Biomedical subjects

R J DeLorenzo

Publications and source records attributed to R J DeLorenzo.

At least 19 recordsLinked to original sources

PubMed2002

In vitro inhibition of pentylenetetrazole and bicuculline-induced epileptiform activity in rat hippocampal pyramidal neurons by aqueous fraction isolated from Delphinium denudatum.

Roots of Delphinium denudatum W. are used for the treatment of epilepsy by traditional healers in subcontinent. Aqueous fraction (AF) isolated from D. denudatum has previously shown significant anticonvulsant activity in in vivo and in vitro models of seizures. We investigated anticonvulsant effects of AF on pentylenetetrazole (PTZ) and bicuculline (BIC)-induced epileptiform activity in primary hippocampal neuronal cultures. Electrophysiological studies on single pyramidal neurons were carried out by using whole-cell current clamp technique. Introduction of AF (0.6 mg/ml) in perfusate blocked PTZ (10 mM) and BIC (100 micro M)-induced epileptiform activity comprising of paroxysmal depolarization shifts (PDS). The PDS were elicited again when AF was removed from perfusate. We conclude that AF contains anticonvulsant compounds that possibly interact with GABA(A) receptor to produce blockade of epileptiform activity. Further studies on isolation of compounds from AF may lead to discovery of new class of anticonvulsants.

Animals↗

PubMed2001

Assessment of the role of CB1 receptors in cannabinoid anticonvulsant effects.

The cannabinoid CB1 receptor has been shown to be the primary site of action for cannabinoid-induced effects on the central nervous system. Activation of this receptor has proven to dampen neurotransmission and produce an overall reduction in neuronal excitability. Cannabinoid compounds like delta9-tetrahydrocannabinol and cannabidiol have been shown to be anticonvulsant in maximal electroshock, a model of partial seizure with secondary generalization. However, until now, it was unknown if these anticonvulsant effects are mediated by the cannabinoid CB1 receptor. Likewise, (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2), a cannabimimetic compound that has been shown to decrease hyperexcitability in cell culture models via the cannabinoid CB1 receptor, has never been evaluated for anticonvulsant activity in an animal seizure model. We first show that the cannabinoid compounds delta9-tetrahydrocannabinol (ED50 = 42 mg/kg), cannabidiol (ED50 = 80 mg/kg), and WIN 55,212-2 (ED50 = 47 mg/kg) are anticonvulsant in maximal electroshock. We further establish, using the cannabinoid CB1 receptor specific antagonist N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A) (AD50 = 2.5 mg/kg), that the anticonvulsant effects of delta9-tetrahydrocannabinol and WIN 55,212-2 are cannabinoid CB1 receptor-mediated while the anticonvulsant activity of cannabidiol is not. This study establishes a role for the cannabinoid CB1 receptor in modulating seizure activity in a whole animal model.

Animals↗

PubMed2001

Long-term alteration of calcium homeostatic mechanisms in the pilocarpine model of temporal lobe epilepsy.

The pilocarpine model of temporal lobe epilepsy is an animal model that shares many of the clinical and pathophysiological characteristics of temporal lobe or limbic epilepsy in humans. This model of acquired epilepsy produces spontaneous recurrent seizure discharges following an initial brain injury produced by pilocarpine-induced status epilepticus. Understanding the molecular mechanisms mediating these long lasting changes in neuronal excitability would provide an important insight into developing new strategies for the treatment and possible prevention of this condition. Our laboratory has been studying the role of alterations in calcium and calcium-dependent systems in mediating some of the long-term neuroplasticity changes associated with epileptogenesis. In this study, [Ca(2+)](i) imaging fluorescence microscopy was performed on CA1 hippocampal neurons acutely isolated from control and chronically epileptic animals at 1 year after the induction of epileptogenesis with two different fluorescent dyes (Fura-2 and Fura-FF) having high and low affinities for [Ca(2+)](i). The high affinity Ca(2+) indicator Fura-2 was utilized to evaluate [Ca(2+)](i) levels up to 900 nM and the low affinity indicator Fura-FF was employed for evaluating [Ca(2+)](i) levels above this range. Baseline [Ca(2+)](i) levels and the ability to restore resting [Ca(2+)](i) levels after a brief exposure to several glutamate concentrations in control and epileptic neurons were evaluated. Epileptic neurons demonstrated a statistically significantly higher baseline [Ca(2+)](i) level in comparison to age-matched control animals. This alteration in basal [Ca(2+)](i) levels persisted up to 1 year after the induction of epileptogenesis. In addition, the epileptic neurons were unable to rapidly restore [Ca(2+)](i) levels to baseline following the glutamate-induced [Ca(2+)](i) loads. These changes in Ca(2+) regulation were not produced by a single seizure and were not normalized by controlling the seizures in the epileptic animals with anticonvulsant treatment. Peak [Ca(2+)](i) levels in response to different concentrations of glutamate were the same in both epileptic and control neurons. Thus, glutamate produced the same initial [Ca(2+)](i) load in both epileptic and control neurons. Characterization of the viability of acutely isolated neurons from control and epileptic animals utilizing standard techniques to identify apoptotic or necrotic neurons demonstrated that epileptic neurons had no statistically significant difference in viability compared to age-matched controls. These results provide the first direct measurement of [Ca(2+)](i) levels in an intact model of epilepsy and indicate that epileptogenesis in this model produced long-lasting alterations in [Ca(2+)](i) homeostatic mechanisms that persist for up to 1 year after induction of epileptogenesis. These observations suggest that altered [Ca(2+)](i) homeostatic mechanisms may underlie some aspects of the epileptic phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.

Animals↗

PubMed2001

Hippocampal neurons exhibit both persistent Ca2+ influx and impairment of Ca2+ sequestration/extrusion mechanisms following excitotoxic glutamate exposure.

Exposure of neurons to glutamate is an essential element of neuronal function, producing transient elevations in free intracellular calcium ([Ca2+]i) that are required for normal physiological processes. However, prolonged elevations in [Ca2+]i have been observed following glutamate excitotoxicity and have been implicated in the pathophysiology of delayed neuronal cell death. In the current study, we utilized indo-1 and fura-2ff Ca2+ imaging techniques to determine if glutamate-induced prolonged elevations in [Ca2+]i were due to persistent influx of extracellular Ca2+ or from impairment of neuronal Ca2+ extrusion/sequestration mechanisms. By experimentally removing Ca2+ from the extracellular solution following glutamate exposure, influx of Ca2+ into the neurons was severely attenuated. We observed that brief glutamate exposures (<5 min, 50 microM glutamate) resulted in a Ca2+ influx that continued after the removal of glutamate. The Ca2+ influx was reversible, and the cell was able to effectively restore [Ca2+]i to resting levels. Longer, excitotoxic glutamate exposures (> or = 5 min) generated a Ca2+ influx that continued for the duration of the recording period (>1 h). This persistent Ca2+ influx was not primarily mediated through traditionally recognized Ca2+ channels such as glutamate receptor-operated channels or voltage-gated Ca2+ channels. In addition to the persistent Ca2+ influx, longer glutamate exposures also produced a lasting disruption of Ca2+ extrusion/sequestration mechanisms, impairing the ability of the neuron to restore resting [Ca2+]i. These data suggest that glutamate-induced protracted [Ca2+]i elevations result from at least two independent, simultaneously occurring alterations in neuronal Ca2+ physiology, including a persistent Ca2+ influx and damage to Ca2+ regulation mechanisms.

Animals↗

PubMed2001

Anticonvulsant activities of the FS-1 subfraction isolated from roots of Delphinium denudatum.

Delphinium denudatum Wall. (Ranunculaceae) is a medicinal herb used for the treatment of epilepsy in the subcontinent. The present study reports the anticonvulsant activities in the maximal electroshock test (MEST) and subcutaneous pentylenetetrazole (PTZ), bicuculline (BIC), picrotoxin (PIC)-induced seizures of the FS-1 subfraction (FS-1) that was obtained by purification of an aqueous fraction isolated from the roots of D. denudatum. In CF 1 mice, FS-1 (600 mg/kg i.p.) exhibited very potent anticonvulsant activity that was comparable to the effects of the well-known antiepileptic drug phenytoin (20 mg/kg) in MEST and protected 100% animals from hind limb tonic extension phase of this model. FS-1 also suppressed PTZ-induced threshold seizure and the loss of the righting reflex with tonic fore and hind limb extension by 100%, similar to the antiepileptic drug valproic acid (350 mg/kg). BIC-induced seizures were suppressed in 80% of the animals. FS-1 exhibited weak anticonvulsant effect on PIC-induced seizures, however, it significantly reduced mortality and delayed the onset of seizures. FS-1 had no effect on strychnine (STN)-induced extensor seizures. The results demonstrate the broad and potent anticonvulsant activity of the compounds in FS-1 of D. denudatum.

Animals↗

PubMed2001

Anticonvulsant activities of ethanolic extract and aqueous fraction isolated from Delphinium denudatum.

Dried roots of Delphinium denudatum Wall. are a popular folk remedy for the treatment of epilepsy in the traditional Unani system of medicine in the sub-continent. We carried out anticonvulsant screening of the ethanolic extract (EE) and aqueous fraction (AF) of this plant utilising the maximal electroshock (MEST) and subcutaneous pentylenetetrazole (scPTZ), bicuculline (scBIC), picrotoxin (scPTX) and strychnine (scSTN) tests for anticonvulsant activity. EE had weak dose-dependent anticonvulsant effects on seizures induced by PTZ and BIC. AF exhibited dose-dependent activity against hind limb tonic extension phase (HLTE) of MEST and comparatively stronger anticonvulsant activity against seizures induced by PTZ and BIC. The results suggest the presence of potent anticonvulsant compounds in AF of D. denudatum and deserve further investigation for isolation of active compounds and elucidation of the mechanism of anticonvulsant action.

Animals↗

PubMed2001

Chronic inhibition of cortex microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the rat pilocarpine model following epileptogenesis.

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca(2+) uptake persists after the establishment of epilepsy, Ca(2+) uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca(2+) uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age-matched controls. The inhibition of Ca(2+) uptake was not due to individual seizures nor an artifact of increased Ca(2+) release from epileptic microsomes. In addition, the decreased Ca(2+) uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg(2+)/Ca(2+) ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the pilocarpine model may underlie some of the long-term plasticity changes associated with epileptogenesis.

Animals↗

PubMed2001

Short-term outcomes of children with febrile status epilepticus.

Febrile status epilepticus (SE) represents the extreme end of the complex febrile seizure spectrum. If there are significant sequelae to febrile seizures, they should be more common in this group. We have prospectively identified 180 children aged 1 month to 10 years who presented with febrile SE over a 10-year period in Bronx, New York, and Richmond, Virginia. They were compared with 244 children who presented with their first febrile seizure (not SE) in a prospective study done in the Bronx. The mean age of the children with febrile SE was 1.92 years, and of the comparison group, 1.85 years. Duration of SE was 30-59 min in 103 (58%), 60-119 min in 43 (24%), and > or =120 min in 34 (18%). Focal features were present in 64 (35%) of cases. There were no deaths and no cases of new cognitive or motor handicap. Children with febrile SE were more likely to be neurologically abnormal (20% vs. 5%; p < 0.001), to have a history of neonatal seizures (3% vs. 0; p = 0.006) and a family history of epilepsy (11% vs. 5%; p = 0.05) and less likely to have a family history of febrile seizures (15% vs. 27%; p = 0.01) than were children in the comparison group. The short-term morbidity and mortality of febrile SE are low. There are differences in the types of children who have febrile SE compared with those who experience briefer febrile seizures. Long-term follow-up of this cohort may provide insight into the relationship of prolonged febrile seizures and subsequent mesial temporal sclerosis.

Age Distribution↗

PubMed2001

Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy.

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.

Animals↗

PubMed2001

Glutamate injury-induced epileptogenesis in hippocampal neurons: an in vitro model of stroke-induced "epilepsy".

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 micromol/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-micromol/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.

Action Potentials↗

PubMed2001

Status epilepticus in older patients: epidemiology and treatment options.

Status epilepticus (SE) is a medical and neurological emergency that has been associated with significant morbidity and mortality. The most widely accepted definition of SE is more than 30 minutes of either continuous seizure activity, or intermittent seizures without full recovery of consciousness between seizures. SE is a major clinical concern in the elderly population, both because it has increased incidence in the elderly compared with the general population, and because of concurrent medical conditions that are more likely to complicate therapy and worsen prognosis in elderly individuals. The incidence of SE in the elderly is almost twice that of the general population at 86 per 100,000 per year. With the anticipated growth of the elderly population, SE is likely to become an increasingly common problem facing clinicians, and an important public health issue. The elderly have the highest SE-associated mortality of any age group at 38%, and the very old elderly (>80 years of age) have a mortality of at least 50%. Acute or remote stroke is the most common aetiology of SE in the elderly. Nonconvulsive SE (NCSE) has a wide range of clinical presentations, ranging from confusion to obtundation. It occurs commonly in elderly patients who are critically ill and in the setting of coma. Electroencephalogram is the only reliable method of diagnosing NCSE. The goal of treatment for SE is rapid cessation of clinical and electrical seizure activity. Most treatment protocols call for the immediate administration of an intravenous benzodiazepine, followed by phenytoin or fosphenytoin. Recent studies suggest that when this initial treatment of SE fails, little is gained by using additional standard drugs. General anaesthetic agents (such as pentobarbital, midazolam, or propofol) should be expeditiously employed, although these treatments have their own potential complications. Intravenous valproic acid is a recent addition to the armamentarium of drugs for the treatment of SE, with a low risk of hypotension, respiratory depression and hypotension, making it a potentially useful drug for the treatment of SE in the elderly. However, further information is needed to establish its role in the overall treatment of SE.

Aged↗

PubMed2000

Chronic inhibition of Ca(2+)/calmodulin kinase II activity in the pilocarpine model of epilepsy.

The development of symptomatic epilepsy is a model of long-term plasticity changes in the central nervous system. The rat pilocarpine model of epilepsy was utilized to study persistent alterations in calcium/calmodulin-dependent kinase II (CaM kinase II) activity associated with epileptogenesis. CaM kinase II-dependent substrate phosphorylation and autophosphorylation were significantly inhibited for up to 6 weeks following epileptogenesis in both the cortex and hippocampus, but not in the cerebellum. The net decrease in CaM kinase II autophosphorylation and substrate phosphorylation was shown to be due to decreased kinase activity and not due to increased phosphatase activity. The inhibition in CaM kinase II activity and the development of epilepsy were blocked by pretreating seizure rats with MK-801 indicating that the long-lasting decrease in CaM kinase II activity was dependent on N-methyl-D-aspartate receptor activation. In addition, the inhibition of CaM kinase II activity was associated in time and regional localization with the development of spontaneous recurrent seizure activity. The decrease in enzyme activity was not attributed to a decrease in the alpha or beta kinase subunit protein expression level. Thus, the significant inhibition of the enzyme occurred without changes in kinase protein expression, suggesting a long-lasting, post-translational modification of the enzyme. This is the first published report of a persistent, post-translational alteration of CaM kinase II activity in a model of epilepsy characterized by spontaneous recurrent seizure activity.

Animals↗

PubMed2000

Chronic DeltaFosB expression and increased AP-1 transcription factor binding are associated with the long term plasticity changes in epilepsy.

NMDA receptor activation during status epilepticus (SE) has previously been shown to be required for epileptogenesis as well as the persistent upregulation of serum response factor (SRF) in the in vivo pilocarpine model of epilepsy. SRF is established as a regulator of the FosB gene which expresses FosB and DeltaFosB components of the AP-1 transcription factor complex. Therefore we investigated whether DeltaFosB expression and AP-1 DNA binding were also persistently elevated in pilocarpine-treated rats which chronically displayed spontaneous seizures. Using hippocampal nuclear extracts, DeltaFosB expression and AP-1 DNA binding were significantly elevated for up to one year in the epileptic animals. The expression of other fos and jun proteins was not persistently altered in epilepsy. Neuronal upregulation of DeltaFosB was correlated with regions of the brain that were involved in seizure generation and propagation. The increase in AP-1 DNA binding was shown to be dependent on NMDA receptor activation during SE. Hippocampal DeltaFosB immunostaining was seen predominately in the neuronal nuclei as opposed to other cell types. The data indicate that recurrent seizures which persistently occur in this model were not responsible for the increased DeltaFosB expression. Chronic DeltaFosB expression in epilepsy may be playing a role in the altered expression of other genes in this model and may be involved in some of the neuronal plasticity changes associated with epileptogenesis.

Animals↗

PubMed2000

Inhibition of calcium/calmodulin kinase II alpha subunit expression results in epileptiform activity in cultured hippocampal neurons.

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the alpha subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.

Animals↗

PubMed2000

Prevalence of nonconvulsive status epilepticus in comatose patients.

BACKGROUND: Nonconvulsive status epilepticus (NCSE) is a form of status epilepticus (SE) that is an often unrecognized cause of coma. OBJECTIVE: To evaluate the presence of NCSE in comatose patients with no clinical signs of seizure activity. METHODS: A total of 236 patients with coma and no overt clinical seizure activity were monitored with EEG as part of their coma evaluation. This study was conducted during our prospective evaluation of SE, where it has been validated that we identify over 95% of all SE cases at the Medical College of Virginia Hospitals. Only cases that were found to have no clinical signs of SE were included in this study. RESULTS: EEG demonstrated that 8% of these patients met the criteria for the diagnosis of NCSE. The study included an age range from 1 month to 87 years. CONCLUSION: This large-scale EEG evaluation of comatose patients without clinical signs of seizure activity found that NCSE is an underrecognized cause of coma, occurring in 8% of all comatose patients without signs of seizure activity. EEG should be included in the routine evaluation of comatose patients even if clinical seizure activity is not apparent.

Adolescent↗

PubMed2000

Status epilepticus results in an N-methyl-D-aspartate receptor-dependent inhibition of Ca2+/calmodulin-dependent kinase II activity in the rat.

Status epilepticus is a major medical emergency that results in significant alteration of neuronal function. Status epilepticus involves seizure activity recurring frequently enough to induce a sustained alteration in brain function. This study was initiated to investigate how status epilepticus affects the activity of calcium and calmodulin-dependent kinase II in the brain. Calcium and calmodulin-dependent kinase II is a neuronally enriched signal transducing system involved in the regulation of neurotransmitter synthesis and release, cytoskeletal function, gene transcription, neurotransmitter receptor function and neuronal excitability. Therefore, alteration of this signal transduction system would have significant physiological effects. Status epilepticus was induced in rats by pilocarpine injection, allowed to progress for 60 min and terminated by repeated diazepam injections. Animals were killed at specific time-points and examined for calcium and calmodulin-dependent kinase II activity. Calcium and calmodulin-dependent kinase II activity was significantly reduced in cerebral cortex and hippocampal homogenates obtained from status epilepticus rats when compared with control animals. Once established, the status epilepticus-induced inhibition of calcium and calmodulin-dependent kinase II activity was observed at all time-points tested following the termination of seizure activity. However, calcium and calmodulin-dependent kinase II activity was not significantly decreased in thalamus and cerebellar homogenates. In addition, status epilepticus-induced inhibition of calcium and calmodulin-dependent kinase II activity was dependent upon activation of N-methyl-D-aspartate subtype of glutamatergic receptors. Thus, status epilepticus induced a significant inhibition of calcium and calmodulin-dependent kinase II activity that involves N-methyl-D-aspartate receptor activation. The data support the hypothesis that inhibition of calcium and calmodulin-dependent kinase II activity may be involved in the alteration of neuronal function following status epilepticus.

Animals↗

PubMed2000

Pilocarpine-induced status epilepticus causes N-methyl-D-aspartate receptor-dependent inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake.

Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus.

Animals↗

PubMed2000

Induction of spontaneous recurrent epileptiform discharges causes long-term changes in intracellular calcium homeostatic mechanisms.

Calcium and calcium-dependent systems have been long implicated in the induction of epilepsy. We have previously observed that intracellular calcium ([Ca2+]i) levels remain elevated in cells undergoing epileptogenesis in the hippocampal neuronal culture (HNC) model. In this study, we employed the hippocampal neuronal culture (HNC) model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs) for the life of the neurons in culture to investigate alterations in [Ca2+]i homeostatic mechanisms that may be associated with the 'epileptic' phenotype. [Ca2+]i imaging fluorescence microscopy was performed on control and 'epileptic' neurons with two different fluorescent dyes ranging from high to low affinities for [Ca2+]i. We measured baseline [Ca2+]i levels and the ability to restore resting [Ca2+]i levels after a brief 2-min exposure to the excitatory amino acid glutamate in control neurons and neurons with SREDs. Neurons manifesting SREDs had statistically significantly higher baseline [Ca2+]i levels that persisted for the life of the culture. In addition, the 'epileptic' phenotype was associated with an inability to rapidly restore [Ca2+]i levels to baseline following a glutamate induced [Ca2+]i load. The use of the low affinity dye Fura-FF demonstrated that the difference in restoring baseline [Ca2+]i levels was not due to saturation of the high affinity dye Indo-1, which was utilized for evaluating the [Ca2+]i kinetics at lower [Ca2+]i levels. Peak [Ca2+]i levels in response to glutamate were the same in both 'epileptic' and control neurons. While [Ca2+]i levels recovered in approximately 30 min in control cells, it took more than 90 min to reach baseline levels in cells manifesting SREDs. Alterations of [Ca2+]i homeostatic mechanisms observed with the 'epileptic' phenotype were shown to be independent of the presence of continuous SREDs and persisted for the life of the neurons in culture. Epileptogenesis was shown not to affect the degree or duration of glutamate induced neuronal depolarization in comparing control and 'epileptic' neurons. The results indicate that epileptogenesis in this in vitro model produced long-lasting alterations in [Ca2+]i regulation that may underlie the 'epileptic' phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.

Animals↗