The specific blockade of the kainate-induced excitatory conductance is consistent with the ability of TPM to reduce neuronal excitability and could contribute to the anticonvulsant efficacy of this drug.
1. Experiments were carried out using intracellular recording techniques on hippocampal neurons maintained in culture to determine if populations of hippocampal neurons could be induced to develop spontaneously recurring epileptiform discharges. This study demonstrates the conversion of normal hippocampal neurons in culture by a brief Mg(2+)-free treatment into a preparation of cells that permanently manifested recurrent, spontaneous seizure discharges. These electrographic seizure discharges illustrated the same electrographic properties seen in human epilepsy and were observed for the life of the culture. 2. The epileptic activity was shown to occur synchronously in populations of neurons and to be controlled by clinically useful anticonvulsant drugs. 3. This new cell culture model of epileptic activity provides a powerful tool to investigate the molecular mechanisms underlying the induction, maintenance, and termination of this "epileptic condition" in vitro and demonstrates that neuronal networks in culture can be transformed to manifest permanently spontaneous recurrent electrographic seizures.
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 ...
The natural insect neuromodulator octopamine (OCT) was released iontophoretically into regions of neuropil in locust metathoracic ganglia. A narrowly-defined site was found on one side of the ganglion at which release caused a prolonged bout of repetitive flex-extend-flex movements of the tibia on the injected side, at a frequency of from 2-3.5 Hz. When a bout had terminated, repetition of the OCT release caused an extremely similar bout to occur, and again with further treatments, indefinitely. OCT iontophoresis at the equivalent site on the contralateral side caused the contralateral flexor to make stepping movements. Two sites were found, in each half of the ganglion, at which similar OCT release evoked a bout of flight motor activity at 10 Hz. The flight bout involved both sides synchronously and nearly equally, except for a slightly greater motor output on the injected side. Evoked bouts lasted from 20 sec to 25 min depending on the preparation and amount of OCT released. At a site in the 6th abdominal ganglion of mature female locusts OCT release suppressed ongoing rhythmic oviposition digging evoked by severing the ventral nerve cord. A number of previously undescribed DUM neurons was encountered and their dendritic patterns, which are distinctive, determined following dye injection. A hypothesis, termed the Orchestration Hypothesis is presented, which considers how modulator neurons such as locust octopaminergic neurons, might be involved in the generation of specific behaviors.
Background and Purpose-Stroke is the major cause of acquired epilepsy. The mechanisms of ischemia-induced epileptogenesis are not understood, but glutamate is associated with both ischemia-induced injury and epileptogenesis in several models. The objective of this study was to develop an in vitro model of epileptogenesis induced by glutamate injury in hippocampal neurons as observed during stroke. Methods-Primary hippocampal cultures were exposed to 5 mol/L glutamate for various durations. Whole-cell current clamp electrophysiology was used to monitor the acute effects of glutamate on neurons and chronic alterations in neuronal excitability up to 8 days after glutamate exposure. Results-A single, 30-minute, 5-mol/L glutamate exposure produced a subset of neurons that died and a larger population of injured neurons that survived. Neuronal injury was characterized by prolonged reversible membrane depolarization, loss of synaptic activity, and neuronal swelling. Surviving neurons manifested spontaneous, recurrent, epileptiform discharges in neural networks characterized by paroxysmal depolarizing shifts and high-frequency spike firing that persisted for the life of the culture. Conclusions-This study demonstrates that glutamate injury produced a permanent epileptiform phenotype expressed as spontaneous, recurrent epileptiform discharges for the life of the hippocampal neuronal culture. These results suggest a novel in vitro model of glutamate injury-induced epileptogenesis that may help elucidate some of the mechanisms that underlie stroke-induced epilepsy.
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.
The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca 2؉ -dependent. The concentration of intracellular free Ca 2؉ ([Ca 2؉ ] i ) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg 2؉ treatment by confocal laser-scanning f luorescent microscopy to directly correlate changes in [Ca 2؉ ] i with alterations in membrane excitability measured by intracellular recording using whole-cell current-clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg 2؉ -induced spike discharges, was prevented in low-Ca 2؉ solutions and was dependent on activation of the N-methyl-D-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA-Ca 2؉ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.Epilepsy or the occurrence of spontaneous recurrent epileptiform discharges (SREDs, seizures) is one of the most common neurological conditions, affecting more than 2% of children and 1% of adults (1). Between 30% and 50% of epilepsy is symptomatic (1, 2), being caused by a known etiology that produces a permanent plasticity change in a previously normal brain, causing recurrent seizures (3). Much of our current knowledge concerning the pathophysiology of epilepsy has been derived from studies of human brain tissue from patients undergoing epilepsy surgery and various animal models of epilepsy (4-7). In several models of epilepsy, SREDs can be induced to occur for the life of the animal or preparation. The process of inducing SREDs or symptomatic epilepsy in previously normal neuronal networks is called epileptogenesis.Calcium is a major second-messenger system that regulates many neuronal processes (8), and alterations in calcium homeostasis have been implicated in the induction of epileptogenesis (9). Furthermore, indirect evidence has suggested that N-methyl-D-aspartate (NMDA) receptor activation may contribute to the induction of altered neuronal excitability in the kindling (10), hippocampal slice (11), and hippocampal neuronal culture (7) models of epilepsy. Thus, it is important to determine whether the induction of epilepsy is clearly dependent on elevated intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and NMDA receptor activation by directly measuring both [Ca 2ϩ ] i and neuronal excitability during epileptogenesis. This investigation was initiated to determine whether the induction of epileptogenesis in a well characterized in vitro hippocampal neuronal culture (HNC) model of epilepsy (7) was calcium-dependent. This model of epilepsy is well suited to study the role of calcium in epileptogenesis, since it utilizes an episode of continuous seizure activity for...
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.
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