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-Mg 2+ 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 EC 50 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 CB 1 receptor since they were blocked by the cannabinoid CB 1 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.

Cannabinoids and Epilepsy

“…The endocannabinoids, methanandamide, and 2-AG reduced neuronal firing in a low Mg 2+ in vitro model of status epilepticus, in a d o s e -d e p e n d e n t m a n n e r ( E C 5 0 1 4 5 ± 4 . 1 5 n M methanandamide, 1.68±0.19 μM 2-AG) [151].…”

The novel antiepileptic drug carisbamate (RWJ 333369) is effective in inhibiting spontaneous recurrent seizure discharges and blocking sustained repetitive firing in cultured hippocampal neurons

“…5, clinically used AEDs including phenytoin (50 μM) and ethosuximide (1 mM) at therapeutic concentrations or higher failed to stop low Mg 2+ -induced in vitro status epilepticus-like activity in this model (Sombati and DeLorenzo, 1995). This type of in vitro continuous seizure discharge has been extremely refractory to anticonvulsant treatment (Blair et al, 2006;Deshpande et al, 2007b;Sombati and DeLorenzo, 1995). We investigated if carisbamate could act differently and block this high frequency spike activity.…”

“…Studies were conducted on primary mixed hippocampal neuronal cultures prepared as described previously with slight modifications (Blair et al, 2006;Deshpande et al, 2007b;Sombati and DeLorenzo, 1995). In brief, hippocampal cells were obtained from 2-day postnatal Sprague-Dawley rats (Harlan, Frederick, MD) and plated at a density of 1 × 10 5 cells/cm 2 onto 35-mm Falcon cell culture dishes (Becton Dickinson and Co., Franklin Lakes, NJ) previously coated with poly-L-lysine (0.05 mg/ml).…”

“…Whole-cell current clamp recordings were performed using previously established procedures (Blair et al, 2006;Deshpande et al, 2007b;Sombati and DeLorenzo, 1995). Briefly, a cell culture dish was mounted on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan) continuously perfused with physiological basal recording solution (pBRS) containing (in mM): 145 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl 2 , 10 glucose, and 0.002 glycine, pH 7.3, and osmolarity adjusted to 325 ± 5 mOsm with sucrose.…”

“…SREDs were induced into neuronal cultures by exposing them for 3-h to a solution containing no added MgCl 2 (low Mg 2+ ) using procedures described previously (Blair et al, 2006;Deshpande et al, 2007b;Sombati and DeLorenzo, 1995). Briefly, after the removal of maintenance media, neurons were gently washed with 3 × 1.5 ml of pBRS (±1 mM MgCl 2 ) and then allowed to incubate in this solution at 37 °C under 5% CO 2 /95% air atmosphere.…”