Calcium-dependent Epileptogenesis in an In Vitro Model of Stroke-induced “Epilepsy”

Laboratory Research

Calcium-dependent Epileptogenesis in an In Vitro Model ofStroke-induced “Epilepsy”

*§David A. Sun, †Sompong Sombati, †Robert E. Blair, and *†‡§Robert J. DeLorenzo

Departments of *Pharmacology and Toxicology, †Neurology, and ‡Biochemistry and Molecular Biophysics, and the §GraduateProgram in Neuroscience, Virginia Commonwealth University, Richmond, Virginia, U.S.A.

Summary: Purpose: Stroke is the most common cause ofacquired epilepsy. The purpose of this investigation was tocharacterize the role of calcium in the in vitro, glutamate inju-ry–induced epileptogenesis model of stoke-induced epilepsy.

Methods: Fura-2 calcium imaging and whole-cell currentclamp electrophysiology techniques were used to measureshort-term changes in neuronal free intracellular calcium con-centration and long-term alterations in neuronal excitability inresponse to epileptogenic glutamate injury (20 �M, 10 min)under various extracellular calcium conditions and in the pres-ence of different glutamate-receptor antagonists.

Results: Glutamate injury–induced epileptogenesis was as-sociated with prolonged, reversible elevations of free intracel-lular calcium concentration during and immediately after injuryand chronic hyperexcitability manifested as spontaneous recur-rent epileptiform discharges for the remaining life of the cul-tures. Epileptogenic glutamate exposure performed in solutionscontaining low extracellular calcium, barium substituted for

calcium, or N-methyl-D-aspartate (NMDA)-receptor antago-nists reduced the duration of intracellular calcium elevation andinhibited epileptogenesis. Antagonism of non–NMDA-receptorsubtypes had no effect on glutamate injury–induced calciumchanges or the induction epileptogenesis. The duration of thecalcium elevation and the total calcium load statistically cor-related with the development of epileptogenesis. Comparableelevations in neuronal calcium induced by non–glutamate re-ceptor–mediated pathways did not cause epileptogenesis.

Conclusions: This investigation indicates that the glutamateinjury–induced epileptogenesis model of stroke-induced epi-lepsy is calcium dependent and requires NMDA-receptor acti-vation. Further, these experiments suggest that prolonged,reversible elevations in neuronal free intracellular calciuminitiate the long-term plasticity changes that underlie the de-velopment of injury-induced epilepsy. Key Words: Calcium—Epileptogenesis—Glutamate NMDA receptor—Stroke.

Epilepsy is one of the most common neurologic dis-orders, affecting an estimated 40 to 50 million peopleworldwide (1). Approximately 50% of all epilepsy caseshave a known cause and are termed acquired epilepsy(2). Epileptogenesis is the process by which normal cen-tral nervous system tissue is transformed into brain tissueprone to the manifestation of spontaneous recurrent sei-zures (3). Neuronal injury in the form of stroke is themost common factor associated with epileptogenesis andacquired epilepsy (4). Despite the important role ofstroke in the development of epilepsy, little is knownregarding the mechanisms by which neuronal injury ini-tiates epileptogenesis in the surviving tissue.

Our laboratory recently developed and characterizedan in vitro model of injury-induced epileptogenesis toreplicate the development of epilepsy as a consequence

of stroke (5). In this model, injury produced by exposureof cultured hippocampal neurons to glutamate resulted ina mixed population of injured neurons that developedexcitotoxic neuronal death and a larger population ofneurons that survived, analogous to the ischemic penum-bra (6). These surviving neurons manifested spontane-ous, recurrent, epileptiform discharges (SREDs) insynchronized neural networks for the remaining life ofthe cultures. This in vitro model of glutamate injury–induced epileptiform activity provides a powerful tool toevaluate the molecular mechanisms mediating stroke-induced epileptogenesis.

The calcium (Ca2+) hypothesis of epileptogenesis sug-gests that alterations in neuronal Ca2+ homeostasis playan essential signaling role in the pathogenesis of epilepsy(7,8). Whereas excessive activation of the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate re-ceptors and irreversible elevations in free intracellularcalcium concentration ([Ca2+]i) have been implicated inexcitotoxic neuronal death (9), NMDAR activation andprolonged but reversible elevations in [Ca2+]i have been

Accepted June 23, 2002.Address correspondence and reprint requests to Dr. R. J. DeLorenzo

at Box 980599, Medical College of Virginia, Virginia CommonwealthUniversity, Richmond, VA 23298, U.S.A. E-mail: [email protected]

Epilepsia, 43(11):1296–1305, 2002Blackwell Publishing, Inc.© 2002 International League Against Epilepsy

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implicated in the induction of epilepsy (8,10–14). Thisstudy was initiated to test the hypothesis that prolongedelevations in [Ca2+]i and NMDAR activation are re-quired for glutamate injury–induced epileptogenesis incultured hippocampal neurons. To this end, we usedfluorescent Ca2+ imaging and electrophysiological tech-niques to characterize the ability of injury induced byglutamate exposure to cause short-term changes in neu-ronal [Ca2+]i and prolonged changes in neuronal excit-ability in the presence of different Ca2+ conditions andvarious glutamate-receptor antagonists.

METHODS

MaterialsUnless otherwise noted, reagents were purchased from

Sigma Chemical Co. (St. Louis, MO, U.S.A.). Sodiumpyruvate, minimum essential medium (MEM) containingEarle’s salts, fetal bovine serum, and horse serum wereobtained from Gibco-BRL (Gaithersburg, MD, U.S.A.).2-Amino-5-phosphonovalerate (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 5-methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine maleate(MK-801), and S-�-methyl-4-carboxyphenylglycine(MCPG) were purchased from Tocris Cookson, Inc. (El-lisville, MO, U.S.A.).

Hippocampal cell culturePrimary mixed hippocampal cultures were prepared

by a modified method of Banker and Cowan (15). Inbrief, hippocampal tissue was dissected from the brainsof 2-day-postnatal Sprague–Dawley rats (Harlan, Fred-erick, MD, U.S.A.), stripped of meninges and vascula-ture, and treated with 0.25% trypsin at 37°C for 30 min.After enzymatic treatment, mechanical trituration of thetissue was performed through a fire-polished Pasteur pi-pette. The resultant single-cell suspension was diluted toa final concentration of 1 × 105 cells/ml in a neuronalfeeding medium [MEM with Earle’s Salts (MEM), 25mM N-2-hydroxyethylpiperazine-N�-2-ethane sulfonicacid (HEPES), 2 mM L-glutamine, 3 mM glucose, 100�g/ml transferrin, 5 �g/ml insulin, 100 �M putrescine, 3nM sodium selenite, 200 nM progesterone, 1 mM sodiumpyruvate, 0.1% ovalbumin, 0.2 ng/ml triiodothyroxine,and 0.4 ng/ml corticosterone] supplemented with 5%horse serum, and plated on confluent glial support layersgrown on poly-L-lysine–coated (0.05 mg/ml) 35-mmplastic culture dishes or Lab-Tek coverglass chambers(Nunc, Naperville, IL, U.S.A.) for 14 days at 37°C in a5% CO2/95% air atmosphere, and fed with glial feedingmedium (MEM, 2 mM L-Glutamine, 3 mM glucose, and10% fetal bovine serum). Twenty-four hours after plat-ing, cultures were treated with 5 mM cytosine arabino-side for 48 h to inhibit nonneuronal growth. Thistechnique prevents overgrowth of the glial elements inthe cultures. Cultures were maintained at 37°C in a 5%

CO2/95% air atmosphere and fed twice weekly with neu-ronal feed supplemented with 20% conditioned medium.Neuronal feed in this modified preparation was supple-mented with 20% conditioned medium (neuronal feedsupplemented with 5% horse serum harvested after 48-hexposure to confluent glial beds). Using conditioned me-dia optimized neuronal culture stability and providedlong-term survival in control and experimental cultures.These mixed cultures were used for experiments from 13days in vitro (DIV) through the life of the cultures (∼21DIV).

Glutamate injury protocolAt 13 DIV, neuronal feed was replaced with a physi-

ologic recording solution (145 mM NaCl, 2.5 mM KCl,10 mM HEPES, 10 mM glucose, 2 mM CaCl2, 1 mMMgCl2, 2 �M glycine, pH 7.3, osmolarity adjusted to 325mOsm with sucrose) in control cultures. This osmolaritywas used as described previously (5) and is commonlyused in tissue-culture electrophysiology. Epileptogenicglutamate injury was produced by exposing cultures torecording solution supplemented with 20 �M glutamatefor 10 min. This treatment produced a neuronal injury,previously defined (5) as neurons surviving the gluta-mate treatment, but manifesting several signs of revers-ible or transient injury, manifested by reversible cellswelling, prolonged depolarization, and altered input re-sistance. These transiently injured neurons developedepileptogenesis (5).

This glutamate treatment was found to produce ∼40%cell death. The surviving 60% of the neurons developedepileptic discharges. These results were essentially iden-tical to the 5 �M glutamate treatment for 30 min ofexposure reported previously (5). In our current cultures,this higher glutamate concentration for a shorter durationwas less influenced by neuronal density and endogenousglutamate levels in the culture media. The seizure pat-terns, cell death, and characterization of epileptic dis-charges were the same as described previously (5). Allexposures were performed at 37°C in a 5% CO2/95% airatmosphere and terminated by three washes with record-ing solution and the addition of fresh neuronal feed. Be-cause 13 DIV is a normally scheduled feeding day, thesecultures maintained the same feeding protocol as naïvecultures.

High extracellular potassium solutions were preparedidentically to physiologic recording solutions except thatKCl was increased from 2.5 to 50 mM and NaCl wasreduced from 145 to 97.5 mM. Treatment of the neuronswith this solution was used to depolarize neurons andinduce calcium influx through non–glutamate-receptorpathways.

ElectrophysiologyWhole-cell current-clamp recordings were performed

on pyramidal neurons by using methods previously de-

GLUTAMATE-INDUCED EPILEPSY REQUIRES Ca2+ 1297

Epilepsia, Vol. 43, No. 11, 2002

scribed in our laboratory (5). In brief, cell-culture me-dium was replaced with recording solution (describedearlier), and cultures were transferred to a heated stage(Brook Industries, Lake Villa, IL, U.S.A.) on a NikonDiaphot inverted microscope (Garden City, NY, U.S.A.).Patch microelectrodes of 3–7 M� resistance were filledwith an internal solution of 140 mM K+ gluconate, 1 mMMgCl2, 10 mM HEPES, 1.1 mM ethylene glycol-bis (�-aminoethyl ether)-N,N,N�,N�-tetraacetic acid, 4 mM Na2

adenosine triphosphate (ATP), 15 mM Tris phosphocre-atine, pH 7.2, with osmolarity adjusted to 310 mOsmwith sucrose.

Recordings were obtained in the whole-cell current-clamp configuration (16) by using an Axopatch 200Aamplifier (Axon Instruments, Foster City, CA, U.S.A.) incurrent-clamp mode. Data were digitized and stored onvideotape by using a Neuro-corder DR-890 (NeurodataInstruments Corp., New York, NY, U.S.A.) and SonyVCR. Data later were played back for analysis to a DashIV chart recorder (Astro-Med Inc., Warwick, RI,U.S.A.).

SREDs were defined as bursts of spike firing at afrequency of �3 Hz for durations of �20 s (5), analo-gous to electrographic seizures (17). Neurons were cat-egorized as “epileptic” on manifestation of two or moreSREDs; otherwise, neurons were categorized as “non-epileptic.” Based on previous experiments, neurons weremonitored for a recording period of �10 min for theexpression of SREDs (5).

Measurement of neuronal [Ca2+]i

Dye loadingChanges in neuronal [Ca2+]i were measured by using

the ratiometric, high-affinity (Kd ≈ 224 nM) fluorescentcalcium indicator, Fura-2 (18). Neurons were loadedwith 1 �M Fura-2 AM (Molecular Probes, Eugene, OR,U.S.A.) dissolved in recording solution (0.1% DMSO)for 1 h at 37°C in a 5% CO2/95% air atmosphere. Dyeloading was terminated with three washes with recordingsolution and incubated an additional 15 min to allowcomplete cleavage of Fura-2 AM.

MicrofluorometryCultures grown on Lab-Tek coverglass chambers

(Nunc) were visualized on an inverted microscope(Olympus IX 70; Olympus America, Melville, NY,U.S.A.) by using a ×20, 0.7 numerical aperture fluoritewater-immersion objective maintained at 37°C with aheated stage (Harvard Apparatus Inc.). Fura-2 was ex-cited with a 75-W xenon arc lamp (Olympus Optical Co.,Shinjuku-ku, Tokyo, Japan) with alternating wave-lengths of 340 and 380 nm filtered through a Sutter FilterWheel (Sutter Instruments Co., Novato, CA, U.S.A.).Fluorescent emission at 510 nm was captured through aFura filter cube (Olympus America) with a dichroic at

400-nm emission by using a cooled digital CCD camera(LSR AstroCam Limited, Cambridge, England).

Ca2+ calibration curvesAn in situ Ca2+ calibration curve was developed to

convert fluorescent ratios to Ca2+ concentrations, as de-scribed by Pal et al. (19) with the equation

�Ca2+�i = Kd �R − Rmin

Rmax − R� �Sf2

Sb2�

where R is the 340/380 ratio at any time; Rmax is themaximal measured ratio in saturating Ca2+ solutions;Rmin is the minimal measured ratio in Ca2+-free solu-tions; Sf2 is the absolute value of the corrected 380-nmsignal at Rmin; Sb2 is the absolute value of the corrected380-nm signal at Rmax; and the Kd is 224 nM (18).

The Temporal Module of the Perkin Elmer Life Sci-ences Imaging Suite, Version 4.0 (Gaithersburg, MD,U.S.A.), was used to control image acquisition and pro-cessing. Image pairs were captured once per minute, andbackground correction for nonspecific fluorescence wasachieved by subtracting images acquired from non–indicator-loaded cultures. A region of interest was des-ignated for each pyramidal neuron in the microscopefield (five to eight neurons). Ratios for each neuron weremeasured from the corresponding region of interest andcalibrated. Individual neurons from multiple experimentswere pooled to determine mean ± SEM. A criterion of[Ca2+]i >400 nM sustained 120 min after glutamate ap-plication was used to define neuronal inability to restoreresting [Ca2+]i. which is known to correlate with delayedexcitotoxic neuronal death (20).

Statistical analysisData are reported as mean ± SEM. One-way analysis

of variance (ANOVA) was used to compare the peakpercentage increase in neuronal [Ca2+]i. Repeated mea-sures analysis of variance (RM ANOVA) was performedon every second data point in Ca2+ imaging experiments.The Tukey test was used for all post hoc multiple com-parisons. Fisher’s Exact test was applied to categoricdata comparing the percentage of neurons made epilepticwith or without drug treatment. The Pearson product–moment correlation was used for all analysis of correla-tion. Statistical analysis was performed by usingSigmaStat 2.0 (Jandel Corp., San Rafael, CA, U.S.A.). p< 0.05 was considered statistically significant for all dataanalysis.

RESULTS

Glutamate injury–induced epileptogenesis wasinhibited by reduced [Ca2+]e

Control neurons displayed spontaneous action poten-tials, excitatory postsynaptic potentials (EPSPs), and in-hibitory post-synaptic potentials (IPSPs) typical ofnormal neuronal activity (n � 37; Figs. 1A and 2A) and

D. A. SUN ET AL.1298


never manifested SREDs (Fig. 3). In contrast, 90% ofneurons surviving an epileptogenic glutamate injury (20�M, 10 min) manifested SREDs �24 h after glutamateexposure (p < 0.001; n � 30; Figs. 1B and 3). As de-scribed previously by our laboratory (5), SREDs werecharacterized by paroxysmal depolarizing shifts (PDSs)and high-frequency spike firing characteristic of electro-graphic seizures (Figs. 1B and 2B) (17).

To test the hypothesis that glutamate injury–inducedepileptogenesis was mediated through a Ca2+-dependentprocess, we injured neurons with the same epileptogenicglutamate exposure (20 �M glutamate, 10 min) in a re-duced [Ca2+]e solution (2.0 mM [Ca2+]e reduced to 0.2mM). Neurons surviving glutamate injury in this low-[Ca2+]e environment exhibited normal levels of activitysimilar to that of controls (Fig. 2C) and never manifestedSREDs (p � 1.0; n � 11; Fig. 3).

Ca2+ is an established second messenger in neurons(21,22). To determine whether Ca2+ had a significantrole as a second-messenger signal in glutamate injury–induced epileptogenesis, epileptogenic glutamate expo-sure was performed in Ca2+-free, barium (Ba2+)-substituted solutions. Although Ba2+ carries a divalentpositive charge across the neuronal membrane through

FIG. 1. Glutamate injury–induced epileptogenesis in culturedhippocampal neurons. A: Representative current-clamp record-ing demonstrating normal spike activity of a control neuron (–64mV membrane potential). Aa: Portion of the trace depicted in A(black bar) displayed at a faster time scale. Note the spontaneousaction potentials, excitatory postsynaptic potentials (EPSPs) andinhibitory PSPs (IPSPs). B: Representative current-clamp re-cording indicative of the epileptiform activity manifested in neu-rons recorded 24 h after glutamate injury (5 µM, 30 min). Thisneuron (–62 mV membrane potential) manifested three sponta-neous, recurrent, epileptiform discharges (SREDs) during a 20-min recording period. The three individual SREDs (indicated byblack bars) of 1.4 min (Ba), 0.72 min (Bb), and 1.3 min (Bc)displayed at a faster time scale. SREDs were characterized byabruptly developing, paroxysmal depolarizing shifts of membranepotential with high-frequency spike firing.

FIG. 2. Whole-cell current-clamp recordings �24 h after epilep-togenic glutamate injury performed in the presence of variousextracellular Ca2+ conditions and glutamate receptor antagonists.A: Control neurons exhibited spontaneous postsynaptic poten-tials, which occasionally reached threshold for the generation ofaction potentials. B: Neurons surviving glutamate injury (20 µM,10 min) manifested spontaneous, recurrent, epileptiform dis-charges (SREDs) characterized by paroxysmal depolarizingshifts (PDSs) and high-frequency spike firing. C, D: Neurons in-jured by glutamate in low-[Ca2+]e (0.2 mM) solutions (C) or Ba2+

substituted for extracellular Ca2+ solutions (D) never manifestedSREDs. E: Glutamate injury performed in the presence of2-amino-5-phosphonovalerate (APV; 50 µM) did not induce epi-leptogenesis. F, G: The non–N-methyl-D-aspartate receptor an-tagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 2 µM; F)and S-�-methyl-4-carboxyphenylglycine (MCPG; 250 µM; G) didnot inhibit glutamate injury–induced epileptogenesis.



conventional routes of Ca2+ entry (23,24), Ba2+ does notactivate Ca2+-sensitive second-messenger pathways(25,26). Extracellular solutions with a 2.0 mM Ba2+ con-centration blocked glutamate injury–induced epilepto-genesis (p � 1.0; n � 11; Figs. 2D and 3). These resultsindicate that glutamate injury–induced epileptogenesisrequired extracellular Ca2+ and activation of intracellularCa2+ pathways, leading to the development of persistentneuronal hyperexcitability.

Glutamate injury–induced epileptogenesis wasinhibited by antagonism of the NMDA receptor

To test the hypothesis that NMDAR activation wasrequired for glutamate injury–induced epileptogenesis,we exposed cultured hippocampal neurons to epilepto-genic glutamate concentrations in the presence of thecompetitive NMDAR antagonist, APV (27). Neurons in-jured in the presence of APV (50 �M) exhibited activitysimilar to that of control neurons (Fig. 2E), never mani-festing SREDs (p � 1.0; n � 10; Fig. 3). Likewise, thenoncompetitive NMDAR antagonist, MK-801 (10 �M)(28) significantly blocked glutamate injury–induced epi-leptogenesis (p � 1.0; n � 10; Fig. 3). Therefore acti-vation of the NMDAR subtype of glutamate receptorswas required for glutamate injury–induced epileptogen-esis.

Antagonism of non-NMDAR subtypes of glutamatereceptors did not inhibit glutamateinjury–induced epileptogenesis

Certain configurations of the �-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate(KA)-receptor subtypes of glutamate receptors also arepermeable to Ca2+ (29). Therefore we tested the role ofAMPA and KA receptors in glutamate injury–inducedepileptogenesis by exposing cultured hippocampal neu-rons to epileptogenic concentrations of glutamate (20�M, 10 min) in the presence of the competitive AMPA-and KA-receptor antagonist, CNQX (2 �M) (30). Ofneurons surviving epileptogenic glutamate injury inCNQX 80% manifested SREDs (Fig. 2F), statisticallygreater than in controls (p < 0.001; n � 10; Fig. 3). Thisrate of epileptogenesis was not statistically differentcompared with glutamate injury in the absence of CNQX(p � 0.584; n � 10).

G protein–coupled metabotropic glutamate receptors(mGluRs) alter neuronal [Ca2+]i through the modulationof intracellular Ca2+ stores, ligand-gated ion channels,and voltage-gated ion channels (31). Therefore weblocked mGluRs with the competitive antagonist, MCPG(250 �M) (32), to investigate the potential role ofmGluR-mediated Ca2+ signaling during epileptogenicglutamate injury. MCPG had no effect on glutamate in-jury–induced epileptogenesis, with 80% of neurons re-corded manifesting SREDs �24 h after glutamate injury,statistically greater than in controls (p < 0.001; n � 10;Figs. 2G and 3). This rate of epileptogenesis was notstatistically different compared with glutamate injury inthe absence of MCPG (p � 0.584, n � 10).

Epileptogenic glutamate injury caused prolonged,reversible increases in neuronal [Ca2+]i

To determine whether the glutamate injury–inducedepileptogenesis was associated with changes in neuronal[Ca2+]i, we monitored neuronal [Ca2+]i before, during,and after epileptogenic glutamate exposure by using theratiometric, high-affinity, calcium indicator Fura-2 (18).Control neurons that were exposed to solution changeswithout glutamate did not manifest elevations in [Ca2+]i,as demonstrated by the representative neuron in Fig. 4Aand the time course in Fig. 5A. In contrast, [Ca2+]i in-creased rapidly on exposure to epileptogenic glutamateconcentrations (Fig. 4B). On washout, the majority ofneurons slowly restored basal Ca2+ levels (Figs. 4B and5B). A subset of neurons (six of 21), however, stillmaintained [Ca2+]i >400 nM, even after 120 min of re-cording (data not shown). Neurons with sustained eleva-tions in [Ca2+]i >400 nM for durations of �120 min werecategorized as having an inability to restore resting[Ca2+]i (IRRC). Because neurons with IRRC undergodelayed excitotoxic neuronal death (20), this subset of

FIG. 3. Glutamate injury–induced epileptogenesis in the pres-ence of various extracellular Ca2+ conditions and glutamate re-ceptor antagonists. Of neurons surviving an epileptogenicglutamate injury (GLU; n = 30), 90% manifested spontaneous,recurrent, epileptiform discharges (SREDs) not observed in con-trols (n = 37). Glutamate injury performed in low-[Ca2+]e (lowCa2+, n = 11), Ba2+ substitution (barium, n = 11), and in thepresence of the competitive 2-amino-5-phosphonovalerate (APV,n = 12) and noncompetitive 5-methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine maleate (MK-801, n = 10)N-methyl-D-aspartate receptor antagonists inhibited the inductionof epileptogenesis. Glutamate injury–induced epileptiform activitywas observed in 80% of neurons injured in the presence of6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; n = 10), as well asin S-�-methyl-4-carboxyphenylglycine (MCPG; n = 10). *p <0.001, Fisher’s Exact test versus control. Data are representedby percentages.



neurons was excluded from statistical analysis. In theremaining neurons, [Ca2+]i was statistically elevated overbasal levels from the time of exposure through 76 min ofrecording (66 GLU; p < 0.05; n � 15, Fig. 5B). Thusglutamate injury–induced epileptogenesis was associatedwith prolonged, reversible increases in neuronal free cal-cium.

Prolonged, reversible increases in neuronal [Ca2+]i

associated with glutamate injury–inducedepileptogenesis were reduced inlow-[Ca2+]e environments

To test the hypothesis that the prolonged [Ca2+]i el-evations observed during the epileptogenic glutamate in-jury played a critical role in the development of theepileptic condition, we measured the [Ca2+]i response ofneurons in low-[Ca2+]e conditions known to inhibit glu-tamate injury–induced epileptogenesis (Fig. 2C). Neuro-nal [Ca2+]i rapidly increased in response to glutamatedespite the low-[Ca2+]e environment (Figs. 4C and 5C).However, [Ca2+]i was rapidly restored in low [Ca2+]e,and neurons did not manifest IRRC. [Ca2+]i was statis-tically elevated over baseline for only 16 min of record-ing (6 min after GLU; p < 0.05; n � 19; Fig. 5C). Thusextracellular Ca2+ contributed to the elevations in [Ca2+]i

observed during glutamate injury–induced epileptogen-esis.


associated with glutamate injury–inducedepileptogenesis were reduced by antagonism of theNMDA receptor

To test the hypothesis that the NMDA receptor medi-ated the prolonged, reversible increases in neuronal[Ca2+]i during epileptogenic glutamate injury, we moni-tored the neuronal [Ca2+]i response to epileptogenic glu-tamate in the presence of APV (50 �M). Similar to thelow-[Ca2+]e experiments, [Ca2+]i rapidly increased in re-sponse to glutamate despite antagonism by APV, as dem-onstrated by the representative neuron in Fig. 4D.Similar to the low-[Ca2+]e experiments, basal [Ca2+]i

FIG. 4. Representative pseudo-color digital images of Fura-2–loaded neurons during epileptogenic glutamate injury in the pres-ence of various extracellular Ca2+ conditions and glutamatereceptor antagonists. A: The representative control neuron didnot undergo changes in [Ca2+]i during the recording period. B–F:In contrast, neuronal [Ca2+]i increased on treatment with gluta-mate in all other experimental conditions, as indicated by thechange in color from blue to red. The representative neuronstreated in nonepileptogenic conditions of low [Ca2+]e (C) andN-methyl-D-aspartate receptor antagonism (D) restored basalneuronal [Ca2+]i within 60 and 30 min, respectively. In contrast,neuronal [Ca2+]i measured in epileptogenic conditions of gluta-mate alone (B) and in the presence of antagonists of AMPA/KAreceptors (E) and mGluRs (F) were elevated for �90 min.

FIG. 5. Alterations in neuronal [Ca2+]i in response to epilepto-genic glutamate injury in the presence of various extracellularCa2+ conditions and glutamate receptor antagonists. A: The[Ca2+]i in control neurons did not change during the recordingperiod (n = 11). B: In contrast, application of epileptogenic glu-tamate (20 µM, 10 min, black bar) caused a significant and per-sistent increase in neuronal [Ca2+]i that was statistically greaterthan basal values for 76 min (n = 15). C, D: Nonepileptogenicglutamate injury (black bar) in low [Ca2+]e (C, n = 19) and2-amino-5-phosphonovalerate (APV; D, n = 16) caused signifi-cant elevations in neuronal [Ca2+]i persisting for only 16 and 28min, respectively. E, F: Similar to glutamate injury alone (B),epileptogenic glutamate injury in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; E, n = 16) and S-�-methyl-4-carboxyphenylglycine (MCPG; F, n = 13) caused significant andpersistent elevations of neuronal [Ca2+]i for 90 and 100 min, re-spectively. *p < 0.05 repeated measures analysis of variance andTukey post hoc test. Data are represented by mean ± SEM.



also was rapidly restored in all neurons recorded (n �16). [Ca2+]i was significantly elevated over basal valuesfor only 28 min (18 min after GLU; p < 0.05; n � 16;Fig. 5D). Experiments performed in the presence ofMK-801 (10 �M) demonstrated similar results (data notshown). In MK-801, [Ca2+]i was significantly elevatedover basal values for only 12 min (2 min after GLU;p < 0.05; n � 17; data not shown), and all neuronsrestored resting [Ca2+]i. Thus activation of the NMDAreceptor was required for the prolonged elevations in[Ca2+]i observed during glutamate injury–induced epi-leptogenesis.


associated with glutamate injury–inducedepileptogenesis were not reduced by antagonism ofnon-NMDAR subtypes

Antagonism of AMPA and KA receptors, as well asantagonism of class I and II mGluRs subtypes had noinhibitory effect on glutamate injury–induced epilepto-genesis (Fig. 3). Likewise, pharmacologic blockade ofAMPA and KA receptors by CNQX (2 �M; Fig. 4E) andpharmacologic blockade of mGluRs by MCPG (250 �M;Fig. 4F) had no inhibitory effect on the prolonged eleva-tions in neuronal [Ca2+]i measured in response to epilep-togenic glutamate exposure. When exposed to glutamateand CNQX, neurons manifested prolonged elevations inneuronal [Ca2+]i (Fig. 5E). Although a subset of neuronsmanifested IRRC (four of 20), the majority of neuronsbuffered the alterations in [Ca2+]i over time. Neuronswere statistically elevated over basal levels, with exclu-sion of neurons manifesting IRRC, for 98 min (88 minafter GLU; p < 0.05; n � 16; Fig. 5E).

Similarly, neurons exposed to glutamate and MCPGmanifested prolonged elevations in neuronal [Ca2+]i (Fig.4F). Of these neurons, 28% manifested IRRC (five of18). The remaining neurons were statistically elevatedover basal level for 100 min (90 min after GLU; p < 0.05;n � 13; Fig. 5F). Thus inhibition of non-NMDAR sub-types of glutamate receptors did not reduce the durationof prolonged [Ca2+]i elevations in glutamate injury–induced epileptogenesis.

The induction of epileptogenesis correlated with theduration of the [Ca2+]i elevation and the total[Ca2+]i load

The experiments described demonstrated that extracel-lular Ca2+ and NMDAR activation were required for theinduction of glutamate injury–induced epileptogenesis.Further, Ca2+ imaging experiments revealed that epilep-togenic glutamate injury was associated with prolongedelevations of neuronal [Ca2+]i. Various parameters ofinjury-induced [Ca2+]i changes during different experi-mental treatments are summarized in Table 1, includingthe duration of statistically elevated [Ca2+]i (minutes),the total [Ca2+]i load as reflected by the area under thecurve (nanomoles per liter × minutes), and the peak in-crease in [Ca2+]i (percentage of basal). The total [Ca2+]i

load associated with the epileptogenic treatments of glu-tamate alone (50,890 ± 9,262 nM × min; n � 15), glu-tamate with CNQX (25,666 ± 5,074 nM × min; n � 16),and glutamate with MCPG (31,731 ± 6,058 nM × min; n� 13) was statistically greater than the total [Ca2+]i loadof the nonepileptogenic treatments of controls (62 ± 34nM × min; n � 11), glutamate in low [Ca2+]i (4,560 ±827 nM × min; n � 19), glutamate with APV (4,416 ±444 nM × min; n � 16), and glutamate with MK-801(520 ± 144 nM × min; n � 17). The peak increase inneuronal [Ca2+]i was largest in the normal epileptogenicglutamate treatment (983 ± 166%; n � 15). Treatmentconditions of controls (18 ± 10%; n � 11), low [Ca2+]e

(476 ± 80%; n � 19), MK-801 (162 ± 45%; n � 17),and CNQX (358 ± 102%; n � 16) were significantly lessthan those in the glutamate injury (p < 0.05; Table 1). Incontrast, the peak increase in the presence of APV (781± 123%; n � 16) and MCPG (521 ± 154%; n � 13) wasnot statistically different from glutamate alone. A statis-tically significant correlation was observed between theinduction of epileptogenesis and the duration of statisti-cally elevated [Ca2+]i (r � 0.95; p < 0.002). In addition,the induction of epileptogenesis and the total change inneuronal [Ca2+]i demonstrated a statistically significantcorrelation (r � 0.94; p < 0.002). In contrast, no statis-tical correlation was observed between epileptogenesis

TABLE 1. Quantification of [Ca2+]i changes during and after glutamate injury and the induction of epileptogenesis in varioustreatment conditions

TreatmentDuration of [Ca2+]i

elevation (min)Total [Ca2+]i load

(nM × min)Peak [Ca2+]i

(% of basal) Epilepsy (%, n)

Control 0 62 ± 64† 18 ± 10† No (%, 37)Glutamate (20 �M) 76 50890 ± 9262* 983 ± 166* Yes (90%, 30)††+Low [Ca2+]e 16 4560 ± 827† 476 ± 80† No (0%, 11)+APV (50 �M) 28 4416 ± 444† 781 ± 123* No (0%, 12)+MK-801 (10 �M) 12 520 ± 144† 162 ± 45† No (0%, 10)+CNQX (2 �M) 98 25666 ± 5074*† 358 ± 102† Yes (80%, 10)††+MCPG (250 �M) 100 31731 ± 6058* 521 ± 154 Yes (80%, 10)††

* p < 0.05 one way ANOVA and Tukey post-hoc test vs. Control.† p < 0.05 one way ANOVA and Tukey post-hoc test vs. Glutamate.†† p < 0.05 Fisher Exact Test vs. Control.



and the peak increase in neuronal [Ca2+]i (r � 0.46; p �0.305).


induced by non–glutamate-mediated mechanismswere not associated with the inductionof epileptogenesis

The previously described results in this study indi-cated that prolonged, reversible elevations in theNMDAR neuronal [Ca2+]i pathway were associated withthe induction of epileptogenesis. To test the hypothesisthat equal non–NMDAR-mediated [Ca2+]i elevations didnot initiate SREDs, we produced a prolonged, reversibleelevation in neuronal [Ca2+]i by using high extracellularpotassium solution to induce neuronal depolarization thatwas of similar duration and equal to or greater than theepileptogenic glutamate-mediated calcium response. Ex-posure of neurons to high extracellular potassium (50mM, 90 min) induced a statistically significant elevationin neuronal [Ca2+]i for the duration of the treatment (Fig.6A). The peak increase in [Ca2+]i during high potassiumexposure (958 ± 194%; n � 13) was not statisticallydifferent from epileptogenic glutamate treatments (GLUalone, GLU + CNQX, or GLU + MCPG). Likewise, thetotal calcium load during high extracellular potassiumexposure (35,508 ± 4,085 nM × min; n � 13) was not

statistically different from the epileptogenic conditionsof glutamate treatment alone and glutamate in the pres-ence of CNQX or MCPG. Although these parameters ofpeak increase and total calcium load for epileptogenicglutamate treatments and non-NMDA, potassium-induced calcium entry were not statistically different,and potassium-induced elevations in neuronal [Ca2+]i

were statistically increased for a similarly prolonged du-ration of 90 min, this treatment condition did not induceepileptogenesis (Fig. 6B; none; n � 10). This resultdemonstrated that the equal non–NMDAR-mediated[Ca2+]i elevations did not produce SREDs.

DISCUSSION

The Ca2+ hypothesis of epileptogenesis suggests thatprolonged elevations in [Ca2+]i play a role in mediatingsome of the long-term plasticity changes associated withepileptogenesis and the persistent manifestation of sei-zure activity (7,8). In addition, activation of the Ca2+-permeable NMDAR has been associated withepileptogenesis (10–14) and has been implicated as amajor source of the epileptogenic Ca2+ signal (8). Theresults of this investigation strongly support this Ca2+

hypothesis of epileptogenesis as an initiating mechanismin glutamate injury–induced epileptogenesis. Glutamateinjury (20 �M, 10 min) causing epileptogenesis in 90%of neurons (n � 30; Figs. 1B, 2B, and 3) was associatedwith prolonged elevations in neuronal [Ca2+]i (Figs. 4Band 5B). This elevation was statistically significant for>1 h (Fig. 5B; Table 1). Similar elevations in neuronal[Ca2+]i were observed when glutamate injury was per-formed in the presence of the AMPA- and KA-receptorantagonist, CNQX, and in the presence of the mGluRantagonist, MCPG (Fig. 5E and F; Table 1). Of neuronsrecorded after epileptogenic glutamate injury in CNQXor MCPG, 80% exhibited epileptiform activity (Figs. 2Fand G and 3). The results provide direct evidence thatpersistent elevations in [Ca2+]e after glutamate exposurecontribute to the development of persistent epileptiformdischarges in the in vitro, glutamate injury–induced epi-leptogenesis model of stroke-induced “epilepsy.”

In contrast, experimental treatment conditions that re-duced the duration of statistically elevated [Ca2+]i

likewise inhibited the development of epilepsy. In low-[Ca2+]e solutions (2.0 mM reduced to 0.2 mM), neuronal[Ca2+]i was statistically elevated over basal levels foronly 16 min (Fig. 5C). Antagonism of the NMDAR withAPV (Fig. 5D) and MK-801 reduced the duration ofstatistically elevated [Ca2+]i to 28 and 12 min, respec-tively (Table 1). Twenty-four hours after glutamate in-jury in these experimental conditions, neuronal activitywas similar to that of controls (Fig. 2A, C, and E), andthe induction of epileptogenesis was completely blocked(Fig. 3). Thus the prolonged elevations in [Ca2+]i asso-

FIG. 6. Prolonged elevations in [Ca2+]i by exposure to high ex-tracellular potassium did not induce epileptogenesis. A: Culturedhippocampal neurons (n = 13) exposed to a high-potassium (50mM, 90 min, black bar) solution underwent statistically significant,reversible elevations in [Ca2+]i. B: A representative whole-cellcurrent-clamp recording from neurons 24 h after exposure tohigh-potassium solutions manifested spontaneous postsynapticpotentials and occasional action potentials similar to those ofcontrol neurons. Neurons exposed to high potassium never mani-fested spontaneous, recurrent, epileptiform discharges (SREDS;n = 10). *p < 0.05 repeated measures analysis of variance andTukey post hoc test. Data are represented by mean ± SEM.



ciated with epileptogenesis required extracellular Ca2+

and NMDAR activation. These data, taken with the posi-tive correlation between the induction of epileptogenesisand the duration of the [Ca2+]i elevation, as well as thepositive correlation between the induction of epilepto-genesis and the total [Ca2+]i load, indicate that Ca2+ in-flux through the NMDAR and prolonged elevations in[Ca2+]i were required for the induction of epileptogenesisby glutamate injury.

As described previously (5), glutamate injury–inducedepileptogenesis required an injury sufficient to produceplasticity changes, but not so severe that it caused celldeath. In the present study, we demonstrated that gluta-mate injury–induced epileptogenesis required parametersof total calcium load and duration of significantly el-evated calcium sufficient to produce long-lasting hyper-excitability, but not sufficient to produce neuronal death.Various combinations of increased duration or total loadof [Ca2+]i could produce a reversible injury sufficient toproduce a reversible injury that induced epileptogenesiswithout reaching the threshold for the induction of IRRCand delayed excitotoxic neuronal death (20). In additionto the increase in [Ca2+]i, the pathway of Ca2+ entry wasshown to be important (Fig. 6). An elevation in [Ca2+]i

induced by exposure to high extracellular potassiumequal in duration and load to the glutamate-induced el-evations did not produced epileptogenesis, demonstrat-ing that the route of Ca2+ entry was also important.

These data suggest a continuum of NMDAR–Ca2+

transduction during neuronal injury ranging from com-plete recovery of function, to neuronal survival with per-sistently altered function and, finally, to neuronal death.Experimental conditions that inhibited the NMDAR–Ca2+ transduction pathway, as indicated by short-duration elevations in [Ca2+]i (low [Ca2+]e, APV, andMK-801; Fig. 5C and D), manifested neither altered neu-ronal function in the form of SREDs (Fig. 2C and E) norneuronal death in the form of IRRC, a short-term hall-mark of excitotoxicity (20). In contrast, experimentalconditions that permitted activation of the NMDAR–Ca2+ transduction pathway (glutamate alone, CNQX, andMCPG), as evidenced by prolonged, but reversible alter-ations in Ca2+ homeostasis, induced epileptogenesis. Inaddition, a small subset of neurons in these epileptogenicexperimental conditions of glutamate, CNQX, andMCPG (29, 20, and 28%, respectively) manifestedNMDAR–Ca2+-dependent IRRC, and underwent de-layed excitotoxic neuronal death, died (20).

Ca2+ is an important second messenger in neurons thattriggers a number of Ca2+-dependent signaling pathwaysincluding the modulation of enzyme systems and genetranscription (21,33,34) that may be altered during epi-leptogenesis. For example, Ca2+/calmodulin kinase II ac-tivity is depressed in a number of whole animal and invitro models of epilepsy (35–39). Alterations in the lev-

els of many messenger RNAs and proteins also havebeen characterized in models of epileptogenesis (40,41).The data in this investigation demonstrate that glutamateinjury performed in Ca2+-free, 2.0 mM extracellular Ba2+

solutions did not induce epileptogenesis (Figs. 2D and 3)and indicate that the Ca2+ influx through the NMDARduring epileptogenic glutamate injury initiated someCa2+-sensitive second-messenger system necessary forthe induction of epilepsy.

Because NMDAR activation, cytoplasmic Ca2+ sig-nals, and nuclear Ca2+ signals can regulate gene tran-scription through multiple Ca2+-activated enzyme andtranscription factor pathways (42–44), the prolongedelevations in neuronal [Ca2+]i and activation of theNMDAR–Ca2+ transduction pathway may underlie po-tential alterations in enzyme regulation and gene expres-sion during glutamate injury–induced epileptogenesis.Long-term modulation of gene expression in epilepsyhas implicated as a molecular mechanism mediating thelong-term plasticity changes associated with epilepto-genesis (40,45,46). Persistent increases in DNA bindingand expression of serum response factor and �fosB havebeen associated with long-term plasticity changes in thepilocarpine model of epilepsy (45,46). Because thesetranscription factors are activated by the NMDA–Ca2+

transduction pathway, it is reasonable to suggest that theNMDAR-mediated Ca2+ changes observed in the gluta-mate injury model in the present study induce epilepto-genesis through the long-term modulation of geneexpression. Future studies will investigate the role ofCa2+-activated gene expression in glutamate injury–induced epileptogenesis.

In summary, brain injuries like stroke have been as-sociated with glutamate excitotoxicity wherein excessiveactivation of the NMDAR leads to an irreversible over-load of calcium and neuronal death (9). This investiga-tion demonstrated that a less severe glutamate exposurethat induced epileptogenesis in surviving neurons alsocauses prolonged elevations in neuronal [Ca2+]i that aredependent on the presence of extracellular Ca2+ and ac-tivation of the NMDAR. In this situation, rather thantriggering excitotoxic neuronal death pathways, calciumactivates Ca2+-regulated pathways that initiate long-termplasticity changes in neurons that ultimately lead to thedevelopment of epilepsy.

Acknowledgment: This study was supported by NIH grantsRO1-NS23350 (R. J. DeLorenzo), P50-NS25630 (R. J. De-Lorenzo), NS07288 (D. A. Sun), and a GLENN/American Fed-eration for Aging Research Grant (D. A. Sun).

REFERENCES

1. Delgado-Escueta AV. Symptomatic lesional epilepsies: introduc-tion. In: Delgado-Escueta AV, Wilson WA, Olsen RW, et al., eds.Jasper’s basic mechanisms of the epilepsies. Philadelphia: Lippin-cott Williams & Wilkins, 1999:433–6.



2. Hauser WA, Hesdorffer DC. Epilepsy: frequency, causes and con-sequences. New York: Demos, 1990.

3. DeLorenzo RJ. The challenging genetics of epilepsy. Epilepsy ResSuppl 1991;4:3–17.

4. Hauser WA, Annegers JF, Kurland LT. Prevalence of epilepsy inRochester, Minnesota: 1940-1980. Epilepsia 1991;32:429–45.

5. Sun DA, Sombati S, DeLorenzo RJ. Glutamate injury-induced epi-leptogenesis in hippocampal neurons: an in vitro model of stroke-induced “epilepsy.” Stroke 2001;32:2344–350.

6. Witte OW, Bidmon HJ, Schiene K, et al. Functional differentiationof multiple perilesional zones after focal cerebral ischemia. JCereb Blood Flow Metab 2000;20:1149–65.

7. Perlin JB, DeLorenzo RJ. Calcium and epilepsy. In: Pedley TA,Meldrum BS, eds. Recent advances in epilepsy. Edinburgh: Chur-chill Livingstone, 1992:15–36.

8. DeLorenzo RJ, Pal S, Sombati S. Prolonged activation of theN-methyl-D-aspartate receptor-Ca2+ transduction pathway causesspontaneous recurrent epileptiform discharges in hippocampalneurons in culture. Proc Natl Acad Sci U S A 1998;95:14482–7.

9. Choi DW. Glutamate receptors and the induction of excitotoxicneuronal death. Prog Brain Res 1994;100:47–51.

10. Dingledine R, McBain CJ, McNamara JO. Excitatory amino acidreceptors in epilepsy. Trends Pharmacol Sci 1990;11:334–8.

11. Rice AC, DeLorenzo RJ. NMDA receptor activation during statusepilepticus is required for the development of epilepsy. Brain Res1998;782:240–7.

12. Croucher MJ, Bradford HF. NMDA receptor blockade inhibitsglutamate-induced kindling of the rat amygdala. Brain Res 1990;506:349–52.

13. Croucher MJ, Bradford HF, Sunter DC, et al. Inhibition of thedevelopment of electrical kindling of the prepyriform cortex bydaily focal injections of excitatory amino acid antagonists. Eur JPharmacol 1988;152:29–38.

14. Stasheff SF, Anderson WW, Clark S, et al. NMDA antagonistsdifferentiate epileptogenesis from seizure expression in an in vitromodel. Science 1989;245:648–51.

15. Banker GA, Cowan WM. Rat hippocampal neurons in dispersedcell culture. Brain Res 1977;126:397–42.

16. Hamill OP, Marty A, Neher E, et al. Improved patch-clamp tech-niques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981;391:85–100.

17. Matsumoto H, Amjone-Marsan C. Cortical cellular phenomena inexperimental epilepsy: interictal manifestations. Exp Neurol 1964;9:286–304.

18. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+indicators with greatly improved fluorescence properties. J BiolChem 1985;260:3440–50.

19. Pal S, Sun D, Limbrick D, et al. Epileptogenesis induces long-termalterations in intracellular calcium release and sequestrationmechanisms in the hippocampal neuronal culture model of epi-lepsy. Cell Calcium 2001;30:285–96.

20. Limbrick DD Jr, Churn SB, Sombati S, et al. Inability to restoreresting intracellular calcium levels as an early indicator of delayedneuronal cell death. Brain Res 1995;690:145–56.

21. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem1987;56:395–433.

22. Berridge MJ. Neuronal calcium signaling. Neuron 1998;21:13–26.23. Nelson MT, French RJ, Krueger BK. Voltage-dependent calcium

channels from brain incorporated into planar lipid bilayers. Nature1984;308:77–80.

24. Mayer ML, Westbrook GL. Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cul-tured central neurones. J Physiol 1987;394:501–27.

25. Olwin BB, Storm DR. Calcium binding to complexes of calmod-

ulin and calmodulin binding proteins. Biochemistry 1985;24:8081–6.

26. Chao SH, Suzuki Y, Zysk JR, et al. Activation of calmodulin byvarious metal cations as a function of ionic radius. Mol Pharmacol1984;26:75–82.

27. Davies J, Watkins JC. Actions of D and L forms of 2-amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate in the catspinal cord. Brain Res 1982;235:378–86.

28. Wong EH, Kemp JA, Priestley T, et al. The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl AcadSci U S A 1986;83:7104–8.

29. Dingledine R, Borges K, Bowie D, et al. The glutamate receptorion channels. Pharmacol Rev 1999;51:7–61.

30. Honore T, Davies SN, Drejer J, et al. Quinoxalinediones: potentcompetitive non-NMDA glutamate receptor antagonists. Science1988;241:701–3.

31. Conn PJ, Pin JP. Pharmacology and functions of metabotropicglutamate receptors. Annu Rev Pharmacol Toxicol 1997;37:205–37.

32. Watkins J, Collingridge G. Phenylglycine derivatives as antago-nists of metabotropic glutamate receptors. Trends Pharmacol Sci1994;15:333–42.

33. Bootman MD, Lipp P, Berridge MJ. The organisation and func-tions of local Ca(2+) signals. J Cell Sci 2001;114:2213–22.

34. West AE, Chen WG, Dalva MB, et al. Calcium regulation ofneuronal gene expression. Proc Natl Acad Sci U S A 2001;98:11024–31.

35. Perlin JB, Churn SB, Lothman EW, et al. Loss of type II calcium/calmodulin-dependent kinase activity correlates with stages of de-velopment of electrographic seizures in status epilepticus in rat.Epilepsy Res 1992;11:111–8.

36. Bronstein JM, Micevych P, Popper P, et al. Long-lasting decreasesof type II calmodulin kinase expression in kindled rat brains. BrainRes 1992;584:257–60.

37. Bronstein JM, Farber DB, Micevych PE, et al. Kindling inducedchanges in calmodulin kinase II immunoreactivity. Brain Res1990;524:49–53.

38. Wasterlain CG, Bronstein JM, Morin AM, et al. Translocation andautophosphorylation of brain calmodulin kinase II in status epilep-ticus. Epilepsy Res Suppl 1992;9:231–8.

39. Blair RE, Churn SB, Sombati S, et al. Long-lasting decrease inneuronal Ca2+/calmodulin-dependent protein kinase II activity in ahippocampal neuronal culture model of spontaneous recurrent sei-zures. Brain Res 1999;851:54–65.

40. DeLorenzo RJ, Morris TA. Long-term modulation of gene expres-sion in epilepsy. Neuroscientist 1999;5:86–99.

41. Bading H. Nuclear calcium-activated gene expression: possibleroles in neuronal plasticity and epileptogenesis. Epilepsy Res 1999;36:225–31.

42. Platenik J, Kuramoto N, Yoneda Y. Molecular mechanisms asso-ciated with long-term consolidation of the NMDA signals. Life Sci2000;67:335–64.

43. Hardingham GE, Chawla S, Johnson CM, et al. Distinct functionsof nuclear and cytoplasmic calcium in the control of gene expres-sion. Nature 1997;385:260–5.

44. Ghosh A, Greenberg ME. Calcium signaling in neurons: molecularmechanisms and cellular consequences. Science 1995;268:239–47.

45. Morris TA, Jafari N, Rice AC, et al. Persistent increased DNA-binding and expression of serum response factor occur withepilepsy-associated long-term plasticity changes. J Neurosci 1999;19:8234–43.

46. Morris TA, Jafari N, DeLorenzo RJ. Chronic �FosB expressionand increased AP-1 transcription factor binding are associated withthe long-term plasticity changes in epilepsy. Mol Brain Res 2000;79:138–49.



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Calcium-dependent Epileptogenesis in an In Vitro Model of Stroke-induced “Epilepsy”