Epilepsy is one of the most common neurological disorders. Although epilepsy can be idiopathic, it is estimated that up to 50% of all epilepsy cases are initiated by neurological insults and are called acquired epilepsy (AE). AE develops in 3 phases: (1) the injury (central nervous system [CNS] insult), (2) epileptogenesis (latency), and (3) the chronic epileptic (spontaneous recurrent seizure) phases. Status epilepticus (SE), stroke, and traumatic brain injury (TBI) are 3 major examples of common brain injuries that can lead to the development of AE. It is especially important to understand the molecular mechanisms that cause AE because it may lead to innovative strategies to prevent or cure this common condition. Recent studies have offered new insights into the cause of AE and indicate that injury-induced alterations in intracellular calcium concentration levels [Ca 2+ ] i and calcium homeostatic mechanisms play a role in the development and maintenance of AE. The injuries that cause AE are different, but they share a common molecular mechanism for producing brain damage -an increase in extracellular glutamate concentration that causes increased intracellular neuronal calcium, leading to neuronal injury and/or death. Neurons that survive the injury induced by glutamate and are exposed to increased [Ca 2+ ] i are the cellular substrates to develop epilepsy because dead cells do not seize. The neurons that survive injury sustain permanent long-term plasticity changes in [Ca 2+ ] i and calcium homeostatic mechanisms that are permanent and are a prominent feature of the epileptic phenotype. In the last several years, evidence has accumulated indicating that the prolonged alteration in neuronal calcium dynamics plays an important role in the induction and maintenance of the prolonged neuroplasticity changes underlying the epileptic phenotype. Understanding the role of calcium as a second messenger in the induction and maintenance of epilepsy may provide novel insights into therapeutic advances that will prevent and even cure AE.

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.

Traumatic brain injury (TBI) survivors often suffer chronically from significant morbidity associated with cognitive deficits, behavioral difficulties and a post-traumatic syndrome and thus it is important to understand the pathophysiology of these long-term plasticity changes after TBI. Calcium (Ca 2+ ) has been implicated in the pathophysiology of TBI-induced neuronal death and other forms of brain injury including stroke and status epilepticus. However, the potential role of long-term changes in neuronal Ca 2+ dynamics after TBI has not been evaluated. In the present study, we measured basal free intracellular Ca 2+ concentration ([Ca 2+ ] i ) in acutely isolated CA3 hippocampal neurons from Sprague-Dawley rats at 1, 7 and 30 days after moderate central fluid percussion injury. Basal [Ca 2+ ] i was significantly elevated when measured 1 and 7 days post-TBI without evidence of neuronal death. Basal [Ca 2+ ] i returned to normal when measured 30 days post-TBI. In contrast, abnormalities in Ca 2+ homeostasis were found for as long as 30 days after TBI. Studies evaluating the mechanisms underlying the altered Ca 2+ homeostasis in TBI neurons indicated that necrotic or apoptotic cell death and abnormalities in Ca 2+ influx and efflux mechanisms could not account for these changes and suggested that long-term changes in Ca 2+ buffering or Ca 2+ sequestration/release mechanisms underlie these changes in Ca 2+ homeostasis after TBI. Further elucidation of the mechanisms of altered Ca 2+ homeostasis in traumatized, surviving neurons in TBI may offer novel therapeutic interventions that may contribute to the treatment and relief of some of the morbidity associated with TBI.