensitization could be overcome by higher concentrations of agonist as indicated by a parallel rightward shift of the concentration response curve from an EC 50 of 2.7 Ϯ 0.3 nM to 320 Ϯ 147 nM for inhibition of [Ca 2ϩ ] i spiking and from 43 Ϯ 17 nM to 4505 Ϯ 403 nM for inhibition of synaptic currents, suggesting that this phenomenon may underlie tolerance. Presynaptic expression of dominant negative G-protein-coupled-receptor kinase (GRK2-Lys220Arg) or ␤-arrestin (319 -418) reduced the desensitization produced by 18-to 24-h pretreatment with 100 nM, Win55,212-2 suggesting that desensitization followed the prototypical pathway for G-protein-coupled receptors. Prolonged treatment with Win55,212-2 produced a modest increase in the EC 50 for adenosine inhibition of synaptic transmission and pretreatment with cyclopentyladenosine produced a slight increase in the EC 50 for Win55,212-2, suggesting a reciprocal ability to produce heterologous desensitization. The long-term changes in synaptic function that accompany chronic cannabinoid exposure will be an important factor in evaluating the therapeutic potential of these drugs and will provide insight into the role of the endocannabinoid system.