Department of Anatomy and Neurobiology, Program in Molecular, Cellular and Integrative Neuroscience, Colorado State University, Fort Collins, Colorado 80523
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ABSTRACT |
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Smith, Bret N. and F. Edward Dudek. Short- and Long-Term Changes in CA1 Network Excitability After Kainate Treatment in Rats. J. Neurophysiol. 85: 1-9, 2001. Neuron loss, axon sprouting, and the formation of new synaptic circuits have been hypothesized to contribute to seizures in temporal lobe epilepsy (TLE). Using the kainate-treated rat, we examined how alterations in the density of CA1 pyramidal cells and interneurons, and subsequent sprouting of CA1 pyramidal cell axons, were temporally associated with functional changes in the network properties of the CA1 area. Control rats were compared with animals during the first week after kainate treatment versus several weeks after treatment. The density of CA1 pyramidal cells and putative inhibitory neurons in stratum oriens was reduced within 8 days after kainate treatment. Axon branching of CA1 pyramidal cells was similar between controls and animals examined in the first week after kainate treatment but was increased several weeks after kainate treatment. Stimulation of afferent fibers in brain slices containing the isolated CA1 region produced graded responses in slices from controls and kainate-treated rats tested <8 days after treatment. In contrast, synchronous all-or-none bursts of spikes at low stimulus intensity (i.e., "network bursts") were only observed in the CA1 several weeks after kainate treatment. In the presence of bicuculline, the duration of evoked bursts was significantly longer in CA1 pyramidal cells weeks after kainate treatment than from controls or those examined in the first week posttreatment. Spontaneous network bursts were also observed in the isolated CA1 several weeks after kainate treatment in bicuculline-treated slices. The data suggest that the early loss of neurons directly associated with kainate-induced status epilepticus is followed by increased axon sprouting and new recurrent excitatory circuits in CA1 pyramidal cells. These changes characterize the transition from the initial acute effects of the kainate-induced insult to the eventual development of all-or-none epileptiform discharges in the CA1 area.
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INTRODUCTION |
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Kainate treatment in rats causes
status epilepticus (i.e., a period of prolonged and repetitive
seizures) and leads to the eventual development of a chronic epileptic
state characterized by an increasing frequency of spontaneous recurrent
seizures weeks after the initial treatment. The kainate-treated rat
thus serves as a model of human temporal lobe epilepsy (TLE)
(Ben-Ari 1985; Lothman and Collins 1981
;
Nadler 1981
). In both kainate-treated rats and TLE
patients, epileptogenesis is associated with loss of neurons in the
hippocampal CA3, CA1, and hilar areas. Increased collaterals of granule
cell axons (i.e., mossy fiber "sprouting") and synaptic
reorganization in the dentate gyrus also occur over several weeks and
months as chronic epilepsy develops with time (Babb et al.
1991
; Ben-Ari 1985
; Ben-Ari et al.
1981
; Buckmaster and Dudek 1997a
,b
;
Cronin and Dudek 1988
; Cronin et al.
1992
; De Lanerolle et al. 1989
; Franck et
al. 1995
; Hellier et al. 1998
; Nadler et
al. 1980
; Sutula et al. 1989
, 1998
; Tauck
and Nadler 1985
). Relatively few studies have examined both
anatomical and electrophysiological changes at different times after
status epilepticus in animal models of TLE (Franck et al.
1988
; Patrylo and Dudek 1998
; Wuarin and
Dudek 1996
).
Several previous studies have provided evidence that the CA1 area of
the hippocampus is a region of both substantial neuronal loss and
increased seizure susceptibility in the epileptic brain. The
combination of axon sprouting of pyramidal cells coupled with selective
neuronal loss in the CA1 area has been hypothesized to be an important
component of the development of TLE. CA1 pyramidal cell density is
often greatly reduced in humans with TLE and in animal models
(Mathern et al. 1995a,b
, 1997
). Selective loss of putative inhibitory neurons has been observed in the CA1 region in the
kainate (Best et al. 1993
; Morin et al.
1998a
) and pilocarpine models of TLE (Hauser and
Esclapez 1996
). Even so, much of the inhibitory circuitry
appears functional weeks after treatment (Esclapez et al.
1997
; Franck et al. 1988
; Nakajima et al.
1991
). Some studies have suggested that inhibition is
transiently decreased immediately after kainate treatment followed by
partial recovery after several weeks (Franck and Schwartzkroin
1985
; Franck et al. 1988
). Electrophysiological
analyses in the isolated CA1 indicated that inhibition is reduced
several weeks after kainate treatment (Meier et al.
1992
). Months after kainate treatment, CA1 pyramidal cells
could generate repetitive, all-or-none bursts of action potentials to
afferent stimulation in the presence of the
GABAA-receptor antagonist, bicuculline (i.e.,
similar to normal CA3 pyramidal cells) (Meier and Dudek
1996
). Early studies indicated an increased number of
excitatory synapses in the CA1 region after kainate treatment
(Nadler et al. 1980
), and reconstruction of
intracellularly labeled CA1 pyramidal cells revealed increased axonal
branching in kainate-treated animals versus controls (Esclapez
et al. 1999
; Perez et al. 1996
), suggesting an
increase in excitatory connectivity in the CA1 due to local axon
reorganization. The temporal relationship between anatomical
alterations and regional changes in excitability is probably a critical
feature of temporal lobe epileptogenesis (see Dudek and Spitz
1997
). However, the time-dependent changes in CA1 network
excitability after kainate treatment have been only briefly discussed
(Franck and Schwartzkroin 1985
; Franck et al.
1988
).
We examined CA1 neuron density, pyramidal cell axonal morphology, and synaptically evoked spike bursts in CA1 pyramidal cells using slices from rats several weeks after kainate treatment, when they had developed spontaneous recurrent motor seizures (i.e., rats with kainate-induced epilepsy), and compared the results to those obtained in slices from rats that received saline injections and from rats studied 3-8 days after kainate treatment. This allowed us to compare immediate anatomical and electrophysiological changes directly associated with kainate-induced status epilepticus to the subsequent alterations that develop over several weeks, when the rats develop spontaneous seizures. We hypothesized that there is a delayed (i.e., time-dependent) increase in synaptic network excitability in the CA1 region after kainate treatment.
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METHODS |
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Kainate treatment
Sprague-Dawley rats (Harlan) were housed under normal 12 h
light/12 h dark cycle and had access to food and water ad libitum. All
procedures were approved by the Colorado State University Animal Care
and Use Committee. Adult male rats (200-250 g) were given
intraperitoneal injections of kainate (5 mg/kg in 150 mM NaCl; Sigma,
St. Louis, MO) every hour for 10 h. Seizure intensity was evaluated
using the scale described by Racine (1972)
.
Low-intensity tonic-clonic seizures, mostly of the forelimbs (stage 3),
usually began by the third injection. Stage 3 seizures generally
progressed into rearing and falling (stage 4-5) after subsequent
injections. Occasional episodes of circling and jumping were also
observed. Stage 4 and 5 seizures were maintained for 4-6 h and then
slowly subsided in most rats, even with more kainate injections. A
similar phenomenon has been seen with another multiple-treatment
kainate-injection protocol (Sarkisian et al. 1997
). In
rats with very frequent or continuous stage 4-5 seizures, we either
eliminated an injection or gave half the dose. The total dose per rat
was 30-50 mg/kg. Each animal had
4 h of recurring seizure activity
before the treatment was stopped. Control rats were injected hourly
with the vehicle in parallel with the kainate-treated rats. All the kainate-treated rats received subcutaneous lactated Ringer (3-5 ml) at
the end of the period of status epilepticus to replenish fluids. The
survival rate was ~80%.
Following the kainate treatment, the behavior of both control and
kainate-treated rats was monitored for 1-2 h/day, 3-5 days/wk (minimum 6 h/wk) over a period of 3-13 mo to confirm that the treatment induced spontaneous, recurrent seizures (i.e., chronic epilepsy). An analysis of seizure activity in kainate-treated rats,
including those animals used in this study, has been published (Hellier et al. 1998). One group of control and two
experimental groups of animals were used in these studies based on the
time after kainate-induced status epilepticus and on development of spontaneous motor seizures in the kainate-treated animals. The control
group consisted of saline-treated rats, which were not observed to have
seizures. One experimental group consisted of animals examined in the
first week after treatment (<8 d) and was therefore defined as a
"short-term" group of kainate-treated rats. These animals underwent
kainate-induced status epilepticus, and no motor seizures were observed
on subsequent days. However, a few seizures could have occurred in some
rats from this group, based on 24-h video monitoring of other animals
in the first week after kainate treatment (Hellier et al.
1999
). Another experimental group of animals was examined 3-13
mo after kainate treatment (mean = 198 ± 17 d) and was
therefore defined as a "long-term" group of kainate-treated rats.
All animals in this group were observed to have had multiple
spontaneous motor seizures in the 30 days prior to recording (mean = 10.6 ± 1.5 seizures). Animals were excluded if seizures were
observed the day of the experiment.
Tissue preparation
Hippocampal slices were prepared and maintained in a manner
similar to that described previously (e.g., Meier et al.
1992). Briefly, rats were anesthetized with pentobarbital
sodium (100 mg/kg ip) and decapitated. Their brains were then rapidly
removed and placed in ice-cold, oxygenated (95%
O2-5% CO2) artificial cerebrospinal fluid (ACSF) for ~1 min. The ACSF contained (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.4 NaH2PO4, 11 mM glucose, 1.3 CaCl2, and 1.3 MgSO4,
pH = 7.3-7.4, with an osmolality of 290-305 mOsm/kg. Brains were
then blocked and mounted on a vibroslicer (Campden Instrument,
Lafayette, IN), and transverse slices of the temporal hippocampus
(400-500 µm) were cut. After isolating the hippocampus from
surrounding tissue, the slices were transferred to a storage chamber
and constantly immersed in perfusion medium warmed to 32-34°C for
1 h prior to placement in an interface-type recording chamber for
electrophysiological recording. On placement in the recording chamber,
the CA1 area was isolated from the CA2/CA3 regions and from the
subiculum with knife cuts. The GABAA antagonist, bicuculline methiodide (30 µM; Sigma), or the glutamate AMPA/kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10-50 µM; Sigma) was added to the ACSF for some specific experiments.
Population and whole cell recording
Field-potential and whole cell patch-clamp recordings were
obtained from pyramidal cells in the isolated CA1. An extracellular electrode containing 1 M NaCl was placed in the pyramidal cell layer.
Field responses were recorded using an Axoclamp 2A amplifier (Axon
Instruments, Foster City, CA). Patch pipettes pulled from thin-wall
(0.45-mm thickness) borosilicate glass capillaries (Garner Glass,
Claremont, CA) were filled with (in mM) 130 K+-gluconate, 1 NaCl, 5 EGTA, 10 HEPES, 1 MgCl2, 1 CaCl2, 3 KOH, and
2-4 ATP; pH = 7.2. Pipettes had open resistances of 2-5 M; seal resistances were typically 1-4 G
and series resistances were
4-19 M
, uncompensated. Whole cell signals were recorded using an
Axopatch 1D amplifier (Axon Instruments). All signals were low-pass
filtered at 2-5 kHz, digitized at 44 kHz (Neuro-corder; Neurodata
Instruments, New York, NY), and stored on videotape.
Once in the whole cell configuration, cells were initially held near the resting membrane potential for 5-10 min to allow equilibration of the recording electrode solution. Electrical stimulation of afferent fibers in stratum radiatum was made with a bipolar electrode constructed from a twisted pair of Teflon-coated, platinum-iridium wires (75-µm diam). An unpaired, two-tailed Student's t-test was used for comparing data between recordings. Numbers are reported as the means ± SE unless otherwise noted.
Tissue staining
Slices adjacent to those from which recordings were made were reserved for histological analysis. At least one slice from most animals was processed for glutamic acid decarboxylase (GAD) immunohistochemistry to identify putative GABAergic neurons. These slices were immersion-fixed overnight in 4% paraformaldehyde in 0.15 M NaHPO4 buffer. Following several rinses in 0.01 M phosphate buffered saline (PBS), the slices were cryoprotected in 30% sucrose in PBS and cut at 20-30 µm on a sliding microtome. After several rinses in PBS, alternate sections were either left untreated for later Nissl-staining with cresyl violet or reacted for GAD immunohistochemistry. These latter sections were immersed in PBS containing 10% normal goat serum and a polyclonal GAD antibody (AB108; Chemicon, Temecula, CA) at a dilution of 1:2000 overnight. After rinsing in PBS, sections were treated with fluorescein-conjugated secondary antisera (goat anti-rabbit, IgG; 1:400) for 4-12 h. Sections were mounted on slides, air-dried, and cover-slipped in Vectashield (Vector Labs, Burlingame, CA) to reduce oxidation and fading of the reaction product. Coverslips were sealed with fingernail polish at the edges. Imunoreactive neurons were visualized on a Zeiss Axiophot microscope and images of the tissue were captured digitally. A section from near the middle of the original slice with well-preserved morphology was selected for analysis of each label. A box drawn around an area 200 × 200 µm was superimposed on the image over the CA1 region that corresponded to the region from which recordings were made in adjacent slices and the images were printed. Semi-quantitative cell counts were made by counting manually all the labeled neurons within the boxed area of a single image. Relative numbers of GAD neurons were independently assessed blind by two persons. For pyramidal neuron assessment, the same blinded investigators counted all neuronal nuclei in the pyramidal cell layer that fell within the grid outline. Counts from the two assessments were averaged. Cell densities were normalized to counts from control tissue.
Neuron reconstruction
Electrodes contained 0.1-0.2% biocytin to label recorded
neurons and verify their location (Horikawa and Armstrong
1988). Following each recording, slices were fixed in 4%
paraformaldehyde in 0.15 M NaPO4 buffer (pH = 7.2-7.4) overnight at 4°C. Following fixation, slices were rinsed
(3 × 5 min) in PBS (pH = 7.4), cryoprotected in PBS
containing 30% sucrose, and sectioned at 30-50 µm on a sliding
microtome. After rinsing in PBS, endogenous peroxidase was removed
(10% methanol/3% H2O2 in
PBS; 60-70 min). The sections were again rinsed in PBS and incubated
overnight in an avidin-biotin-horseradish peroxidase complex (ABC elite
kit; Vector Labs) in PBS (1:100; pH = 7.3) containing 0.1% Triton
X-100. The reaction product was visualized with diaminobenzidine at a
concentration of 0.06% with 0.003%
H2O2 in 0.01 M PBS (pH = 7.4) to confirm the location of the recorded neuron, and the tissue
was subsequently dehydrated in alcohols and mounted in permount. Axonal
morphology was examined by reconstructing serial sections using
computer-assisted neuron-drawing software (Neurolucida;
Microbrightfield, Colchester, VT). Axon reconstructions were limited to
neurons in slices from which only a single neuron was recorded. In
neurons from each experimental group, total axon length and number of
branches were quantified by the Neurolucida software. Dendrites,
identified by the presence of spines, were not reconstructed for this study.
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RESULTS |
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Data were obtained from 28 rats in the long-term group (i.e.,
examined >3 mo after kainate treatment), 9 kainate-treated rats in the
short-term group (i.e., used 8 days posttreatment), and 13 age-matched control animals (i.e., received vehicle injections). Anatomical analyses were performed on a subset of the animals from
which electrophysiological results were obtained.
Anatomical studies
PYRAMIDAL CELL LOSS. Nissl staining was performed on slices adjacent to those from which recordings were obtained to determine if the number of pyramidal cells was reduced after kainate treatment (Fig. 1, A-C). Pyramidal cell density was examined in controls (n = 7), short-term animals (n = 7), and long-term (n = 14). In tissue from short-term animals, CA1 pyramidal cell density was 81 ± 7% of that in age-matched control animals (P < 0.05) and was 69 ± 7% of control in long-term rats. Cell counts in both long- and short-term animals differed from controls (P < 0.05) but not from each other (P > 0.05).
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INHIBITORY NEURON LOSS.
To determine the relative decrease in the number of putative inhibitory
neurons in the CA1 region after status epilepticus in kainate-treated
rats, we examined sections from adjacent slices for GAD
immunoreactivity (Fig. 1, D-F). The density of
GAD-immunoreactive neurons in the short-term group was significantly
reduced in stratum oriens (n = 5; 54 ± 24% of
control; P < 0.03). Similar to previous reports on
interneuron density (Best et al. 1993; Hauser and
Esclapez 1996
; Morin et al. 1998a
), the density
of GAD-immunoreactive neurons in the long-term group (n = 7) was also significantly reduced in stratum oriens (37 ± 18%
of control; P < 0.03) but not in stratum radiatum
(93 ± 11% of control; P > 0.05). The numbers of
GAD-immunoreactive neurons in stratum oriens was reduced in both
experimental groups, but there was no difference between short- and
long-term animals (P > 0.05).
AXON SPROUTING.
Axon sprouting in the dentate gyrus (i.e., mossy fiber sprouting) has
been associated with an increased propensity for seizure activity in
previous studies using this animal model (Buckmaster and Dudek
1999; Hellier et al. 1998
; Wuarin and
Dudek 1996
). Axon sprouting has also been demonstrated in CA1
pyramidal neurons from kainate-treated rats (Esclapez et al.
1999
; Perez et al. 1996
). To corroborate that
there were differences in the axon morphology of CA1 pyramidal cells
between the three groups we used, we reconstructed digitally the axons
of a few of the biocytin-filled CA1 pyramidal cell neurons from which
we recorded. We found that axons of CA1 pyramidal cells were more
highly branched (P < 0.05) in slices from long-term
animals than in either age-matched controls or short-term rats (Fig.
2). The mean axon length from four
neurons in control rats was 1,105 ± 213 µm, with 8 ± 2 branches in stratum oriens of the isolated CA1 slice. In neurons from
short-term animals (n = 3), the mean axon length was
1,346 ± 474 µm and the number of branches was 9 ± 2. In
neurons from long-term rats (n = 4), the mean axon
length was 2,505 ± 531 µm (P < 0.05 vs. both
other groups) and branch number was 30 ± 6 (P < 0.05 vs. both other groups). Axon length and number of branch points
was greater in the long-term animals than in either short-term or
control rats with short-term and control animals having comparable axon
measurements.
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Electrophysiological studies
EVOKED RESPONSES: FIELD-POTENTIAL RECORDINGS.
Extracellular field-potential recordings were made in the CA1 pyramidal
cell layer, which had been isolated from CA3 and the subiculum by knife
cuts. Field recordings were obtained in 27 isolated CA1 slices from 16 kainate-treated rats in the long-term group, 9 slices from 5 kainate-treated rats in the short-term group, and 12 slices from 8 saline-treated control rats. A low-intensity stimulus applied to the
fibers in stratum radiatum resulted in a single population spike in the
CA1 region of control rats (Fig. 3A). The amplitude of the
spike increased as the stimulus intensity was increased, and
occasionally a second population spike developed at stimulation
intensities greater than three times that required for population spike
generation. As in slices from control animals, a stimulus at threshold
intensity evoked a single population spike in the CA1 pyramidal cell
layer in kainate-treated animals from the short-term group. Unlike
controls, increasing the stimulus intensity resulted in a graded
increase in spike number (8 spikes) in addition to an increase in the
amplitude of the first spike in this short-term group (Fig.
3B).
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WHOLE CELL RECORDINGS.
Whole cell patch-clamp recordings were made from pyramidal cells in
hippocampal slices of the isolated CA1 area from control rats
(n = 17) and short-term (n = 17) and
long-term (n = 38) groups of kainate-treated rats.
Average resting membrane potential and input resistance were similar
between the three groups (control, 65 ± 1 mV, 139 ± 24 M
; short-term,
64 ± 2 mV, 140 ± 18 M
; long-term,
65 ± 1 mV, 136 ± 13 M
; P > 0.05).
Afferent stimulation (minimally twice the minimum intensity for spike
generation) resulted in a single action potential in slices from
control animals (Fig. 4A1). In
animals from the short-term group, afferent stimulation at twice
threshold intensity resulted in a short burst of action potentials
(Fig. 4A2). Similar afferent stimulation resulted in a
longer burst of spikes (mean 8 ± 1 spikes; 85 ± 9 ms
duration) in CA1 pyramidal cells from kainate-treated rats in the
long-term group (Fig. 4A3). As in the field recordings,
qualitative "all-or-none" burst responses were also seen with low
stimulation intensities.
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EVOKED RESPONSES: BICUCULLINE. In the presence of the GABAA-receptor antagonist, bicuculline (30 µM), the typical response in slices from control rats was a short burst of action potentials (40-170 ms; 10 ± 2 spikes; Fig. 4B1). A brief burst was also generated by similar stimulation in bicuculline-treated slices from rats in the short-term group. This burst was of somewhat longer duration in the presence of bicuculline than in these same slices recorded in normal ACSF (70-220 ms; 12 ± 2 spikes; Fig. 4B2). The duration of evoked bursts was similar for slices from animals in control and short-term groups when tested in the presence of bicuculline (P > 0.05).
A brief "all-or-none" burst was often generated in slices from animals in the long-term group, regardless of stimulus intensity, but addition of bicuculline increased burst duration to as much as 2,500 ms (mean, 920 ± 405 ms) in these animals (Fig. 4B3). The response morphology was characterized by an initial burst of action potentials followed by a period of inactivation prior to repolarization of the membrane. As in normal ACSF, similar responses were seen at threshold stimulus intensities, with the principal difference being the variable latency to burst onset at lower stimulus intensity. The bursts observed in slices from the long-term group in the presence of bicuculline were significantly longer than those in either of the other groups under the same recording conditions (P < 0.05).SPONTANEOUS ACTIVITY.
In an earlier study in kainate-treated rats, 1 of 53 isolated CA1
slices (i.e., CA3 input was cut) exhibited spontaneous bursting activity in the presence of bicuculline (Meier and Dudek
1996). To determine if spontaneous, synchronized bursting in
the CA1 region developed after kainate treatment, we examined
population and cellular activity in the isolated CA1 region in the
presence of bicuculline (30 µM). Spontaneous bursts were not observed
when bicuculline was added to normal ACSF in isolated slices from
controls (0 of 10 slices from 10 animals) or in slices from the
short-term group (0 of 7 slices from 7 animals). Under identical
conditions, four of six slices from six animals in the long-term group
developed spontaneous bursts of activity after 10-30 min. The mean
interburst interval was 57 ± 28 s in these slices.
Spontaneous bursting was observed in disinhibited slices from the
long-term group but not short-term or control groups.
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DISCUSSION |
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Shortly after kainate-induced status epilepticus, the number of inhibitory neurons and pyramidal cells was reduced in the CA1 region. Graded bursts to afferent stimulation were present in these same animals. Network bursts in the isolated CA1 region became apparent after a period of months, a time when increased axon branching and spontaneous seizures were also noted. These results are consistent with the hypothesis that axon sprouting is a reactive event that occurs over time in the CA1 region after neuronal injury and leads to an increased propensity for network bursts.
Compared with single injections, the multiple-kainate-injection
protocol we used results in a prolonged period of status epilepticus and consistently robust mesial temporal sclerosis and to the
development of a chronic state of recurrent seizures (Patrylo
and Dudek 1998; Wuarin and Dudek 1996
). The
robust nature of the lesion may also account for the consistency of
some measurements of slice excitability we employed. A previous study
showed that practically all of the long-term kainate-treated rats used
in the present experiments developed epilepsy (Hellier et al.
1998
). By examining the CA1 area at two time points (i.e., 3-8
days and 3-13 mo) after treatment, we aimed to differentiate between
immediate effects of kainate-induced status epilepticus in animals that
likely would have developed spontaneous seizures (i.e., short-term
group) versus the effects that developed over the next several weeks
and were observed in animals that had spontaneous recurrent seizures
(i.e., long-term group). We performed multiple anatomical and
physiological analyses on tissue from the same animals in which the
behavior had been determined. Our results suggest that the transition
from kainate-induced status epilepticus to development of chronic
epilepsy involves neuron loss in the first week posttreatment, probably
including an immediate decrease in inhibition, followed by increased
axon collateralization and accompanying enhanced network excitability in the rat CA1 area.
Reduced synaptic inhibition
Both anatomical and electrophysiological studies have suggested
that kainate-induced status epilepticus leads to a short-latency and
long-lasting (months) depressionbut not elimination
of synaptic inhibition in the CA1 area, and the present experiments further support
this hypothesis. Although some examinations of GAD immunoreactivity have suggested that intraventricular kainate injections do not reduce
the number of GABAergic "basket" neurons (Franck et al. 1988
), other studies using GAD (Morin et al.
1998a
) or parvalbumin (Best et al. 1993
) have
shown that inhibitory neurons are lost in stratum oriens after kainate
treatment. As in the latter studies (Best et al. 1993
;
Morin et al. 1998a
), we also found that the number of
putative inhibitory neurons was reduced in stratum oriens but not
stratum radiatum. Furthermore our results indicate that a significant
loss occurs shortly after systemic kainate treatment.
The observation that orthodromically evoked bursts were graded with
stimulus intensity in animals from the short-term group further
suggested that inhibition was functionally decreased (Crepel et
al. 1997). Several studies have indicated that stimulation of
afferent fibers in stratum radiatum results in brief bursts of spikes
in the CA1 area in slices from kainate-treated rats (Ashwood et
al. 1986
; Esclapez et al. 1999
; Franck
and Schwartzkroin 1985
; Franck et al. 1988
;
Meier et al. 1992
; Nakajima et al. 1991
; Perez et al. 1996
; Williams et al. 1993
).
The observation that blocking the remaining
GABAA-receptor mediated inhibition with bicuculline led to more robust bursts indicates that the
kainate-induced depression of inhibition was only partial. The
immediate kainate-induced loss of inhibitory interneurons in stratum
oriens and the concomitant depression of synaptic inhibition were not
sufficient to reveal the all-or-none, network-mediated bursts, even
after bath application of bicuculline. Months after kainate treatment,
when the animals were clearly epileptic by virtue of displaying
spontaneous recurrent seizures, such network bursts were observed in
this and a previous study (Meier and Dudek 1996
). This
finding is consistent with the hypothesis suggested previously that
inhibition is diminished yet sufficient to suppress epileptiform
activity in CA1 pyramidal cells (Esclapez et al. 1997
;
Franck and Schwartzkroin 1985
; Franck et al.
1988
). However, the partial loss of inhibition may contribute to locally enhanced excitability in the CA1 area. In the short-term, kainate-induced status epilepticus therefore results in reduced inhibition but not a complete breakdown of CA1 inhibitory circuitry.
Increased excitation
Intracellular staining has revealed that CA1 pyramidal cells show
an increase in axon collaterals a few weeks after kainate treatment
(Esclapez et al. 1999; Perez et al.
1996
). In the present study we also observed an increase in
axon collaterals weeks after kainate treatment, but this increase was
not apparent days afterward (i.e., in the short-term group).
Simultaneous intracellular and extracellular recordings revealed that
bicuculline-treated slices containing only the CA1 area could generate
robust, all-or-none burst discharges that were driven by EPSPs many
weeks after kainate treatment (Meier and Dudek 1996
),
which is widely considered to be a property of interconnected
excitatory networks (Gutnick et al. 1982
; Miles
and Wong 1983
; Miles et al. 1988
; Smith
et al. 1999
; Traub and Wong 1982
). We have
corroborated this result in the isolated CA1 of animals examined months
after kainate treatment and found that all-or-none brief network bursts
can be initiated even when residual inhibition is not blocked with
bicuculline. The present data extend previous research suggesting a
time-dependent increase in propensity for seizure-like events, possibly
mediated by new recurrent excitatory circuits. These new circuits
probably increase in number over time. By inference, the number of
previous seizures may influence synaptic excitability if successive
seizures contribute to the severity of the lesion.
The bursts in CA1 pyramidal cells from control and short-term groups
were similar in the presence of bicuculline. That is, little evidence
for increased excitatory connectivity was observed shortly after
kainate treatment when changes in inhibition were equalized by receptor
blockade. However, some slices from animals in the short-term group,
but not controls, could generate network bursts of low frequency in
nominally Mg2+-free ACSF that contained
bicuculline. There may be several explanations for this activity. One
possibility is a rapid alteration of NMDA receptors (Turner and
Wheal 1991). In this scenario, the relatively few excitatory
connections that normally exist between CA1 pyramidal cells
(Christian and Dudek 1988
; Deuchars and Thomson
1996
) might be functionally augmented by postsynaptic receptor
modifications. Activity in the isolated CA1 used in this and previous
studies (Meier and Dudek 1996
; Meier et al.
1992
) indicates that activation of CA3 neurons are not
necessary to generate network bursts in epileptic rats. Postsynaptic
NMDA receptor sensitivity would be expected to increase continuously if
this single mechanism explains the enhancement of network excitability
we observed over time.
An additional possibility is that a few axons begin to sprout soon
after the initial kainate treatment (i.e., days), allowing expression
of the bursting activity in a minority of slices. Axon sprouting of
gradual but immediate onset might contribute to the increasing
propensity for network activity over time and also might contribute to
the variability in the latent period to observed seizure onset seen in
these animals (Hellier et al. 1998). In a recent
examination of identically treated animals, seizures were observed with
continuous 24-h video monitoring in some animals within a week after
kainate treatment and after a short (i.e., 1-2 d) latent period
(Hellier et al. 1999
). The increased propensity for
network bursting in the long-term group could be derived from increases, over time, in axon collateralization and new excitatory synapse formation, which could begin relatively soon after kainate treatment. However, it should be noted that neither this nor previous studies have provided direct evidence that newly sprouted recurrent CA1
axon collaterals increase connectivity between CA1 pyramidal cells and
contribute to the increased excitability. In fact, electrophysiological studies using dual recordings found little or no evidence for increased
excitatory synaptic connectivity between pairs of CA1 pyramidal cells
in kainate-treated rats (Esclapez et al. 1999
; Nakajima et al. 1991
). Although anatomical studies have
suggested that the new axon collaterals might contact pyramidal
neurons, they also suggested that the new terminals could increase
excitatory input to specific subtypes of surviving inhibitory
interneurons (Morin et al. 1998b
). Regardless of
remaining inhibition, enhanced excitatory connectivity could allow
generation of all-or-none network bursts. This would be especially
apparent when the surviving inhibition is suppressed as can occur with
rises in extracellular K+ levels related to
increased firing of large populations of neurons at the beginning of a
seizure (Traynelis and Dingledine 1988
). Nonsynaptic
mechanisms may also play an important role in seizure generation when
extracellular K+ concentration is varied. Our
data suggest that network burst development is temporally related to
excitatory axon collateralization but not cell loss. They are therefore
consistent with the hypothesis that excitatory axon sprouting and
synaptic reorganization result in an increased propensity for
seizure-like activity in the CA1 area. Whether these new synaptic
connections directly enhance local excitatory or inhibitory network
interactions remains uncertain.
Cellular hypothesis of TLE
Probably the oldest view of alterations in seizure susceptibility during the process of chronic epileptogenesis is based on the balance of synaptic inhibition and excitation. The studies described here, and many others, lead to the hypothesis that status epilepticus and other forms of neuronal injury cause a rapid but variable decrease in synaptic inhibition in several cortical areas. The consequent axonal sprouting and formation of new recurrent excitatory circuits, probably also in multiple neuronal systems, provides the substrate for local recruitment of network bursts among numerous populations of cortical neurons. The probability of network burst generation would be expected to depend on the density of recurrent excitatory circuits, which would increase over the days, weeks, months, and even years following an injury, and might be further increased by the seizures themselves. Finally, the "masking" effect of persistent inhibition in some cortical areas could hypothetically explain why the prolonged interictal periods are seizure-free. The evaluation of these interrelated hypotheses will require further quantitative and multidisciplinary analyses, plus the merging of molecular, cellular, and network analyses of local synaptic circuits in many cortical areas throughout the development of epilepsy.
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ACKNOWLEDGMENTS |
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We thank T. Sampson and M. Higgins for technical assistance, Drs. J. Hellier and P. Dou for assistance with animal preparation, and Dr. J.-P. Wuarin for comments on the manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-16683 (F. E. Dudek).
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FOOTNOTES |
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Present address and address for reprint requests: B. N. Smith, Dept. of Cell and Molecular Biology, 2000 Percival Stern Hall, Tulane University, New Orleans, LA 70118 (E-mail: bnsmith{at}tulane.edu).
Received 7 June 2000; accepted in final form 18 September 2000.
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