The cannabinoid anticonvulsant effect on pentylenetetrazole-induced seizure is potentiated by ultra-low dose naltrexone in mice

Summary

Cannabinoid compounds are anticonvulsant since they have inhibitory effects at micromolar doses, which are mediated by activated receptors coupling to G_i/o proteins. Surprisingly, both the analgesic and anticonvulsant effects of opioids are enhanced by ultra-low doses (nanomolar to picomolar) of the opioid antagonist naltrexone and as opioid and cannabinoid systems interact, it has been shown that ultra-low dose naltrexone also enhances cannabinoid-induced antinociception. Thus, concerning the seizure modulating properties of both classes of receptors this study investigated whether the ultra-low dose opioid antagonist naltrexone influences cannabinoid anticonvulsant effects. The clonic seizure threshold was tested in separate groups of male NMRI mice following injection of vehicle, the cannabinoid selective agonist arachidonyl-2-chloroethylamide (ACEA) and ultra-low doses of the opioid receptor antagonist naltrexone and a combination of ACEA and naltrexone doses in a model of clonic seizure induced by pentylenetetrazole (PTZ). Systemic injection of ultra-low doses of naltrexone (1 pg/kg to 1 ng/kg, i.p.) significantly potentiated the anticonvulsant effect of ACEA (1 mg/kg, i.p.). Moreover, the very low dose of naltrexone (500 pg/kg) unmasked a strong anticonvulsant effect for very low doses of ACEA (10 and 100 μg/kg). A similar potentiation by naltrexone (500 pg/kg) of anticonvulsant effects of non-effective dose of ACEA (1 mg/kg) was also observed in the generalized tonic–clonic model of seizure. The present data indicate that the interaction between opioid and cannabinoid systems extends to ultra-low dose levels and ultra-low doses of opioid receptor antagonist in conjunction with very low doses of cannabinoids may provide a potent strategy to modulate seizure susceptibility.

Introduction

Cannabinoids have important functions in a myriad of physiological and pathophysiological processes such as mood, appetite, emesis control, memory, spatial coordination muscle tone and analgesia, which are mediated primarily through the central cannabinoid CB₁ receptor (Pertwee, 1995, Hao et al., 2000). Among the numerous effects induced by endogenous and exogenous cannabinoids are also included modulatory effects on seizure susceptibility (Chesher and Jackson, 1974, Wallace et al., 2001, Wallace et al., 2002, Shafaroodi et al., 2004, Blair et al., 2006). Recent evidence also point towards a cannabinoid CB₁ receptor-mediated anticonvulsant effect against the maximal electroshock model of grand-mal seizure (Wallace et al., 2001, Wallace et al., 2002), the rat pilocarpine model of acquired epilepsy (Wallace et al., 2003), the in vitro hippocampal neuronal culture models of acquired epilepsy and status epilepticus (Blair et al., 2006) and the pentylenetetrazole (PTZ) model of myoclonic seizures in mice (Shafaroodi et al., 2004). In addition, our previous data suggested a tonic protective role for CB₁ receptors against myoclonic seizure induced by PTZ (Shafaroodi et al., 2004). Although there is convincing evidence that cannabinoids modulate seizure susceptibility via the CB₁ receptor, the exact mechanisms underlying this effect have not been completely understood.

The cannabinoid CB₁ receptor is the most abundant G protein-coupled receptor (GPCR) in the mammalian brain (Wallace et al., 2002). The CB₁ receptor has initially been shown to couple via pertussis toxin-sensitive G_i/o G proteins to, for example, inhibit adenylyl cyclase (Demuth and Molleman, 2006, Bonhaus et al., 1998). However, it has recently been demonstrated that the CB₁ receptor appears to be intrinsically active and possibly coupled to more than one type of G protein. In this regard, stimulation of cAMP was observed in rat cultured striatal neurons and in CB₁-transfected CHO cells in response to the CB₁ agonist HU-210 (Glass and Felder, 1997, Felder et al., 1998). This effect was the CB₁ receptor antagonist rimonabant (SR 141716A)-sensitive but did require the presence of forskolin, suggesting that stimulation of cAMP could be due to the possible CB₁ receptor coupling with G_s proteins. Calandra et al. (1999) even reported a CB₁ receptor-mediated increase in cAMP in Chinese hamster ovary cells by low doses of agonist, but in the absence of any co-stimulant. Collectively the data strongly suggests that CB₁ receptors may be dually coupled to both G_s and G_i/o proteins in some systems but the physiological significance of this has not been completely understood.

Opioid receptors as well as cannabinoid CB₁ receptors belong to the super family of GPCRs (Saidak et al., 2006). Opioids, like cannabinoids, exert a wide range of their effects through coupling to inhibitory G_i/G_o proteins (Williams et al., 2001). On the other hand, it has been shown that very low doses of opioids can selectively activate an excitatory G_s protein-coupled signaling pathway (Shen and Crain, 1997, Crain and Shen, 1995, Crain and Shen, 2000). Ultra-low doses of a opioid receptor antagonist has been shown to block this sensitive excitatory mechanisms and unmask a potent inhibitory tone induced by opioids at concentrations much lower than their minimal effective doses in some tests such as antinociception. In this regard, several studies reported the significant potentiation of opioid-induced antinociception or anticonvulsion by ultra-low doses of opioid receptor antagonists (Levine et al., 1988, Crain and Shen, 1995, Gan et al., 1997, Cruciani et al., 2003, Honar et al., 2004). On the other hand, several studies have revealed a number of functional interactions between cannabinnoid and opioid systems (Manzanares et al., 1999, for review), such as modulation of seizure threshold (Shafaroodi et al., 2004), which might be a result of shared signal transduction mechanisms (Childers et al., 1992) as well as the release of endogenous opioids by cannabinoid agonists (Valverde et al., 2001). In our recent study, we reported that ultra-low doses of the cannabinoid CB₁ receptor antagonist AM251 could enhance the anticonvulsant effect of ultra-low dose cannabinoid agonist on clonic seizures in mice (Gholizadeh et al., 2007). On the other hand, it was shown that ultra-low doses of naltrexone interestingly enhance the cannabinoid-induced antinociception (Paquette and Olmstead, 2005). Therefore, it would be reasonable if one expect that ultra-low doses of opioid receptor antagonists could influence the effects of cannabinoids on seizure susceptibility.

In view of the fact that the anticonvulsant effect of cannabinoids is suspected to be mediated through inhibitory G proteins (Howlett et al., 2004), we hypothesized that (1) blocking the sensitive excitatory effects of the opioid receptor by ultra-low doses of the opioid antagonist naltrexone may allow potentiation of the inhibitory component of the CB1 receptor signaling and the associated anticonvulsant effect. To address this question, we assessed the clonic seizure threshold induced by i.v. injection of GABA receptor antagonist PTZ. Furthermore, we hypothesized that (2) combination of ultra-low doses of naltrexone with very low and non-effective doses of the specific CB1 agonist agonist arachidonyl-2-chloroethylamide (ACEA) might produce an anticonvulsant effect. It should be noted that this model of PTZ exerts its convulsant effect through specific interaction with the GABAA-gated chloride ionophore, resulting in the hyperexcitability of epileptogenic centers in the forebrain. This paradigm represents an animal model of myoclonic seizures and is very sensitive to changes in seizure susceptibility (Löscher et al., 1991). We also hypothesized that (3) interaction between low doses of cannabinoid receptor agonist and ultra-low doses of opioid receptor antagonist in modulation of seizure might extend to other model of seizures. Therefore, we used another distinct and clinically relevant experimental model of seizure, namely latency for the onset and the incidence of the generalized tonic–clonic seizures after acute intraperitoneal (i.p.) injection of a relatively high dose of PTZ, which is a model of grand-mal seizure (Löscher et al., 1991, Kupferberg, 2001).