Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on February 9, 2006; DOI: 10.1124/jpet.105.100354
0022-3565/06/3173-1072-1078$20.00
JPET 317:1072-1078, 2006
NEUROPHARMACOLOGY
Activation of the Cannabinoid Type-1 Receptor Mediates the Anticonvulsant Properties of Cannabinoids in the Hippocampal Neuronal Culture Models of Acquired Epilepsy and Status Epilepticus
Robert E. Blair, Laxmikant S. Deshpande, Sompong Sombati, Katherine W. Falenski,Billy R. Martin, and Robert J. DeLorenzoDepartments of Neurology (R.E.B., S.S., R.J.D.), Pharmacology and Toxicology (L.S.D., K.W.F., B.R.M., R.J.D.), and Molecular Biophysics and Biochemistry (R.J.D.), Virginia Commonwealth University, Richmond, Virginia
Received December 20, 2005; accepted February 7, 2006.
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Abstract |
Cannabinoids have been shown to have anticonvulsant properties, but no studies have evaluated the effects of cannabinoids in the hippocampal neuronal culture models of acquired epilepsy (AE) and status epilepticus (SE). This study investigated the anticonvulsant properties of the cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol[1,2,3 de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2)in primary hippocampal neuronal culture models of both AE and SE. WIN 55,212-2 produced dose-dependent anticonvulsant effects against both spontaneous recurrent epileptiform discharges (SRED) (EC50 = 0.85 µM) and SE (EC50 = 1.51 µM), with total suppression of seizure activity at 3 µM and of SE activity at 5 µM. The anticonvulsant properties of WIN 55,212-2 in these preparations were both stereospecific and blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 1 µM), showing a CB1 receptor-dependent pathway. The inhibitory effect of WIN 55,212-2 against low Mg2+-induced SE is the first observation in this model of total suppression of SE by a selective pharmacological agent. The clinically used anticonvulsants phenytoin and phenobarbital were not able to abolish low Mg2+-induced SE at concentrations up to 150 µM. The results from this study show CB1 receptor-mediated anticonvulsant effects of the cannabimimetic WIN 55,212-2 against both SRED and low Mg2+-induced SE in primary hippocampal neuronal cultures and show that these in vitro models of AE and SE may represent powerful tools to investigate the molecular mechanisms mediating the effects of cannabinoids on neuronal excitability.
Since the isolation and purification of the psychotropically active constituent 
9-tetrahydrocannabinol from Cannabis in the 1960s (reviewed in Mechoulam, 2000
), a number of studies have shown the anticonvulsant effects of cannabinoids in a variety of experimentally induced seizure models that include maximal electroshock-induced convulsions, electrical kindling, chemoconvulsants, and audiogenic and photogenic seizures (Corcoran et al., 1973; Karler et al., 1974; Wada et al., 1975; Consroe and Wolkin, 1977; Chiu et al., 1979; Wallace et al., 2001, 2002; Shafaroodi et al., 2004). In addition, several reports have been published on the clinical use of cannabinoids as antiepileptic agents in humans (reviewed in Consroe, 1998). Thus, it is important to elucidate the molecular mechanisms mediating the anticonvulsanteffects of cannabinoids.A major advance in the field of cannabinoid research came with the discovery and cloning of receptor proteins that bind cannabinoids with high affinity (reviewed in Mechoulam, 2000). Within the central nervous system, the Gi/o protein-coupled cannabinoid type-1 (CB1) receptor is widely distributed and is the primary mediator of the physiological and psychotropic effects of cannabinoids in the brain (Devane et al., 1988; Matsuda et al., 1990; Herkenham et al., 1991; Howlett, 1995). In recent years, a better understanding of the endocannabinoid system has led to the development of highly specific synthetic compounds that have been instrumental in the pharmacological evaluation of cannabinoid receptor-mediated regulation of synaptic transmission (Howlett et al., 2004). Utilizing the maximal electroshock (MES)-induced seizure model, studies from this laboratory provided the first evidence that both cannabinoid and endocannabinoid compounds produced anticonvulsant effects through activation of the CB1 receptor (Wallace et al., 2001, 2002). Additionally, the active cannabimimetic compound WIN 55,212-2 was shown to totally suppress spontaneous recurrent epileptiform discharges (SRED; seizures) via CB1 receptor activation in the rat pilocarpine model of acquired epilepsy (AE) (Wallace et al., 2003). This study further showed that endocannabinoids,acting through the CB1 receptor, were essential for maintaining tonic inhibition of seizure frequency and duration in this in vivo model of AE (Wallace et al., 2003). Although these in vivo models of acute seizure and AE are useful for studying the anticonvulsant effects of CB1 receptor activation on intact systems (Wallace et al., 2001, 2002, 2003), they are limited in their ability to carry out sophisticated molecular techniques needed to study underlying cellular mechanisms. Thus, it is important to utilize well established in vitro neuronal preparations that are more amenable to sophisticated electrophysiological and molecular biological procedures to evaluate the cellular mechanisms underlying the anticonvulsant properties of cannabinoids. The hippocampal neuronal culture (HNC) model of AE is a well established model that exhibits SRED for the life of the neurons in culture (Sombati and DeLorenzo, 1995) and has been shown by our laboratory and others to manifest many of the electrophysiological and molecular properties of intact animal models of AE (Kim and Rhim, 2004; Delorenzo et al., 2005). It is also important to evaluate the anticonvulsant effects of cannabinoids against continuous seizure activity, status epilepticus (SE), a major neurological emergency that is often resistant to conventional anticonvulsant treatments (Delorenzo et al., 2005). The well established HNC model of SE (Sombati and DeLorenzo, 1995) has been widely used to evaluate the effects of SE on neuronal cell physiology and molecular changes (Pal et al., 1999; Blair et al., 2004; Mangan and Kapur, 2004; Delorenzo et al., 2005). Thus, the HNC models of AE and SE may serve as valuable tools for elucidating the cellular mechanisms underlying the anticonvulsant properties of cannabinoids because these in vitro models are amenable to experimental manipulation and allow for direct analysis of neurons undergoing SRED and SE in culture (Sombati and DeLorenzo, 1995; Churn et al., 2000; Blair et al., 2004; Delorenzo et al., 2005).
In the current study, we set out to investigate the effects of the cannabimimetic WIN 55,212-2 on seizure activity in the in vitro HNC models of AE and SE (Sombati and DeLorenzo, 1995). Whole-cell current-clamp (WCC) analysis was utilized to directly evaluate the effect of WIN 55,212-2 on SRED and SE and to determine whether a CB1 receptor-dependent pathway was involved. The results show that WIN 55,212-2 was effective in terminating SRED and SE in a stereoselective manner. In addition, these anticonvulsant effects of WIN 55,212-2 were mediated through activation of the CB1 receptor. This study shows that the HNC models of AE and SE provide powerful tools to further elucidate the cellular mechanisms underlying the effect of cannabinoids on seizure activity and neuronal excitability.
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