D-Tetrahydrocannabinol Acts as a Partial Agonist to Modulate Glutamatergic Synaptic Transmission between Rat Hippocampal Neurons in Culture

D-Tetrahydrocannabinol (D-THC) is the principal psychoactive ingredient in marijuana. We examined the effects of DTHC 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. D-THC produced a concentration-dependent inhibition of spike frequency with an EC50 of 20 6 4 nM and a maximal inhibition of 41 6 3%. Thus, D-THC was potent, but had low intrinsic activity. D-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. D-THC attenuated the inhibition produced by a full cannabinoid receptor agonist, (1)-[2,3-dihydro-5methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate (Win 55212-2), indicating that D-THC is a partial agonist. The effect of D-THC on synaptic currents was also studied. 6-Cyano-2,3dihydroxy-7-niroquiinoxaline (CNQX)-sensitive excitatory postsynaptic currents were recorded from cells held at 270 mV in the whole-cell configuration of the patch-clamp and elicited by presynaptic stimulation with an extracellular electrode. Win 55212-2 and D-THC inhibited excitatory postsynaptic current (EPSC) amplitude by 96 6 2% and 57 6 4%, respectively. Excitatory postsynaptic current amplitude was reduced to 75 6 5% in the presence of both drugs, demonstrating that D-THC is a partial agonist. The psychotropic effects of D-THC may result from inhibition of glutamatergic synaptic transmission. The modest physical dependence produced by D-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. D-Tetrahydrocannabinol is the principal psychoactive ingredient in marijuana. D-THC produces euphoria, sedation, hypoactivity, hypothermia, hypotension and bradycardia (Abood and Martin, 1992; Lake et al., 1997). Dronabinol, D-THC in sesame oil, has been used clinically to stimulate appetite and reduce nausea in patients undergoing chemotherapy for cancer and AIDS (Plasse et al., 1991). D-THC also appears to have other useful clinical attributes including analgesic, antiglaucoma, and antiepileptic properties (Howlett, 1995; Adams and Martin, 1996). The effects of D-THC are mediated by cannabinoid receptors that are distributed throughout the central nervous system (Herkenham et al., 1990; Tsou et al., 1998) and are present at high density on the presynaptic terminals of glutamatergic synapses (Twitchell et al., 1997). Cannabinoid receptors are members of the G-protein-coupled receptor family (Matsuda et al., 1990) and act via inhibitory G proteins (Childers et al., 1993) to activate K channels (Deadwyler et al., 1993; Henry and Chavkin, 1995; Mackie et al., 1995) and inhibit Ca channels (Mackie and Hille, 1992; Twitchell et al., 1997; Shen and Thayer, 1998). The activation of these receptors by cannabimimetic drugs attenuates glutamatergic neurotransmission by acting presynaptically to inhibit the release of glutamate (Shen et al., 1996). The cannabinoid neuromodulatory system exhibits an extensive pharmacology with several endogenous lipids proposed as ligands (Devane et al., 1992) as well as a number of synthetic cannabinoid (Johnson and Melvin, 1986) and aminoalkylindole (D’Ambra et al., 1992) derivatives that vary in potency, efficacy and stereoselectivity. In radioligand binding assays, compounds with affinities that range from subnanoThis work was supported by grants from the National Institute on Drug Abuse (DA07304, DA09293) and the National Science Foundation (IBN9723796). M. S. was supported by NIDA Training Grant DA07097. ABBREVIATIONS: D-THC, D-tetrahydrocannabinol; NMDA, N-methyl-D-aspartate; Win55212–2 (R enantiomer), (1)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate; SR141716, N-piperidino-5-(4chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide; EPSC, excitatory postsynaptic current; CP55940, [1a,2b(R)5a]-(2)-5(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol. 0026-895X/99/010008-06$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 55:8–13 (1999). 8 at A PE T Jornals on Jauary 7, 2018 m oharm .aspeurnals.org D ow nladed from molar to micromolar have been described (Devane et al., 1988; Herkenham et al., 1990). Some of the putative endogenous ligands as well as some of the cannabinoid derivatives behave as partial agonists in receptor mediated inhibition of Ca channels, G protein activation and synaptic transmission (Mackie et al., 1993; Pan et al., 1996; Shen et al., 1996; Sim et al., 1996b; Burkey et al., 1997a). In some behavioral assays, the maximal effects of D-THC were less than other cannabimimetic drugs, suggesting that it acted as a partial agonist (Compton et al., 1992). The stereoisomers of the aminoalkylindole Win55212-2 differ by over 100-fold in activity for inhibition of electrically stimulated mouse vas deferens (D’Ambra et al., 1992). The effects of D-THC on excitatory synaptic transmission have not been described. In this report, we show that D-THC is a potent inhibitor of glutamatergic synaptic transmission, although it exhibits partial inhibition at maximal concentrations. The ability of this drug to reduce, but not block, excitatory neurotransmission explains some of its behavioral effects. Materials and Methods Materials were obtained from the following companies: Win55212-2 and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX), RBI, Natick, MA; N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716), Sanofi Recherche, Montpellier Cedex, France; D-THC and all other reagents, Sigma Chemical