1Bloorview Epilepsy Research Laboratory, 2Playfair Neuroscience Unit, 3Department of Medicine (Neurology) and 4Department of Physiology, University of Toronto, Toronto, Ontario M5T 2S8, Canada
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ABSTRACT |
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Pelletier, Marc R., Jehangir S. Wadia, Linda R. Mills, and Peter L. Carlen. Seizure-induced cell death produced by repeated tetanic stimulation in vitro: possible role of endoplasmic reticulum calcium stores. Seizures may cause brain damage due to mechanisms initiated by excessive excitatory synaptic transmission. One such mechanism is the activation of death-promoting intracellular cascades by the influx and the perturbed homeostasis of Ca2+. The neuroprotective effects of preventing the entry of Ca2+ from voltage-dependent Ca2+ channels, NMDA receptors, and non-NMDA receptors, is well known. Less clear is the contribution to excitotoxicity of Ca2+ released from endoplasmic reticulum (ER) stores. We produced epileptiform discharges in combined entorhinal cortex/hippocampus slices using repeated tetanic stimulation of the Schaffer collaterals and assessed cell death after 1, 3, or 12-14 h with gel electrophoresis of genomic DNA and immunohistologically using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) staining. We manipulated ER Ca2+ stores using two conventional drugs, dantrolene, which blocks the Ca2+ release channel, and thapsigargin, which blocks sarco-endoplasmic reticulum Ca2+-ATPases resulting in depletion of ER Ca2+ stores. To monitor epileptogenesis, and to assess effects attributable to dantrolene and thapsigargin on normal synaptic transmission, extracellular potentials were recorded in stratum pyramidale of the CA1 region. Repeated tetanic stimulation reliably produced primary afterdischarge and spontaneous epileptiform discharges, which persisted for 14 h, the longest time recorded. We did not observe indications of cell death attributable to seizures with either method when assessed after 1 or 3 h; however, qualitatively more degraded DNA always was observed in tetanized slices from the 12- to 14-h group compared with time-matched controls. Consistent with these data was a significant, fourfold, increase in the percentage of TUNEL-positive cells in CA3, CA1, and entorhinal cortex in tetanized slices from the 12- to 14-h group (16.5 ± 4.4, 33.7 ± 7.1, 11.6 ± 2.1, respectively; means ± SE; n = 7) compared with the appropriate time-matched control (4.1 ± 2.2, 7.3 ± 2.0, 2.8 ± 0.9, respectively; n = 6). Dantrolene (30 µM; n = 5) and thapsigargin (1 µM; n = 4) did not affect significantly normal synaptic transmission, assessed by the amplitude of the population spike after 30 min of exposure. Dantrolene and thapsigargin also were without effect on the induction or the persistence of epileptiform discharges, but both drugs prevented seizure-induced cell death when assessed with gel electrophoresis. We suggest that Ca2+ entering a cell from the outside, in addition to the Ca2+ contributed from ryanodine-sensitive stores (i.e., Ca2+-induced Ca2+ release), may be necessary for seizure-induced cell death.
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INTRODUCTION |
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Pathology of the brain attributable to epileptic seizures has been
reported as a consequence of prolonged febrile seizures (Aicardi
and Chevrie 1970; Verity et al. 1993
),
complex partial seizures (Babb et al. 1984
;
Duncan and Sagar 1987
), and status epilepticus
(Corsellis and Bruton 1983
; Lothman
1990
). Seizure-induced brain damage results in death of
susceptible cell types and fibrillary gliosis, which occurs typically
in hippocampus but also in other structures including dentate gyrus,
cerebellum, amygdala, and neocortex (reviewed in Honavar and
Meldrum 1997
). Consistent with these clinical observations are
studies describing brain damage produced by experimentally induced
seizures (Ben-Ari 1985
; Cavazos et al.
1994
; Lothman and Collins 1981
; Meldrum
et al. 1973
; Sloviter and Damiano 1981
;
Thompson et al. 1996
; Vicedomini and Nadler 1990
).
Substantial evidence implicates cellular cascades initiated by the
influx and the perturbed intracellular homeostasis of Ca2+
in excitotoxic cell death (Choi 1988; Meldrum and
Garthwaite 1990
; Nicotera and Orrenius 1992
).
The neuroprotective effects of preventing the entry of Ca2+
from a variety of sources including voltage-dependent Ca2+
channels (VDCC) (Siesjo 1990
; Siesjo and
Bengtsson 1989
), N-methyl-D-aspartate (NMDA) receptors (Clifford et al. 1989
;
Garthwaite and Garthwaite 1989
), and non-NMDA receptors
(Brorson et al. 1994
; Penix and Wasterlain
1994
) is well known. Less clear is the contribution to cell
death of Ca2+ released from endoplasmic reticulum (ER) stores.
Neuronal Ca2+ signaling mediated by stores associated with
the ER regulates a wide variety of processes including excitability, synaptic plasticity, and gene transcription (reviewed in Alkon et al. 1998; Berridge 1998
). Two types of
intracellular stores identified in neurons and associated with the ER
are the inositol 1,4,5-trisphosphate (IP3)-and the
ryanodine-sensitive stores (Ehrlich et al. 1994
;
Henzi and MacDermott 1992
; McPherson et al.
1991
). The two major classes of receptor/channel complexes that
mediate Ca2+ release from ER stores share considerable
structural and molecular similarity (Coronado et al.
1994
; McPherson et al. 1991
; Mignery et
al. 1990
). Thus far, four IP3 receptor
(IP3R1-4) and three ryanodine receptor
(RYRI-III) isoforms have been identified (reviewed in
Berridge 1993
; Simpson et al. 1995
). The
molecular diversity is due to the existence of separate genes, and for
type 1 IP3Rs, alternative splicing.
The refilling state of the stores determines whether they function as a
sink or a source of Ca2+. When stores are replete,
Ca2+ entry can promote the release of Ca2+ from
ryanodine-sensitive stores, referred to as Ca2+-induced
Ca2+ release (CICR), which amplifies and prolongs the
cytosolic Ca2+ transient (Clapham 1995;
Irving et al. 1992
; Simpson et al. 1995
). Prolonged depolarization effectively activates CICR as observed in
sympathetic ganglion neurons (Kuba et al. 1992
) and
cerebellar purkinje cells (Llano et al. 1994
) and with
epileptiform discharges in both hippocampal slices (Albowitz et
al. 1997
) and cultured hippocampal neurons (Segal and
Manor 1992
).
We produced epileptiform discharges, similar to stimulus train-induced
bursting (STIB) (Stasheff et al. 1985), with repeated tetanic stimulation in entorhinal cortex/hippocampus slices and manipulated ER Ca2+ stores using two conventional drugs,
dantrolene and thapsigargin. Dantrolene blocks the Ca2+
release channel and is cytoprotective in hepatoma 1C1C7 cells (Dypbukt et al. 1990
), cultured cortical neurons
(Frandsen and Schousboe 1991
), and CA1 pyramidal neurons
(Wei and Perry 1996
). Thapsigargin inhibits the sarco-ER
Ca2+-ATPase (SERCA) pumps that participate in refilling the
stores resulting in an increase in cytosolic Ca2+, which
persists for several minutes (Thastrup et al. 1990
;
Thomas and Hanley 1994
; Treiman et al.
1998
). Both apoptosis of thymocytes (Jiang et al.
1994
; Waring and Beaver 1996
) and
neuroprotection of sympathetic neurons (Lampe et al.
1992
) and cultured cerebellar granule cells (Levick et
al. 1995
; Lin et al. 1997
) have been reported
for thapsigargin. In this paper, we demonstrate seizure-induced cell
death in vitro, which was prevented by both dantrolene and thapsigargin.
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METHODS |
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Slice preparation
Male Wistar rats (30-65 days of age) were anesthetized with
2-Bromo-2-chloro-1,1,1-trifluoroethane (Halothane; Halocarbon Laboratories, River Edge, NJ) and then decapitated. The brain was
removed rapidly and placed for 1 min in ice-cold, oxygenated (95%
O2-5% CO2) sucrose-based artificial
cerebrospinal fluid (ACSF) containing (in mM): 210 sucrose, 26 NaHCO3, 3.5 KCl, 1 CaCl2, 4 MgCl2,
1.25 NaH2PO4, and 10 glucose. Because of the
long duration of these experiments, to promote slice viability we used
a sucrose-based, high Mg2+-containing ACSF during the
preparation of slices to reduce dissection-induced damage and
Na+-dependent excitotoxicity (Rafiq et al.
1993
; Rasmussen and Aghajanian 1990
). The brain
was hemisected with a midsagittal cut, then the cerebellum and the
forebrain were removed. Finally, the dorsal cortex was cut parallel to
the longitudinal axis, and the remaining block of brain was glued
dorsal surface down, caudal end facing the blade, to an aluminum chuck,
which was secured at a 12° angle. To ensure reliability of the
cutting angle, a line subtending a 12° angle from the front corner of
the chuck was scored on the side of the chuck. Brain slices (500 µm)
comprising both the entorhinal cortex and the hippocampus then were
prepared using a Vibratome. We employed this method of slice
preparation because sustained epileptiform discharges are provoked
reliably in slices prepared with this orientation (Jones and
Heinemann 1988
; Rafiq et al. 1993
;
Walther et al. 1986
), which might be attributable to
intrinsic reverbatory circuitry in the entorhinal cortex and the
frequency-dependent transfer of this neuronal activity to the
hippocampus via the dentate gyrus (Iijima et al. 1996
).
The ACSF for the incubation period and the recording of
electrophysiological responses contained (in mM) 125 NaCl, 26 NaHCO3, 5 KCl, 2 CaCl2, 0.5 MgCl2,
1.25 NaH2PO4, and 10 glucose. After a minimum
of 1 h, slices were transferred to an interface-type chamber and
perfused continuously (2-4 ml/min) with oxygenated ACSF. Time-matched,
untetanized control slices were included in the recording chamber with
slices receiving tetanic stimulation. Therefore the only difference in
the treatment of the slices was the application or the absence of
tetanic stimulation. A maximum of four slices (2 tetanized/2 control)
were used from an individual rat.
Electrophysiology
To monitor epileptogenesis in the slice, extracellular responses
were recorded in the stratum pyramidale of CA1 with NaCl-filled (150 mM; 2-4 M) microelectrodes. Orthodromic responses were evoked via a
bipolar stimulating electrode (enamel-insulated nichrome wire; 125 µm
diam) placed in the stratum radiatum. Signals were recorded, amplified,
and filtered (3 kHz) with an Axoclamp 2A amplifier in bridge mode.
Input/output relations were determined by varying the amplitude of
100-µs pulses and acquired using the CLAMPEX program of pClamp
version 6.0 software (Axon Instruments, Foster City, CA). The slice
then was tetanized once every 10 min: 100-Hz, 2-s train duration at
twice the threshold intensity for evoking a population spike of 1 mV,
total of 10 episodes. To permit full recovery of synaptic function
after evoking a primary afterdischarge (PAD), orthodromic responses
were not evoked between episodes of tetanic stimulation
(Anderson et al. 1990
). Experiments also were recorded
on video tape (Instrutech Corporation; VR-10) and digitized using
software (WCP V1.2) provided by Dr. John Dempster, University of
Strathclyde. Seizure-induced cell death was assessed at three time
points; 1, 3, and 12-14 h after the 10th tetanization. Orthodromic
responses were evoked at these time points, and only slices where
orthodromic responses could be evoked were included in the analysis.
Experiments were conducted at 34°C.
Gel electrophoresis
The integrity of DNA from tetanized and time-matched control
slices was determined by size fractionation in agarose gels. Slices
designated for gel electrophoresis were stored at 80°C. Genomic DNA
was isolated from slices using a conventional protocol (Easy-DNA Kit,
Invitrogen, San Diego, CA). Two slices, from the same rat and in the
same condition, were pooled. Briefly, cells were lysed with lysis
buffer then proteins and lipids were precipitated. DNA was extracted
with Tris-saturated phenol/chloroform (1:1) and centrifuged at 13,000 g for 20 min followed by reextraction with chloroform only
and further centrifuging. The DNA then was precipitated with ethanol,
resuspended in tris ethylenediamine tetraacetic acid (TE; 10:1, pH
8.0) buffer, then treated with DNase-free RNase (40 µg/ml).
The amount of DNA in each sample (10-25 µg) was determined with a
spectrophotometer (Perkin Elmer Lambda 3B). Samples were heated at
65°C for 10 min, centrifuged for 2 min, then put on ice for 1 min
before loading. Approximately 5 µg of DNA from each sample was
electrophoresed through 2.0% agarose in Tris borate buffer. DNA was
visualized by ethidium bromide staining and photographed under UV
illumination. For a positive control, organotypic hippocampal slice
cultures were prepared as described previously (Perez Velazquez
et al. 1997
; Stoppini et al. 1991
) and exposed
for 3 or 6 h to actinomycin D (5 µg/ml), a potent inducer of
apoptosis in a variety of cells (Martin et al. 1990
).
Immunohistochemistry
Slices designated for immunohistochemistry were fixed in 10%
buffered formaldehyde, embedded in paraffin, then resectioned (5 µm).
Sections were taken 100 µm below the slice surface then mounted on
glass slides. In situ labeling of new 3'-OH DNA ends generated by DNA
fragmentation was performed using terminal deoxynucleotidyl transferase
(TdT)-mediated deoxyuridine 5'-triphosphate (dUTP) nick end labeling
(TUNEL; in situ cell death detection kit, Boehringer Mannheim).
Briefly, sections were deparaffinized, rehydrated through a graded
series of ethanols, then washed in phosphate-buffered saline (PBS).
Sections then were treated with Proteinase K (20 µg/ml; Boehringer
Mannheim) for 30 min at room temperature and then washed with PBS.
Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide
in methanol for 30 min at room temperature. Positive control sections
were treated with DNaseI (1 µg/ml; Pharmacia Biotech) for 10 min at
room temperature. DNaseI was dissolved by gentle inversion in DN
reaction buffer that contained 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, and 50 µg/ml BSA. Sections were washed with PBS
and TdT-fluorescein was added to the sections, which then were covered
with Parafilm and incubated in a humidified chamber for 60 min at
37°C. Negative control sections were incubated without TdT. After
washing with PBS, fluorescein-labeled DNA strand breaks were analyzed
under a confocal microscope (Biorad MRC600; excitation wavelength, 488 nm). If fluorescent cells were observed, an antifluorescein antibody
FAB fragment conjugated with horse-radish peroxidase was applied to the
slice, covered with Parafilm, and incubated in a humidified chamber for
30 min at 37°C. Labeling was then visualized with metal-enhanced
diaminobenzidine (Boehringer Mannheim). Some sections were
counterstained lightly with cresyl violet and coverslipped for
assessment of morphology of TUNEL-positive cells.
TUNEL-positive cells were counted either visually or with the use of Imagetool version 1.28 software (University of Texas Health Science Center in San Antonio) by scorers that were blind to the treatment condition. The two methods of scoring produced close to identical results and the interrater reliability was r = 0.92. Scoring was done in five regions of the section and were defined as follows: DG, both superior and inferior blades of the granule cell layer; hilus (H), area lying within the two blades of the DG and delimited by a line connecting the ends of the blades of the DG; CA3, stratum pyramidale commencing from a line connecting the blades of the DG to a line level with the superior blade of the DG; CA1, stratum pyramidale (excluding CA2) extending to the subiculum; and entorhinal cortex (EC), a region the width of the DG extending from the hippocampal fissure to the peripheral limit of the section. Cells were counted and expressed as a percentage of the average number of cells per region determined from slices (n = 4) fixed and stained with hematoxylin/eosin immediately after sectioning with the Vibrotome.
Drugs
Dantrolene sodium (30 µM; Sigma) and thapsigargin (1 µM; Sigma) were dissolved in dimethyl sulfoxide (DMSO). Actinomycin D-mannitol (5 µg/ml; Sigma) was dissolved in purified water. All drugs were stored frozen as aliquots in stock solutions. Final concentration of DMSO was 0.05%.
Statistics
Differences in the percentage of TUNEL-positive cells were determined using a 5 (region) × 2 (tetanized/control) factorial ANOVA (SPSS version 8.0). Post hoc analyses were conducted with the Scheffé test. To assess the effect of dantrolene and thapsigargin on normal synaptic transmission, population spikes were evoked at a frequency of 0.05 Hz and after a stable baseline had been achieved (typically 15 min), the drugs were applied. Three responses, at the end of the baseline period and after 30 min of drug application, were averaged and the population spike amplitude was measured using the CLAMPFIT program of pClamp version 6.0 software (Axon Instruments). Differences in population spike amplitude attributable to the application of drugs were determined using a paired t-test. Significance was determined at P < 0.05. Results are presented as the means ± SE.
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RESULTS |
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Electrophysiology
PADs were produced reliably by tetanic stimulation in all
experiments. As illustrated in Fig. 1,
PAD increased progressively in duration and amplitude with repeated
tetanization. The duration of PAD after the 10th tetanic stimulation
ranged from 40 to 140 s. In contrast to previous reports using
this preparation (Rafiq et al. 1993, 1995
),
where secondary afterdischarge (SAD) was observed in 85% of
experiments, we observed SAD in only 29% of experiments. Consistent
with intense, prolonged epileptiform activity, we observed episodes of
spreading depression (SD) after tetanic stimulation in 46% of
experiments, which consisted of a negative DC potential shift (10-25
mV) during which time the slice was synaptically quiescent. Spontaneous
epileptiform discharges typically emerged during the recovery from SD.
A representative record of SD is presented in Fig. 1B.
|
Input/output relations included four to five stimulation intensities:
weak stimulation produced a small-amplitude, positive-deflecting, postsynaptic potential (PSP) that increased in amplitude with increasing stimulation intensity. As seen in Fig.
2A, before tetanization, suprathreshold stimulation for evoking a population spike produced a
single population spike. After the 10th tetanization, responses evoked
at all intensities were of greater amplitude and longer duration
compared with control responses, with multiple population spikes,
characteristic of epileptiform responses (Fig. 2B). We could
record epileptiform responses in tetanized slices for 14 h, the
longest time assessed. Representative responses from a long-duration
recording are presented in Fig. 2C. We also recorded spontaneous epileptiform discharges from slices that received tetanic
stimulation. Spontaneous epileptiform discharges were observed as early
as after the second tetanization but typically were observed after the
fifth tetanization and persisted for the duration of the experiment
(
14 h). A representative record of spontaneous epileptiform
discharges is presented in Fig. 2D.
|
In contrast to the responses evoked in slices receiving tetanic
stimulation, responses evoked from time-matched slices not receiving
tetanic stimulation were not epileptiform and were similar to the
responses evoked at the beginning of the experiment. In the record
presented in the inset in Fig. 2C, a
small-amplitude, second population spike is seen after 13 h.
Additionally, spontaneous epileptiform discharges were never recorded
in control slices. These experiments demonstrate evoked and spontaneous
epileptiform discharges were produced reliably in entorhinal
cortex/hippocampus slices with repeated tetanic stimulation, responses
evoked in time-matched controls not receiving tetanic stimulation were
not epileptiform, and both epileptiform responses from tetanized slices and physiological responses from time-matched control slices, could be
recorded for 14 h after the tetanization protocol, the longest time
we measured.
Gel electrophoresis
The integrity of DNA was preserved from slices assessed at
the early time points, 1 h (tetanized, n = 4;
time-matched control, n = 4) and 3 h (tetanized,
n = 4; time-matched control, n = 4) after the tetanization protocol (data not shown). In contrast, we
observed indications of internucleosomal DNA degradation in 9/11 slices
receiving repeated tetanic stimulation and assessed 12-14 h later.
Because endogenous Ca2+-dependent endonucleases cleave DNA
in regular 180- to 200-bp pieces, the classical profile of apoptosis in
gel electrophoresis is a regular pattern of banding referred to as
"DNA laddering" (Wyllie et al. 1980). The results
of the DNA fragmentation assay we performed did not produce the typical
banding pattern consistent with apoptosis but one more representative
of necrosis. The results of the DNA fragmentation assay we employed may
have been obscured by the overwhelming presence of intact DNA from
normal cells. The appearance of degraded DNA in our study ranged from a
smear of low-molecular-weight fragments (<100-400 bp) to a more
diffuse smear and a few discernable bands. We observed no degradation (8/11), or qualitatively less (3/11), degradation of DNA isolated from
12 to 14 h time-matched control slices compared with tetanized slices. A representative example of the degradation in the DNA isolated
from tetanized slices after 12-14 h is presented in Fig. 3. Interestingly, in the long-duration
experiments where DNA degradation was not observed (2/11), repeated
tetanic stimulation potentiated greatly the amplitude of the population
spike but did not produce epileptiform responses, suggesting that the
damage we observed was a direct result of the sustained epileptiform
discharges and not attributable to the persistent presence of the
stimulating electrode.
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Immunohistochemistry
To determine whether the degraded DNA we observed with
electrophoresis was expressed as region-specific cell death, as
reported previously for the CA3 region after intraamygdaloid infusion
of kainate (Pollard et al. 1994), we assessed the
percentage of TUNEL-positive cells in each of the five regions
described above. TUNEL-positive cells were not observed in sections
prepared from slices 1 and 3 h after the tetanization protocol
(data not shown); this is consistent with the absence of degraded DNA
we observed after gel electrophoresis in slices from these groups.
However, when the percentage of TUNEL-positive cells was quantified
from sections prepared from the late time point group (12-14 h), there
was a significantly greater percentage of TUNEL-positive cells in CA3, CA1, and EC when the tetanized group (16.5 ± 4.4, 33.7 ± 7.1, 11.6 ± 2.1, respectively; n = 7) was
compared with the appropriate time-matched control group (4.1 ± 2.2, 7.3 ± 2.0, 2.8 ± 0.9, respectively; n = 6). Although there was an increase in TUNEL-positive cells in both
the DG and the H in tetanized slices compared with time-matched controls, due to variability, the differences failed to reach significance in these regions. Some of the morphological features we
observed in the TUNEL-positive cells were characteristic of apoptosis,
including well-delimited chromatin aggregates located peripherally on
the nuclear membrane and cell shrinkage; however, we did not observe
membrane blebbing or apoptotic bodies. TUNEL-positive cells were
observed in small clusters or individually. TUNEL-positive cells were
observed typically, but not exclusively, in the principal cell body
layer. For example, in the DG, TUNEL-positive cells ranged from the
infragranular layer to the most distal border of the granule cell
layer. Representative examples of TUNEL-positive cells are presented in
Fig. 4A. A summary of the
percentage of TUNEL-positive cells in each of the five regions of the
section assessed after 12-14 h is presented in Fig. 4B.
|
Pharmacological manipulation of ER Ca2+ stores
Having demonstrated seizure-induced cell death in entorhinal
cortex/hippocampus slices, we then were interested in testing the
hypothesis that Ca2+ released from ER stores contributes to
seizure-induced cell death. We, therefore, repeated the long-duration
(12-14 h) experiments and applied two conventional drugs known to
modify the functioning of ER Ca2+ stores, dantrolene and
thapsigargin. Dantrolene or thapsigargin was applied for 30 min before
the commencement of tetanic stimulation to enable the assessment of
their effect on normal synaptic transmission, which was determined by
the amplitude of the population spike evoked with maximal stimulation.
The drugs were present for the duration of the experiments. Dantrolene
(30 µM) was without significant effect on normal synaptic
transmission, something that has been reported previously
(Obenaus et al. 1989; O'Mara et al.
1995
). The amplitude of the population spike after 30 min of
dantrolene exposure was 104.3 ± 9.8% of control
(n = 5). Dantrolene also was without effect on the
induction or the maintenance of epileptiform discharges. That is, there
were no differences compared with experiments where no drugs were
applied in any feature of epileptiform discharges, e.g., PAD,
epileptiform responses evoked with single 100-µs shocks, spontaneous
epileptiform discharges. The amplitude of the population spike, when
measured at the end of the 30 min application of thapsigargin (1 µM)
was 133.2 ± 31.7% of control (n = 4), which
failed to reach significance due to the variability. Thapsigargin also
had no effect on the induction or the maintenance of epileptiform
discharges. Electrophysiological responses evoked in the presence of
dantrolene and thapsigargin are presented in Fig.
5, A and B,
respectively.
|
Gel electrophoresis
DNA isolated from slices that had been tetanized and exposed to
dantrolene (n = 5) or thapsigargin (n = 4) demonstrated no indications of degradation. This was true also for
time-matched control slices. To rule out the possibility that our
observation of the absence of degraded DNA in slices exposed to
dantrolene and thapsigargin was a false negative, we simultaneously
electrophoresed DNA isolated from organotypic hippocampal slice
cultures exposed to actinomycin-D (5 µg/ml), a potent inducer of
apoptosis in a variety of cells (Martin et al. 1990). A
representative example of the gel electrophoresis of DNA from these
experiments is presented in Fig. 6.
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DISCUSSION |
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Repeated tetanic stimulation in entorhinal cortex/hippocampus
slices produced both evoked and spontaneous epileptiform activity, which we could record for 14 h. We assessed seizure-induced cell death using gel electrophoresis of genomic DNA and immunohistologically by counting TUNEL-positive cells. Indications of cell death by either
method were not observed when assessed at 1 and 3 h after the
tetanization protocol. In contrast, we observed degraded DNA isolated
from tetanized slices in the 12- to 14-h group, which ranged in
appearance from a smear of low-molecular-weight DNA fragments
(<100-400 bp) to a more diffuse smear accompanied by a few
discernable bands. The amount of degraded DNA from time-matched controls was either below the threshold detection limit or
qualitatively less compared with tetanized slices. Consistent with the
observation of degraded DNA from tetanized slices in the 12-14 h group
was the presence of TUNEL-positive cells in sections prepared from this
group. We observed a significant fourfold increase in the percentage of
TUNEL-positive cells in CA3, CA1, and EC from tetanized slices compared
with the respective time-matched control.
Cell death
There are two fundamental and discernable patterns of
cell death, necrosis and apoptosis (reviewed in Buja et al.
1993; Robbins 1994
). Necrosis occurs after an
exogenous insult and manifests as cell swelling, or rupture,
denaturation, and coagulation of cytoplasmic proteins, breakdown of
cell organelles, and a significant inflammatory response. Apoptosis,
described first by Kerr, Wyllie, and Currie (1972)
, is a
feature of normal development in both the central and the peripheral
nervous system, may require de novo gene transcription, and drives the
elimination of cells during embryogenesis (Stewart 1994
;
White 1996
). In addition to its physiological role,
apoptosis also has been observed in a variety of pathological conditions (Thompson 1995
).
There is a complex relation between experimentally induced
seizures and cell death. Cell death produced by limbic motor seizures induced by intraamygdaloid injection of kainate has been described previously to have features of both necrosis and apoptosis
(Charriaut-Marlangue et al. 1996; Pollard et al.
1994
; Represa et al. 1995
). Conversely, status
epilepticus produced by systemic injection of pilocarpine produced cell
death that was described as being solely necrotic (Fujikawa
1996
). Additionally, kindling can give rise to both apoptosis
and proliferation of DG neurons (Bengzon et al. 1997
). Because both necrosis and apoptosis often are described in pathology and share some features, distinguishing between the type of cell death
sometimes can be difficult. Convergent evidence from different methods
of analysis is required to differentiate between apoptosis and necrosis
(Charriaut-Marlangue and Ben-Ari 1995
). Additionally, differences in the type of cell death produced, and other features such
as regional susceptibility, might be attributable to the method
employed for inducing the seizures.
Once we established seizure-induced cell death in vitro, characterization between necrosis and apoptosis was not the focus of this investigation, and on the basis of these data, we cannot with confidence discriminate which form of cell death predominated. Additionally, although we observed that the majority of TUNEL-positive were located in the principal cell body layers, because we did not use immunohistochemical methods to differentiate between neurons and glia, we cannot address the differential susceptibility of neurons compared with nonneuronal cells. Therefore we use the term "cell death." The results of the gel electrophoresis were more reminiscent of the random DNA cleavage that occurs in necrosis. Nevertheless, some of the morphological features we observed in the TUNEL-positive cells are characteristic of apoptosis, including well-delimited chromatin aggregates located peripherally on the nuclear membrane and cell shrinkage. TUNEL-positive cells were present in small clusters or individually, typically, but not exclusively, in principal cell body layers. However, membrane blebbing or apoptotic bodies were not seen.
Mesial temporal sclerosis associated with complex partial seizures
typically involves the CA1 region (Honavar and Meldrum 1997). We observed the greatest percentage of TUNEL-positive
cells in the CA1 region. We may have observed DNA laddering if the DNA from the CA1 region alone was electrophoresed. This would require pooling the CA1 region from several slices to yield a sufficient quantity of DNA. If these cells are dying due to apoptosis and if this
process is dependent on the synthesis of macromolecules (Wyllie
et al. 1984
), blockade of this synthesis might be of
therapeutic relevance. Cleavage of DNA by endogenous nucleases is an
event that occurs well after the cell has committed itself to death (Mesner et al. 1995
). Therefore we suggest that
assessment of earlier occurring apoptotic events, such as release of
cytochrome C (Yang et al. 1996
), caspace activation
(Miura et al. 1993
), or translocation of plasma membrane
phosphatidylserine (Martin 1995
), between 3 and 12 h, is necessary for a more refined temporal assessment of the induction
of seizure-induced apoptosis in vitro.
Mechanisms of seizure-induced cell death
Seizures are characterized cellularly by synchronized synaptic
activity, which results in excessive glutamate release. Prolonged overactivation of postsynaptic glutamate receptors is neurotoxic, and
this form of cell death is referred to as "excitotoxicity" (Olney and Sharpe 1969).
Illustrative of this relation is the recent report that homozygous mice
deficient in the glutamate transporter GLT-1 presented with lethal
spontaneous seizures and an exacerbation of the seizure-induced cell
death compared with wild type (Tanaka et al. 1997).
Excitotoxicity is a biphasic process and includes both a rapid and a
delayed component (Meyer 1989
; Randall and Thayer
1992
). The early damage is the result of an influx of
Na+, Cl
, and H2O with osmolysis.
The delayed cell death is secondary to an increase in intracellular
Ca2+, which is thought to initiate a variety of deleterious
consequences including activation of phospholipase A2, which leads to
the production of arachidonic acid and free radicals, endonuclease
fragmentation of DNA, and production of nitric oxide, which contributes
to inhibition of mitochondrial oxidative phosphorylation (Choi
1988
; Dugan and Choi 1994
; Meldrum and
Garthwaite 1990
).
Role of ER Ca2+ stores
Ca2+ released from ER stores participates in a variety
of physiological processes including gene transcription, protein
synthesis, cell differentiation, and synaptic plasticity
(Berridge 1998; Henzi and MacDermott
1992
). This source of Ca2+ also is recognized as
being important for pathological events. Blockade of Ca2+
release from ER stores with dantrolene has been reported previously to
not significantly affect normal synaptic transmission (Obenaus et al. 1989
; O'Mara et al. 1995
); this is
consistent with what we observed. The relation of ER Ca2+
stores to long-term potentiation (LTP) is more complex. The induction of LTP has been reported to be blocked by both thapsigargin
(Harvey and Collingridge 1992
) and dantrolene
(Obenaus et al. 1989
). Conversely, O'Mara et al.
(1995)
reported that dantrolene inhibited long-term depression
and depotentiation but was without effect on LTP. This discrepancy is
likely attributable to differences in experimental conditions such as
the concentration of dantrolene (20 or 50 µM), the duration of the
perfusion period (180 or 20 min), or the synapses that were tetanized
(Schaffer collateral to CA1 vs. medial perforant path to DG). Although
we did not systematically assess the effect of dantrolene and
thapsigargin on LTP, we did not observe any differences in the
epileptiform activity induced by repeated tetanic stimulation
attributable to these drugs. Our observation that dantrolene prevented
seizure-induced cell death is consistent with the reports of many
others; however, the neuroprotection we observed for thapsigargin was
somewhat unexpected. Thapsigargin has been reported to produce multiple
effects including inhibition of protein synthesis, cell proliferation,
tumor promotion, and at low concentrations (i.e., nanomolar), apoptosis
(Treiman et al. 1998
). In our hands, thapsigargin did
not produce apoptosis in control slices and did not exacerbate
seizure-induced cell death in tetanized slices. Indeed, thapsigargin
prevented seizure-induced cell death (compare Fig. 3 with Fig. 6).
Maintenance of the filling state of intracellular stores is
attributable to the dynamic interplay between the leak of
Ca2+ out of the store via the leak channel, SERCAs, and
capacitative Ca2+ influx (Thomas and Hanley
1994). Blockade of SERCAs by thapsigargin produces a biphasic
elevation of cytosolic Ca2+ influx (Treiman et al.
1998
). The initial increase (15-120 s) is due to release of
Ca2+ from stores via the leak channel (Thomas and
Hanley 1994
), but its persistence (minutes) is attributable to
capacitative Ca2+ influx (Putney 1986
),
which is thought to be carried by the Ca2+-release
activated Ca2+ current (ICRAC)
(Hoth and Penner 1992
; Penner et al.
1993
). Before tetanization we did observe a
thapsigargin-induced increase in population spike amplitude; this might
be attributable to the initial passive leak of Ca2+. We
applied thapsigargin at a concentration of 1 µM. At micromolar concentrations, thapsigargin should maximally discharge the contents of
the store (Thomas and Hanley 1994
) but also might block
capacitative Ca2+ influx (Mason et al.
1991
), Ca2+ channels (Shmigol et al.
1995
) and protein synthesis (Paschen et al.
1996
). Additionally, chronic exposure to thapsigargin may have
increased the capacity of endogenous Ca2+ buffers
(Petersen et al. 1993
).
The maximum probability of opening for the IP3-sensitive
store occurs at 200 nM free Ca2+, with sharp decreases on
either side of the maximum. In contrast, the ryanodine-sensitive store
is maximally responsive to concentrations of Ca2+ ranging
from 1 to 100 µM (Bezprozvanny et al. 1991). Because IP3-sensitive stores are inhibited by concentrations of
free intracellular Ca2+ only slightly higher than
physiological levels, the most likely candidate for the source of ER
Ca2+ that would participate in Ca2+-dependent
cell death cascades is the ryanodine-sensitive store; however,
cross-talk between the two stores has been described whereby
Ca2+ released from IP3-sensitive stores can
induce CICR (Simpson et al. 1995
).
Type I RYRs, or skeletal muscle RYRs, are located on the sarcoplasmic
reticulum, and proximal to dihydropyridine-sensitive voltage-operated
Ca2+ channels on T-tubule infoldings of the plasma
membrane, which together form the functional unit underlying
excitation-contraction coupling. Type I RYRs also are present in
cerebellar Purkinje neurons. Type II, or cardiac muscle RYRs, are
ubiquitous in the brain, whereas type III RYRs are restricted to
specific brain regions such as CA1 stratum pyramidale, caudate, and
dorsal thalamus (Furuichi et al. 1994). Although RYR
isoforms are expressed differentially in a variety of tissues, more
than one isoform may be coexpressed in both neurons (Furuichi et
al. 1994
) and in nonneuronal cells (reviewed in Sutko
and Airey 1996
). Functional differences between receptor
isoforms are understood only poorly; however, differential regulation
of types 1 and 3 IP3R by cytosolic Ca2+ has
been described recently (Cardy et al. 1997
). Although
only speculative, types II and III RYRs appear to be the most likely candidates to participate in the seizure-induced, CICR-dependent cell
death we observed.
We did not employ the use of blockers of glutamatergic transmission or
VDCCs, and neither dantrolene nor thapsigargin affected the
induction or maintenance of epileptiform discharges. Therefore the
entry of extracellular Ca2+ into the cell due to the
prolonged epileptiform depolarizations was unperturbed. We interpret
our results as being consistent with the hypothesis that
Ca2+-dependent excitotoxicity requires both the entry of
Ca2+ and the release of Ca2+ from ER stores,
resulting in the initiation, the amplification, or the persistent
functioning of Ca2+-dependent death cascades. A direct test
of this hypothesis would require the comparison of
[Ca2+]I during epileptiform discharges (e.g.,
PAD) in the presence and the absence of CIRC blockers followed by an
assessment of cell death. Our hypothesis is consistent with
Frandsen and Schousboe (1991, 1993
), who postulate that
Ca2+ derived from internal stores participates in
NMDA-receptor-mediated excitotoxicity they observed in cultured
cortical neurons. Additionally, Ca2+-dependent
excitotoxicity might be dependent on the source of Ca2+
influx (Tymianski et al. 1993
). The source specificity
hypothesis requires the spatial constraint of proximity of
Ca2+-dependent cell death nanomachinery and NMDA receptors.
Intracellular Ca2+ stores associated with the ER are
distributed throughout neurons (Sharp et al. 1993
). In
the dendrites of CA1 pyramidal neurons, the ER terminates in various
forms in the dendritic spines, is referred to as the spine apparatus
(Spacek and Harris 1997
; Svoboda et al.
1996
), and sometimes comes into contact with the postsynaptic density (Spacek and Harris 1997
). Additionally, in CA1
pyramidal neurons, where we observed the greatest percentage of
TUNEL-positive cells, there is a greater density of ryanodine receptors
compared with IP3 receptors (Sharp et al.
1993
). The smaller volume of dendrites compared with soma might
promote the interaction between ryanodine-sensitive ER stores, NMDA
receptors, and the Ca2+-dependent nanomachinery required
for excitotoxicity.
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ACKNOWLEDGMENTS |
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We thank F. Vidic and Drs. Sukriti Nag, Jim Eubanks, Joe Francis, Jose-Luis Perez Velazquez, Marina Frantseva, and Moshe Kushnir for technical assistance and critical comments.
This work was supported by the Canadian Networks of Centers of Excellence and the Bloorview Epilepsy Program (to M. R. Pelletier and P. L. Carlen) as well as the Natural Science and Engineering Research Council of Canada (to L. R. Mills).
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FOOTNOTES |
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Address for reprint requests: M. R. Pelletier, Playfair Neuroscience Unit, Bloorview Epilepsy Research Laboratory, The Toronto Hospital (Western Division), MCL12-413, 399 Bathurst St., Toronto, ON M5T 2S8, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 October 1998; accepted in final form 15 January 1999.
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REFERENCES |
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