Several lines of evidence suggest that cannabinoid compounds are anticonvulsant. However, the anticonvulsant potential of cannabinoids and, moreover, the role of the endogenous cannabinoid system in regulating seizure activity has not been tested in an in vivo model of epilepsy that is characterized by spontaneous, recurrent seizures. Here, using the rat pilocarpine model of epilepsy, we show that the marijuana extract ⌬ 9 -tetrahydrocannabinol (10 mg/kg) as well as the cannabimimetic, 4, significantly increased both seizure duration and frequency. In some animals, CB 1 receptor antagonism resulted in seizure durations that were protracted to a level consistent with the clinical condition status epilepticus. Furthermore, we determined that during an short-term pilocarpine-induced seizure, levels of the endogenous CB 1 ligand 2-arachidonylglycerol increased significantly within the hippocampal brain region. These data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of the CB 1 receptor. Furthermore, Western blot and immunohistochemical analyses revealed that CB 1 receptor protein expression was significantly increased throughout the CA regions of epileptic hippocampi. By demonstrating a role for the endogenous cannabinoid system in regulating seizure activity, these studies define a role for the endogenous cannabinoid system in modulating neuroexcitation and suggest that plasticity of the CB 1 receptor occurs with epilepsy.

Organophosphate (OP) compounds are among the most lethal chemical weapons ever developed and are irreversible inhibitors of acetylcholinesterase. Exposure to majority of OP produces status epilepticus (SE) and severe cholinergic symptoms that if left untreated are fatal. Survivors of OP intoxication often suffer from irreversible brain damage and chronic neurological disorders. Although pilocarpine has been used to model SE following OP exposure, there is a need to establish a SE model that uses an OP compound in order to realistically mimic both acute and long-term effects of nerve agent intoxication. Here we describe the development of a rat model of OP-induced SE using diisopropylfluorophosphate (DFP). The mortality, behavioral manifestations, and electroencephalogram (EEG) profile for DFP-induced SE (4 mg/kg, sc) were identical to those reported for nerve agents. However, significantly higher survival rates were achieved with an improved dose regimen of DFP and treatment with pralidoxime chloride (25 mg/kg, im), atropine (2 mg/kg, ip), and diazepam (5 mg/kg, ip) making this model ideal to study chronic effects of OP exposure. Further, DFP treatment produced N-methyl-D-aspartate (NMDA) receptor-mediated significant elevation in hippocampal neuronal [Ca(2+)](i) that lasted for weeks after the initial SE. These results provided direct evidence that DFP-induced SE altered Ca(2+) dynamics that could underlie some of the long-term plasticity changes associated with OP toxicity. This model is ideally suited to test effective countermeasures for OP exposure and study molecular mechanisms underlying neurological disorders following OP intoxication.

Alterations in hippocampal neuronalneuronal plasticity ͉ pilocarpine model ͉ calcium homeostasis ͉ seizure E pilepsy is one of the most common neurological disorders (1), and Ϸ40% of epilepsies are acquired, meaning that the epileptic condition is acquired through an injury to the nervous system (2, 3). Epileptogenesis is the process by which an injury such as status epilepticus (SE), stroke, or traumatic brain injury produces long-term plasticity changes in neurons, resulting in spontaneous recurrent seizures [acquired epilepsy (AE)] in previously normal brain tissue (4-6). AE develops in three phases: injury (brain insult), epileptogenesis (latency), and, finally, chronic epilepsy (spontaneous recurrent seizure) (7). The molecular basis for developing AE is still not completely understood. However, there is growing evidence from the SE and glutamate injury-induced models of AE that elevated intracellular calcium concentration ([Ca 2ϩ ] i ) and altered Ca 2ϩ -homeostatic mechanisms (Ca 2ϩ dynamics) may play a role in the development of AE (6,(8)(9)(10)(11)(12)(13). In addition, altered Ca 2ϩ dynamics have been observed in the hippocampus of chronic epileptic animals as long as 1 year after the induction of seizures in the in vivo pilocarpine model of AE (14). This model of AE shares many of the clinical and pathophysiological characteristics of partial-complex or temporal-lobe epilepsy in humans (14-19). The hippocampus has been shown to be the focus for many of the plasticity, pathophysiological, and epileptogenic alterations in the pilocarpine model of AE (14-19). Thus, if Ca 2ϩ is involved as a second messenger in the inductions and maintenance of AE in the pilocarpine model, it would be expected that Ca 2ϩ dynamics should be altered immediately after SE and in the three phases of the development of AE.This study was undertaken to determine whether hippocampal neuronal Ca 2ϩ dynamics are altered immediately after SE and in the three phases of the development of AE.Ca 2ϩ dynamics were evaluated in acutely isolated CA1 hippocampal, frontal, and occipital neurons at several time points during the injury, epileptogenesis, and chronic-epilepsy phases of AE. The effects of NMDA receptor inhibition by (ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801) on both the development of seizures and Ca 2ϩ dynamics were determined. Comparisons of sham (salinetreated), pilocarpine without SE, and pilocarpine with SE but without AE control animals with SE animals with AE indicated that Ca 2ϩ dynamics were significantly altered during the development of AE and that both changes in Ca 2ϩ dynamics and the development of AE could be blocked by inhibition of the NMDA receptor during SE. The results demonstrate that altered Ca 2ϩ dynamics were associated with the development of AE and that inhibition of these changes in Ca 2ϩ dynamics was associated with the inhibition of the development of AE. The results provide direct evidence that Ca 2ϩ dynamics are significantly altered during epileptogenesis and ...

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(ϩ)- [2,3-dihydro-5-methyl-3-[(morpholinyl) (WIN 55, 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) (EC 50 ϭ 0.85 M) and SE (EC 50 ϭ 1.51 M), with total suppression of seizure activity at 3 M and of SE activity at 5 M. 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-(piperi-, showing a CB1 receptor-dependent pathway. The inhibitory effect of WIN 55,212-2 against low Mg 2ϩ -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 Mg 2ϩ -induced SE at concentrations up to 150 M. The results from this study show CB1 receptor-mediated anticonvulsant effects of the cannabimimetic WIN 55,212-2 against both SRED and low Mg 2ϩ -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., 2001Wallace et al., , 2002Shafaroodi 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 anticonvulsant effects of cannabinoids. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.100354.ABBREVIATIONS: CB1, cannabinoid type 1; MES, maximal electroshock; WIN 55, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride; pBRS, physiological bath recording solution; PDS, paroxysmal depolarization shift(s); AEA, arachidonylethanolamine; DSI, depolarization-induced suppression of inhibition; DSE, depolarization-induced suppression of excitation.

Activation of the cannabinoid type 1 (CB1) receptor, a major G-protein-coupled receptor in brain, acts to regulate neuronal excitability and has been shown to mediate the anticonvulsant effects of cannabinoids in several animal models of seizure, including the rat pilocarpine model of acquired epilepsy. However, the long-term effects of status epilepticus on the expression and function of the CB1 receptor have not been described. Therefore, this study was initiated to evaluate the effect of status epilepticus on CB1 receptor expression, binding, and G-protein activation in the rat pilocarpine model of acquired epilepsy. Using immunohistochemistry, we demonstrated that status epilepticus causes a unique "redistribution" of hippocampal CB1 receptors, consisting of specific decreases in CB1 immunoreactivity in the dense pyramidal cell layer neuropil and dentate gyrus inner molecular layer, and increases in staining in the CA1-3 strata oriens and radiatum. In addition, this study demonstrates that the redistribution of CB1 receptor expression results in corresponding functional changes in CB1 receptor binding and G-protein activation using [3H] R+-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl](1-napthalen-yl)methanone mesylate (WIN55,212-2) and agonist-stimulated [35S]GTPgammaS autoradiography, respectively. The redistribution of CB1 receptor-mediated [35S]GTPgammaS binding was 1) attributed to an altered maximal effect (Emax) of WIN55,212-2 to stimulate [35S]GTPgammaS binding, 2) reversed by the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), 3) confirmed by the use of other CB1 receptor agonists, and 4) not reproduced in other G-protein-coupled receptor systems examined. These results demonstrate that status epilepticus causes a unique and selective reorganization of the CB1 receptor system that persists as a permanent hippocampal neuronal plasticity change associated with the development of acquired epilepsy.