Endocannabinoids block status epilepticus in cultured hippocampal neurons

Abstract

Status epilepticus is a serious neurological disorder associated with a significant morbidity and mortality. Antiepileptic drugs such as diazepam, phenobarbital and phenytoin are the mainstay of status epilepticus treatment. However, over 20% of status epilepticus cases are refractory to the initial treatment with two or more antiepileptic drugs. Endocannabinoids have been implicated as playing an important role in regulating seizure activity and seizure termination. This study evaluated the effects of the major endocannabinoids methanandamide and 2-arachidonylglycerol (2-AG) on status epilepticus in the low-Mg²⁺ hippocampal neuronal culture model. Status epilepticus in this model was resistant to treatment with phenobarbital and phenytoin. Methanandamide and 2-AG inhibited status epilepticus in a dose-dependent manner with an EC₅₀ of 145 ± 4.15 nM and 1.68 ± 0.19 μM, respectively. In addition, the anti-status epilepticus effects of methanandamide and 2-AG were mediated by activation of the cannabinoid CB₁ receptor since they were blocked by the cannabinoid CB₁ receptor antagonist AM251. These results provide the first evidence that the endocannabinoids, methanandamide and 2-AG, are effective inhibitors of refractory status epilepticus in the hippocampal neuronal culture model and indicate that regulating the endocannabinoid system may provide a novel therapeutic approach for treating refractory status epilepticus.

Introduction

Status epilepticus is a life-threatening neurological disorder associated with a significant morbidity and mortality (Delorenzo, 2006, DeLorenzo et al., 1995, Hauser and Hesdorffer, 1990). Seizure events that last greater than 30 min or intermittent seizures without regaining consciousness lasting for 30 min or longer are classified as status epilepticus (DeLorenzo et al., 1995, Hauser and Hesdorffer, 1990). Status epilepticus has been shown to cause significant neuronal damage especially in the limbic system in both animals and man (Drislane, 2000). Benzodiazepines including diazepam and lorazepam are the initial drugs of choice for treatment of status epilepticus (Treiman et al., 1998). Despite their effectiveness, at least 1/3 of the patient population with generalized convulsive status epilepticus is refractory to benzodiazepines (Chen and Wasterlain, 2006, Treiman et al., 1998). In addition, benzodiazepines such as diazepam rapidly develop pharmacoresistance and lose their effectiveness in treating status epilepticus as the seizure duration increases (Goodkin and Kapur, 2003). Other antiepileptic drugs that can be administered intravenously, such as phenobarbital, divalproex and phenytoin, have also been effective in treating status epilepticus (Chen and Wasterlain, 2006). However, despite aggressive treatment with these agents, over 20% of status epilepticus cases are refractory to treatment with two or more medications (Mayer et al., 2002, Treiman et al., 1998). These cases of status epilepticus are referred to as refractory status epilepticus since they do not respond to the first two major antiepileptic agents used to treat status epilepticus. Due to the high mortality associated with prolonged seizure activity from status epilepticus (Towne et al., 1994), refractory status epilepticus is treated aggressively with the induction of coma using pentobarbital, midazolam and propofol (Chen and Wasterlain, 2006).

The endocannabinoid system has recently been implicated as an important endogenous mechanism for terminating seizures (Lutz, 2004, Smith, 2005, Wallace et al., 2003). This system is comprised of G protein-coupled cannabinoid receptors (CB₁ and CB₂), endogenous cannabinoids (endocannabinoids) and the associated enzymatic systems involved in their synthesis, transport and degradation (Mackie, 2006). Cannabinoids, such as marijuana and other derivatives, have been used since antiquity for the treatment of seizures (Adams and Martin, 1996) and have also been shown to possess anticonvulsant properties (Karler et al., 1974). Search for the endogenous ligands for the cannabinoid receptors first led to the discovery of anandamide (Devane et al., 1992) and then 2-arachidonylglycerol (2-AG) (Stella et al., 1997). It has been shown that physiological or pathological stimulation of neurons stimulate endocannabinoid synthesis that regulate neuronal excitability by activating presynaptic cannabinoid CB₁ receptors and ultimately inhibiting neurotransmitter release in a retrograde fashion. Endocannabinoids are thought mediate their anticonvulsant effects by activating cannabinoid CB₁ receptors. We also recently demonstrated that synthetic cannabinoid CB₁ receptor agonist WIN 55,212-2 blocked both status epilepticus and acquired epilepsy in hippocampal neuronal cultures (Blair et al., 2006). The hippocampus is rich in cannabinoid CB₁ receptors and is also known to be involved in generation of seizures. Despite the powerful neuromodulatory and anticonvulsant effects of cannabinoid CB₁ receptor activation in regulating seizure termination, the role of endocannabinoids in modulating status epilepticus remains unexplored.

This study employed patch clamp electrophysiology to evaluate the role of the endocannabinoids methanandamide and 2-AG in blocking electrographic status epilepticus using the well-characterized hippocampal neuronal culture model of status epilepticus. Removal of Mg²⁺ from culture medium induces tonic high-frequency epileptiform discharges in hippocampal neurons in culture and represents a well-characterized in vitro model of status epilepticus that has been routinely used to carry out biochemical, electrophysiological and molecular investigations on status epilepticus (DeLorenzo et al., 1998, Delorenzo et al., 2005, Sombati and DeLorenzo, 1995). The spike frequency and epileptiform discharges manifested in this model are essentially identical to the electrographic features of status epilepticus observed with in vivo animal models and in human status epilepticus and represent an excellent model to study status epilepticus and refractory status epilepticus (Delorenzo et al., 2005, Mangan and Kapur, 2004, Sombati and DeLorenzo, 1995, Goodkin et al., 2005). The results from this study indicate that endocannabinoids inhibit status epilepticus by activating cannabinoid CB₁ receptors. While conventional antiepileptic drugs were ineffective in completely blocking status epilepticus at the high micromolar range, the endocannabinoids were very potent and prevented status epilepticus at nanomolar concentrations. The results suggest that development of novel therapeutic agents that modulate the endocannabinoid system could offer unique pharmacological approaches for the treatment of status epilepticus.