Assessment of the role of CB₁ receptors in cannabinoid anticonvulsant effects

Abstract

The cannabinoid CB₁ receptor has been shown to be the primary site of action for cannabinoid-induced effects on the central nervous system. Activation of this receptor has proven to dampen neurotransmission and produce an overall reduction in neuronal excitability. Cannabinoid compounds like Δ⁹-tetrahydrocannabinol and cannabidiol have been shown to be anticonvulsant in maximal electroshock, a model of partial seizure with secondary generalization. However, until now, it was unknown if these anticonvulsant effects are mediated by the cannabinoid CB₁ receptor. Likewise, (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2), a cannabimimetic compound that has been shown to decrease hyperexcitability in cell culture models via the cannabinoid CB₁ receptor, has never been evaluated for anticonvulsant activity in an animal seizure model. We first show that the cannabinoid compounds Δ⁹-tetrahydrocannabinol (ED₅₀=42 mg/kg), cannabidiol (ED₅₀=80 mg/kg), and WIN 55,212-2 (ED₅₀=47 mg/kg) are anticonvulsant in maximal electroshock. We further establish, using the cannabinoid CB₁ receptor specific antagonist N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A) (AD₅₀=2.5 mg/kg), that the anticonvulsant effects of Δ⁹-tetrahydrocannabinol and WIN 55,212-2 are cannabinoid CB₁ receptor-mediated while the anticonvulsant activity of cannabidiol is not. This study establishes a role for the cannabinoid CB₁ receptor in modulating seizure activity in a whole animal model.

Introduction

Despite marijuana's illegal status in the United States, individuals both here and abroad report its use to be therapeutic in the treatment of a variety of ailments, including epilepsy Hollister, 1983, Adams and Martin, 1996. Approximately 1% of Americans have epilepsy and 30% of these patients are refractory to conventional antiepileptic drug treatments (Zarrelli et al., 1999). Cannabinoid compounds have been used as a natural remedy for seizures for nearly 2000 years (Adams and Martin, 1996). In 1974, Karler et al. found that Δ⁹-tetrahydrocannabinol, the primary psychoactive compound in marijuana, displayed anticonvulsant properties in maximal electroshock-induced tonic–clonic convulsions (Karler et al., 1974). The non-psychoactive marijuana constituent, cannabidiol, was also shown to be protective in this seizure model (Karler et al., 1973). Since this initial research, several cannabimimetic compounds have been synthesized and evaluated in vitro for their effects on neuronal hyperexcitability. (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2) attenuated low-Mg²⁺ induced burst-firing in hippocampal culture (Shen and Thayer, 1999). In addition, the endogenous ligands anandamide and 2-Arachidonylglycerol were found to decrease the amplitude of stimulation-induced population spikes, as well as attenuate low-Mg²⁺-induced epileptiform discharges in rat hippocampal slice preparation (Ameri and Simmet, 2000). The mechanism underlying this dampening of excitability is believed to involve the inhibition of presynaptic excitatory neurotransmitter release Shen and Thayer, 1999, Takahashi and Linden, 2000, of which glutamate is the most ubiquitous.

Cannabinoids are known to bind two G protein-coupled 7-transmembrane spanning receptors, CB₁ and CB₂ Matsuda et al., 1990, Munro et al., 1993. Cannabinoid receptor CB₁ is the type preferentially expressed in brain and is known to mediate the psychoactive effects of cannabinoids. The classic tetrad Δ⁹-tetrahydrocannabinol-induced behaviors; ataxia, catalepsy, analgesia and hypothermia show susceptibility to block by the selective cannabinoid CB₁ receptor antagonist, pyrazole compound, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A) Reche et al., 1996, Fields and Meng, 1998, Smith et al., 1998. Similarly, cannabinoid effects on excessive neuronal excitability, in vitro, are inhibited by pretreatment with SR141716A (Rinaldi-Carmona et al., 1994), indicating a cannabinoid CB₁ receptor-mediated mechanism. Conversely, cannabidiol does not bind the cannabinoid CB₁ receptor with reasonable affinity (Thomas et al., 1998) and does not produce Δ⁹-tetrahydrocannabinol like behaviors that are blockable by the antagonist. Although the anticonvulsant activities of Δ⁹-tetrahydrocannabinol and cannabidiol in the maximal electroshock model have been recognized for many years, it has not been investigated as to whether the anticonvulsant activity of these compounds is conferred by cannabinoid CB₁ receptor activation. Furthermore, cannabimimetic compounds such as WIN 55,212-2 dampen neuronal hyperexcitability in cultured neurons (Shen et al., 1996), but have never been evaluated for their anticonvulsant activity in whole animals. Therefore, the purpose of this study was to evaluate the cannabinoid compounds Δ⁹-tetrahydrocannabinol, cannabidiol, and WIN 55,212-2 for anticonvulsant activity in the maximal electroshock model and determine if their protective activity is cannabinoid CB₁ receptor-mediated.