Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523
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ABSTRACT |
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Wuarin, Jean-Pierre and F. Edward Dudek. Excitatory Synaptic Input to Granule Cells Increases With Time After Kainate Treatment. J. Neurophysiol. 85: 1067-1077, 2001. Temporal lobe epilepsy is usually associated with a latent period and an increased seizure frequency following a precipitating insult. After kainate treatment, the mossy fibers of the dentate gyrus are hypothesized to form recurrent excitatory circuits between granule cells, thus leading to a progressive increase in the excitatory input to granule cells. Three groups of animals were studied as a function of time after kainate treatment: 1-2 wk, 2-4 wk, and 10-51 wk. All the animals studied 10-51 wk after kainate treatment were observed to have repetitive spontaneous seizures. Whole cell patch-clamp recordings in hippocampal slices showed that the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) in granule cells increased with time after kainate treatment. This increased excitatory synaptic input was correlated with the intensity of the Timm stain in the inner molecular layer (IML). Flash photolysis of caged glutamate applied in the granule cell layer evoked repetitive EPSCs in 10, 32, and 66% of the granule cells at the different times after kainate treatment. When inhibition was reduced with bicuculline, photostimulation of the granule cell layer evoked epileptiform bursts of action potentials only in granule cells from rats 10-51 wk after kainate treatment. These data support the hypothesis that kainate-induced mossy fiber sprouting in the IML results in the progressive formation of aberrant excitatory connections between granule cells. They also suggest that the probability of occurrence of electrographic seizures in the dentate gyrus increases with time after kainate treatment.
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INTRODUCTION |
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Two important
characteristics of temporal lobe epilepsy are 1) a latent
period between the insult and the appearance of chronic seizures and
2) a progressive increase in the probability of seizure occurrence with time after the insult. Many types of injuries seem
capable of producing a permanently increased susceptibility of the
brain to generate seizures, and several hypothetical mechanisms have
been proposed to contribute to or be responsible for the epileptogenic
process. Although the nature of this hypothetical process is still
unclear, synaptic reorganization following axonal sprouting has been
proposed to play a central role in the generation of epileptic activity
(Dudek and Spitz 1997; McNamara 1999
).
The progressive formation of recurrent excitatory circuits is an
attractive hypothesis for temporal lobe epilepsy and post-traumatic
epilepsy, because this hypothetical mechanism would seem likely to be
associated with a latent period and an increase in chronic seizure frequency.
Kainate treatment can result in chronic epilepsy (Franck
1993; Sperk 1994
). The seizure frequency usually
increases with time after treatment (Hellier et al.
1998
). Rats treated with kainate typically display neuron loss
in the hippocampus (particularly in CA1, CA3, and the hilus) and
sprouting of the mossy fibers into the inner molecular layer (IML) of
the dentate gyrus (Babb et al. 1991
; Ben-Ari
1985
; Buckmaster and Dudek 1997a
,b
;
Houser 1992
). Several lines of evidence suggest that in
the kainate model, as well as in other models of temporal lobe epilepsy
(e.g., pilocarpine and kindling models), axonal sprouting in the IML
forms recurrent excitatory connections between granule cells.
Antidromic stimulation of the granule cells in slices from
kainate-treated rats showing Timm stain in the IML could produce bursts
of action potentials (Tauck and Nadler 1985
),
particularly when the inhibition was reduced with bicuculline
(Cronin et al. 1992
). Epileptiform bursts, both
spontaneous and in response to electrical stimulation of the hilar
region, were associated with Timm stain in the IML in slices from
kainate-treated rats recorded after chronic epilepsy was established
(Patrylo and Dudek 1998
; Wuarin and Dudek
1996
). More direct evidence in support of the hypothesis that
sprouting of mossy fibers produces excitatory synapses between granule
cells was provided by the demonstration that electrical stimulation of
the mossy fibers at the level of the CA3 area could evoke
excitatory postsynaptic currents (EPSCs) in granule cells
from pilocarpine-treated rats (Okazaki et al. 1999
).
Also, electrical stimulation of the outer blade in slices from
kainate-treated rats evoked excitatory postsynaptic potentials (EPSPs)
in granule cells recorded in the inner blade (Lynch and Sutula
2000
), corroborating the anatomical observation that sprouted
axons from granule cells in the outer blade can cross the hilus and
project into the IML of the inner blade in kainate-treated rats
(Sutula et al. 1998
). Studies using microstimulation
with glutamate also strongly support the hypothesis that excitatory
connections are formed between granule cells after kainate and
pilocarpine treatment. Microdrops of glutamate applied in the granule
cell layer evoked repetitive EPSPs in granule cells from rats with
kainate-induced epilepsy (Lynch and Sutula 2000
; Wuarin and Dudek 1996
), and laser photostimulation of
the granule cell layer could evoke single EPSCs in granule cells of
pilocarpine-treated rats (Molnar and Nadler 1999
). Thus
several lines of in vitro experimentation using electrophysiological
techniques support the hypothesis that the epileptogenic process in
different animal models of temporal lobe epilepsy is associated with
the formation of new recurrent excitatory circuits between dentate
granule cells.
Although there is a relatively large body of data suggesting that new excitatory synapses are formed between granule cells in models of temporal lobe epilepsy, little is known about the changes in excitatory synaptic input that occur in granule cells with time after the initial insult. The present study aimed to test the general hypothesis that kainate treatment produces a continuous formation of new mossy fiber axons in the IML, which creates new excitatory synapses between granule cells and results in a progressive increase in the excitatory input to granule cells and in the probability of epileptiform activity. To test this hypothesis, three groups of animals were defined as a function of time after treatment: 1) the first 2 wk, 2) 2-4 wk, and 3) 10-51 wk (i.e., animals observed to have spontaneous recurrent seizures). We found that with time after kainate treatment 1) the intensity of the Timm stain in the IML increased, 2) the frequency and amplitude of spontaneous EPSCs also increased, 3) photostimulation of the granule cell layer revealed more excitatory interactions between granule cells, and 4) in bicuculline (30 µM) and high [K+]o (6 mM), epileptiform activity was evoked only in slices from animals 10-51 wk after kainate treatment. These results support the hypothesis that kainate treatment triggers the progressive formation of an excitatory network between granule cells, which results in an increased probability of epileptiform activity when inhibition is simultaneously compromised.
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METHODS |
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Kainate treatment and chronic seizures
Adult male rats (Sprague-Dawley; 150-250 g; Harlan) were
injected with kainate (5 mg/kg in 150 mM NaCl ip) or saline every hour.
Motor seizures generally appeared after three to four injections. The
treatment was continued until the animals had class IV/V seizures for
3 h (Ben-Ari 1985
; Racine 1972
). Thus
the total dose of kainate per rat was 20-50 mg/kg. At the end of the
treatment, all animals received lactated Ringer (1-4 ml sc). Both
kainate- and saline-injected rats were subsequently observed for 6-8
h/wk until the time of the slice experiments. Only class III-V
seizures were recorded. These seizures represent unequivocal departure
from normal rat behavior, and thus the observer was not blinded to the
treatment. A total of 48 kainate- and 25 saline-injected control rats
were divided into 3 groups as a function of time after treatment.
Fifteen animals injected with kainate and 7 animals injected with
saline were used 1-2 wk after treatment. The same number of kainate- and saline-injected animals was used 2-4 wk after treatment. Eighteen kainate-injected animals were maintained until after chronic epilepsy was established (10-51 wk), and 11 rats were maintained for 17-72 wk
days after saline injection. No seizures were detected in any of the
control rats. The delay between kainate treatment and the appearance of
behavioral seizures (class III-V) was 4-23 wk (average, 10.8 ± 1.5 wk, mean ± SE, n = 14 animals), when
examined 6-8 h/wk (Fig. 1). The total
number of observed seizures per animal was 6-48 (average, 25.1 ± 3.9). All the animals in the group maintained 10-51 wk after kainate
treatment had spontaneous recurrent seizures for >3 wk by the time
they were used for the slice experiments. Although no seizure was
observed during the first 3 wk after kainate treatment with 6-8 h/wk
direct observation, a recent study using 24-h video monitoring showed
that 5/26 kainate-treated rats had at least 1 motor seizure during the
first week posttreatment (see Hellier et al. 1999
). This
result indicates that chronic spontaneous seizures (i.e., epilepsy)
following kainate treatment may occur earlier than suggested by the
data from 6-8 h/wk direct observation.
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Slice preparation and recording methods
Kainate-treated and saline-injected rats were anesthetized with
pentobarbital sodium (50 mg/kg ip), and their brains were dissected and
placed for 30-60 s in oxygenated (95% O2-5%
CO2), ice-cold artificial cerebrospinal fluid
(ACSF) containing (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 1.3 MgSO4, 1.4 NaH2PO4, 1.3 CaCl2, and 11 glucose, pH 7.4. The brains were
bisected, and each half was glued on the stage of a vibratome (Campden
Instruments, Lafayette, IN). Four to six 200- to 300-µm-thick slices,
mostly from the middle third of the hippocampus, were cut parallel to
the base of the brain. Slices were trimmed to isolate the hippocampus
and incubated for 1-2 h in oxygenated ACSF at room temperature
(21-23°C) before being transferred into the recording chamber.
Slices were continuously perfused (2 ml/min) with oxygenated ACSF (10 ml, recirculated) containing caged glutamate [L-glutamic
acid, -(
-carboxy-2-nitrobenzyl) ester (250 µM), Molecular
Probes, Eugene, OR]. Whole cell recordings were obtained at
21-23°C. Pipettes were pulled (P-87 Flaming-Brown pipette puller,
Sutter Instruments, Novato, CA) from borosilicate glass capillaries
(KG-33, Garner Glass, Claremont, CA), and filled with a solution
containing (in mM) 140 K-gluconate, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 NaCl, 1 CaCl2, 1 MgCl2, 5 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 4 magnesium
ATP, pH 7.2. Open resistance was 2-4 M
. Whole cell currents were
amplified with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 2 kHz, and digitized at 44 kHz for storage on
videotapes (Neuro-Corder, Neurodata Instruments, New York, NY). Data
were analyzed off-line with sampling rates 5-10 kHz (pClamp 6, Axon
Instruments). The spontaneous EPSCs were detected by defining two
levels with horizontal cursors. One cursor was positioned approximately
in the middle of the baseline noise and the second cursor outside of
the peak noise. Currents with amplitude larger than 50% of the
difference between the two levels were detected (Fetchan, pClamp6, Axon
Instruments). Every detected event was examined and only EPSCs,
characterized by a typical fast raising phase and slow decay phase,
were included in the analysis. The amplitude of each EPSC was measured
by placing a cursor manually at the peak of the raising phase.
Recording and analysis of the electrophysiological data were done
without knowledge of the treatment. Cumulative amplitude distributions
of EPSCs were compared using the Kolmogorov-Smirnov (KS) two-sample
test. The significance of the maximum difference between two cumulative amplitude distributions was determined using a
2 distribution (Siegel 1956
).
The
2 test was also used to test for
differences in the effects of the flash photolysis of caged glutamate
between the different treatment groups. Student-Newman-Keuls ANOVA was
used to assess differences in EPSC frequency. Data are expressed as
means ± SE.
Flash photolysis of caged glutamate
A xenon flash lamp (Chadwick-Helmuth, El Monte, CA) was used to
uncage glutamate (Callaway and Katz 1993). The flash of
ultraviolet (UV) light was transmitted through the epifluorescence
attachment of an upright Optiphot microscope (Nikon), mounted
upside-down. A high-numerical aperture, oil-immersion objective (×40,
Nikon) focused the flash of UV light approximately 200 µm into the
tissue. The optical components were mounted on an X-Y stage to move the objective underneath the transparent bottom (coverslip) of the recording chamber. Location of the objective under the slice (i.e., the
location of the photostimulation) was determined with a HeNe laser,
mounted on the epi-fluorescence attachment and aimed directly through
the objective into the tissue. The light produced by the laser was
aligned with the center of the field and focused to a small spot
(25-50 µm). Slices were viewed with a monochrome charge-coupled
device (CCD) camera (Cohu, San Diego, CA). A video monitor was used to
determine the location of the spot of laser light and of each recorded
cell in the slice (approximated as the point of entry in the tissue of
the recording electrode). The distance between recorded cells and the
spot of laser light was measured with a calibrated grid superimposed on
the video monitor. Preliminary experiments showed that the spatial
resolution of the photostimulation was dependent on the intensity of
the flash and also on the concentration of the caged glutamate. With a
flash intensity of 50-100 mJ and a concentration of caged glutamate of
250 µM, the spatial resolution was approximately 100 µm. Therefore to optimize the probability of finding local circuits,
photostimulations were applied at sites 150 µm apart, throughout the
entire extent of the granule cell layer. To determine whether the
increase in synaptic input produced by flash photolysis of caged
glutamate was mediated by action potential firing, control experiments
were done with tetrodotoxin (TTX) in the CA3 area where the presence of
recurrent excitatory connections between pyramidal cells has been
established (Miles and Wong 1986
). In all the pyramidal
cells tested (n = 7, from 4 untreated rats), TTX (2 µM) blocked the increase in excitatory synaptic evoked by
photostimulation (Fig. 2). This supports
earlier work using glutamate microdrops (Christian and Dudek
1988a
) that changes in EPSCs frequency produced by photolysis of caged glutamate are due to firing in presynaptic cells evoked by
glutamate.
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Staining of mossy fiber sprouting
A modified Timm histological procedure was used to label the
zinc-containing axons of the granule cells (Babb et al.
1991; Patrylo and Dudek 1998
). Slices were
processed with the Timm stain for sulfide precipitation of zinc and
counterstained with cresyl violet. Slices treated with the Timm
procedure included slices that were placed in fixative directly after
the dissection, and slices processed after completion of the
electrophysiological experiments. All the sections were coded, and the
intensity of the Timm stain in the IML was graded with blind procedures
according to the rating scale of Tauck and Nadler
(1985)
. The grades of all the sections were added and divided
by the total number of sections to determine a Timm score for each animal.
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RESULTS |
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The Timm stain and whole cell patch-clamp recordings, combined with flash photolysis of caged glutamate, were used to test the following specific hypotheses: 1) the intensity of the Timm stain in the IML increases with time after kainate treatment, 2) the amplitude and frequency of spontaneous EPSCs increase with time after kainate treatment, 3) the number of granule cells responding to locally applied flash photolysis of caged glutamate increases with time after treatment, and 4) in conditions of reduced inhibition [i.e., bicuculline (30 µM) and [K+]o (6 mM)], the probability of epileptiform bursts of action potentials increases with time after treatment.
Progression of staining in the IML with time after treatment
To test the hypothesis of a progressive increase of the Timm
stain in the IML, sections obtained from the three
post-kainate-treatment and post-saline-injection groups were processed
with the Timm's method and counterstained with cresyl violet. A total
of 3,648 sections was processed and scored on a 0-3 rating scale
(Tauck and Nadler 1985) (Fig.
3). A small proportion (4-7%) of the
sections from the control animals displayed a thin, sometimes
discontinuous band of black reaction product in the IML (i.e., grade
1). During the first 2 wk after kainate treatment, most sections (71%)
did not show any Timm stain in the IML. However, a substantial
proportion of sections (28%) already showed some degree of Timm stain
in the IML (grade 1 and grade 2), and surprisingly, a small fraction (1%) had robust staining of the IML (grade 3). Between 2 and 4 wk
after kainate treatment, 14% of sections did not show any Timm stain
in the IML. Most sections (75%) showed Timm stain in the IML of grade
1 and grade 2. The proportion of sections showing robust Timm stain in
the IML (grade 3) increased substantially (11%), compared with the
results obtained during the first 2 wk. Of the sections obtained from
the animals 10-51 wk after kainate treatment (average, 190 ± 14 days), only 1% did not show any detectable Timm stain in the IML. In
this group, most sections (67%) showed robust Timm stain in the IML
(grade 3). The overall Timm score (see METHODS) for all
sections obtained from control animals was 0.05 ± 0.01. For the
sections obtained from kainate-treated animals during the first 2 wk
after treatment, the Timm score was 0.36 ± 0.13. This score
increased to 1.33 ± 0.09 in sections obtained 2-4 wk after
treatment and to 2.6 ± 0.1 in sections from rats 10-51 wk after
treatment (average, 190 ± 14 days). These data suggest that Timm
stain in the IML is already apparent 2 wk after kainate treatment, and
that its intensity increases with time after the kainate-induced
injury.
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Spontaneous EPSCs
To test the hypothesis that the amplitude and frequency of spontaneous EPSCs increase with time after kainate treatment, whole cell recordings were obtained from granule cells at resting membrane potential (Fig. 4). The amplitude and frequency of EPSCs in granule cells from kainate-treated and age-matched control groups were measured during 120-s periods for each cell and compared at three different times after treatment. Recordings were obtained from 8 granule cells in 7 slices from 6 kainate-treated rats 1-2 wk after treatment, 9 granule cells in 6 slices from 6 kainate-treated rats 2-4 wk after treatment, and 10 granule cells in 7 slices from 7 rats 10-51 wk after kainate treatment (average, 162 ± 30 days). Recordings from control, saline-injected animals were obtained from five granule cells in five slices from five animals 1-2 wk after injection, five granule cells in five slices from five animals 2-4 wk after injection, and six granule cells in five slices from five animals 17-72 wk after injection (average, 233 ± 47 days).
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Amplitude of spontaneous EPSCs
Cumulative amplitude distributions showed a shift toward larger
EPSC amplitude in granule cells with time after kainate treatment (Fig.
5, A and B).
Comparisons between cumulative amplitude distributions for spontaneous
EPSCs in granule cells from kainate-treated animals using the KS test
(2 samples, 1-tailed) revealed a highly significant increase in the
EPSC amplitude between all three groups of animals treated with kainate
(P < 0.001 for all comparisons between kainate-treated groups, 2 test). In Fig. 5, A and
B, the EPSCs from the control groups were lumped in one
plot. However, statistical comparisons with controls were made between
kainate-treated animals and aged-matched controls. Comparison between
experimental and control groups showed highly significant differences
in all three groups (P < 0.001 for comparisons between
all kainate-treated groups and aged-matched controls, KS test, 2 samples, 1-tailed). To test for a potential change in the EPSC
amplitude with age in the control groups, cumulative probability plots
were constructed for granule cells from each control group and compared
with each other (Fig. 5C). The KS test (2 samples, 2-tailed)
showed no significant difference in the EPSC amplitude between any of
the control groups (P > 0.08 for all comparisons
between control groups). This result suggests that EPSC amplitude is
larger in the kainate-treated animals than in age-matched controls and
that this increase is progressive and begins within the first 2 wk
after the insult produced by kainate treatment.
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Frequency of spontaneous EPSCs
The average frequency of spontaneous EPSCs in granule cells from control animals did not differ significantly with time after saline injection (P > 0.05; Fig. 6). The average frequency of spontaneous EPSCs 1-2 wk after kainate treatment was not significantly different from any of the control groups (P > 0.05). The difference in the average frequency of spontaneous EPSCs between the groups 2-4 wk and 10-51 wk (average, 162 ± 30 days) after kainate treatment was not significant (P > 0.05), but the average frequency in both groups was significantly higher than the frequency in all the other groups (P < 0.05). These data suggest that the increased frequency of EPSCs observed in granule cells from kainate-treated rats was induced by kainate and was not related to the age of the animals since the frequency of EPSCs in granule cells from control animals did not change significantly with time after saline injection. This result is consistent with the hypothesis that granule cells from kainate-treated rats receive input from more excitatory synapses, and/or from more active excitatory synapses than granule cells from saline-injected control animals.
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Relationship between Timm stain in the IML and properties of spontaneous EPSCs
To determine whether the average amplitude and frequency of EPSCs in granule cells from rats studied 10-51 wk after kainate treatment were correlated with the intensity of the Timm stain in the IML, a score for the Timm stain was obtained for each animal (see METHODS), and the average EPSC amplitude and average EPSC frequency were plotted as a function of the Timm score (Fig. 7). Animals with lower Timm score generally had lower amplitude and lower frequency EPSCs, whereas animals with higher Timm score showed a wide range of results for both frequency and amplitude. Several granule cells from animals with high Timm score showed EPSC amplitude and frequency similar to those of granule cells from animals with low Timm score. However, EPSCs of high frequency and large amplitude were seen only in granule cells from animals with high Timm score. Linear regressions showed a significant correlation between Timm score and average EPSC amplitude (Pearson R = 0.69, P < 0.05) and between Timm score and average EPSC frequency (Pearson R = 0.66, P < 0.05). This result supports the hypothesis that increased intensities of Timm stain in the IML are associated with an overall increase of the excitatory synaptic input to granule cells.
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Photostimulation of the granule cell layer
In hippocampal slices with Timm stain in the IML, local
stimulation of the granule cell layer with glutamate microdrops has been shown to evoke repetitive EPSPs in most granule cells (64%) (Wuarin and Dudek 1996). In the present study, we tested
the hypothesis that the proportion of granule cells responding to
glutamate microstimulation with repetitive EPSCs would increase with
time after treatment. Specifically, we hypothesized that 1)
glutamate microstimulation of the granule cell layer would produce
repetitive EPSCs in relatively few granule cells during the first 2 wk
after treatment, 2) this response should be observed in a
larger proportion of granule cells 2-4 wk after treatment, and
3) glutamate microstimulation should evoke repetitive EPSCs
in more granule cells from animals 10-51 wk after kainate treatment
than in either of the other two groups.
Photostimulations were applied every 150 µm throughout the entire extent of the granule cell layer, and the postsynaptic response in granule cells was determined with whole cell recordings at resting membrane potential. Photostimulations produced either no change in the spontaneous EPSCs, or they evoked repetitive EPSCs (Fig. 8). This response varied from a few EPSCs (e.g., Fig. 8B) to periods of repetitive EPSCs lasting several hundreds of milliseconds (Fig. 9). Repeated stimulations of the same area in the granule layer produced consistent responses (Fig. 9). The ratio of the number of cells showing evoked excitatory synaptic responses to the total number of cells tested was determined for each group. A total of 53 granule cells from 23 saline-injected rats were tested with flash photolysis of caged glutamate (Fig. 10). Only one granule cell from an animal belonging to the group 2-4 wk after saline injection (n = 18 granule cells, 7 rats) showed repetitive EPSCs in response to photostimulation. No granule cell showed a change in EPSCs in response to photostimulation 1-2 wk (n = 18 granule cells, 7 rats) and 17-72 wk after saline injection (average, 337 ± 47 days, n = 17 granule cells, 9 rats). Control groups were not significantly different from each other (P = 0.31); therefore they were pooled for comparison. Ninety-two granule cells from 46 kainate-treated rats were tested with photostimulation (Fig. 10). The proportion of granule cells responding to photostimulation of the granule cell layer with repetitive EPSCs 1-2 wk after kainate treatment was not significantly different from controls (3/29 granule cells, 15 rats, P = 0.085). However, this proportion increased significantly to 32% during the period 2-4 wk after treatment (10/31 granule cells, 15 rats, P = 0.04). In the animals 10-51 wk after kainate treatment (average, 202 ± 19 days), photostimulation of the granule cell layer evoked repetitive EPSCs in 66% of the granule cells tested (21/32 granule cells, 16 rats, P = 0.008). This result supports the hypothesis that the number of functional excitatory connections between granule cells increases with time after kainate treatment.
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Photostimulation-evoked epileptiform bursts
In conditions of reduced inhibition and increased
excitability, hippocampal slices from rats 4-13 mo after kainate
treatment have been shown to generate epileptiform bursts of action
potentials in response to electrical stimulation of the hilar region
(Patrylo and Dudek 1998; Wuarin and Dudek
1996
). In the present study we tested the hypothesis that, in
conditions of reduced inhibition, the probability of evoking
epileptiform bursts of action potentials in the dentate gyrus would be
relatively low during the first few weeks after kainate treatment
compared with several months after treatment (i.e., when the animals
had become epileptic). To evoke epileptiform bursting in the dentate
gyrus, we used photostimulation of the granule cell layer in the
presence of bicuculline (30 µM) to reduce inhibition, and high
[K+]o (6 mM) to increase
the probability of multisynaptic interactions. Current-clamp recordings
of the electrical activity of granule cells were obtained in the whole
cell configuration. In these conditions, photostimulation did not evoke
bursts of action potentials in any of the granule cells recorded from
saline-injected animals (0/8 granule cells, 7 rats). Four granule cells
from 3 kainate-injected animals 1-2 wk after treatment and 13 granule
cells from 7 kainate-injected rats 2-4 wk after treatment were tested
with photostimulation applied throughout the granule cell layer.
Photostimulation of the granule cell layer did not evoke bursts of
action potentials in any of the granule cells tested during the first 4 wk after kainate treatment. In contrast, photostimulation of the
granule cell layer evoked epileptiform bursts of action potentials in all the granule cells tested from rats 10-51 wk after kainate treatment (average, 209 ± 11 days, 11/11 granule cells, 7 rats; Fig. 11). Bursts of action potentials
of similar duration were recorded regardless of the distance between
the photostimulations and the recorded cell (e.g., recorded granule
cell in the inner blade and photostimulation applied in the outer
blade). Hyperpolarization of the recorded cells decreased the number of
action potentials, but did not block the bursts (not shown). These
observations suggest that photostimulation-evoked firing of a
relatively small fraction of the granule cell population in hippocampal
slices from rats 10-51 wk after kainate treatment can induce the
entire population of granule cells to fire epileptiform bursts when
inhibition is depressed. This experimental approach revealed bursting
activity only in slices from animals 10-51 wk after kainate treatment
and not in slices from kainate-treated rats tested within the first 4 wk after treatment, suggesting that the dentate gyrus becomes more
susceptible of generating epileptiform activity with time after
treatment.
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DISCUSSION |
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The present study provides several lines of evidence supporting the hypothesis that kainate treatment induces the progressive formation of new excitatory synapses between granule cells leading to an increased probability of epileptiform activity in the granule cell layer. The intensity of the Timm stain in the IML increased with time after treatment; this change in mossy fiber sprouting was correlated with an increase of both amplitude and frequency of spontaneous EPSCs in granule cells. The number of granule cells showing repetitive EPSCs in response to photostimulation of the granule cell layer increased dramatically with time after kainate treatment, and epileptiform bursts were observed only in slices with robust Timm stain in the IML from animals tested 10-51 wk after treatment.
Increased excitatory input
The first main finding of this study is the demonstration of a progressive increase in amplitude and frequency of spontaneous EPSCs with time after kainate treatment. A significant shift toward larger amplitude was already observed within 2 wk after kainate treatment. This trend continued during the 2- to 4-wk period after kainate treatment. In the animals studied 10-51 wk after kainate treatment, most EPSCs were larger than 10 pA. The frequency of spontaneous EPSCs in granule cells from both controls and kainate-treated animals was relatively low; therefore summation was unlikely to influence significantly the amplitude measurement of the EPSCs. No significant change in the frequency of EPSCs was observed during the first 2 wk after kainate treatment, but the frequency of spontaneous EPSCs more than doubled 2-4 wk after kainate treatment and was even higher in the animals 10-51 wk after treatment. No significant change in EPSC amplitude or frequency was observed with time after saline injection, indicating that the increase in excitatory input to granule cells from kainate-treated animals was not related to animal age but was caused by the kainate treatment.
Since kainate treatment has been shown to produce extensive cell loss
in the hilus (e.g., Buckmaster and Dudek 1997b) and also
probably in the entorhinal cortex (Eid et al. 1999
),
part of the excitatory input to granule cells is expected to be lost early after treatment, and thus one might expect a decrease in the
frequency of EPSCs during the first few days after the treatment. The
absence of a significant difference in the frequency of EPSCs between
controls and rats tested during the first 2 wk after kainate treatment
may result from a sampling problem (i.e., too few EPSCs sampled).
Alternatively, as synaptic terminals from dying hilar neurons
disappear, some of them may be replaced relatively rapidly by new
synapses from sprouted mossy fibers. The presence of large-amplitude EPSCs during the first 2 wk after kainate treatment supports the idea
that new synapses may be formed early after the kainate-induced injury.
Mossy fiber sprouting
Timm stain in the IML was detectable as early as 2 wk after
treatment and was robust in most sections from rats 10-51 wk after kainate treatment. Recent studies have shown that mossy fiber sprouting
can occur within the first 2 wk after kainate treatment, and its
expression seems more pronounced in the temporal pole of the
hippocampus (Buckmaster and Dudek 1997b;
Cantallops and Routtenberg 1996
; Hellier et al.
1999
; Simpson et al. 1997
). Data derived from
the pilocarpine model showed EPSCs of higher frequency and amplitude in
granule cells from slices with more intense Timm stain in the IML
(Simmons et al. 1997
). Sutula et al.
(1998)
showed that in longitudinal slices from kainate-treated
rats, sprouted axons in the inner molecular layer can extend 600-700
µm in the septotemporal axis. Corroborating this in vitro study,
reconstruction of the axonal arbor of granule cells from
kainate-treated rats recorded in vivo and labeled with biocytin
revealed that the septotemporal span of sprouted axons in the IML was
600 µm (Buckmaster and Dudek 1999
). We therefore
hypothesized that the spontaneous EPSCs detected in granule cells from
kainate-treated rats originated not only in neurons located in the
slice but also from sprouted axons originating from granule cells
outside of the slice, which were cut during the preparation of the
slice. In consequence, we used a method to evaluate the intensity of
the Timm in the IML stain throughout the hippocampus. We found that
granule cells with spontaneous EPSCs of small amplitude and low
frequency were from animals with low Timm score and that higher EPSC
frequency and amplitude were generally associated with higher Timm
score. Synaptic projections cut during the slice preparation may
explain the few granule cells with low-frequency and small-amplitude
EPSCs recorded in slices from animals with high Timm score. It is also
likely that synaptic reorganization induced by kainate treatment did
not result in increased excitatory input to all the granule cells.
Our observation that changes in the EPSCs were correlated with
increased mossy fiber sprouting in the IML supports the notion that
this progressive increase in excitatory input to granule cells is due,
at least in part, to mossy fiber sprouting.
Focal stimulation
The second result of this study is that the number of granule
cells responding to photostimulation of the granule cell layer with
repetitive EPSCs increased with time after kainate treatment. This
approach was based on research by Katz and co-workers (Callaway and Katz 1993; Dalva and Katz 1994
; Katz
and Dalva 1994
) demonstrating that photostimulation of caged
glutamate can be used to map local neuronal circuitry in mammalian
brain slices. Glutamate is known to activate selectively the
somatodendritic area, but does not generate action potential firing
when applied to axons (Christian and Dudek 1988a
).
Previous work has shown that glutamate microdrops applied in the
granule layer of hippocampal slices from kainate-treated rats can evoke
repetitive EPSPs in granule cells (Lynch and Sutula 2000
; Wuarin and Dudek 1996
), and minimal laser
photostimulation of the granule cell layer from pilocarpine-treated
rats could evoke single EPSCs in granule cells (Molnar and
Nadler 1999
). Taken together, these studies strongly support
the hypothesis that both kainate and pilocarpine treatments result in
the formation of an extensive excitatory network. In the present study,
we showed that the mossy fiber sprouting in the IML, as well as the
amplitude and frequency of spontaneous EPSCs, increased with time after kainate treatment. We next tested whether there was a parallel increase
in the density of the putative excitatory network between granule
cells. With flash photostimulation combined with whole cell patch-clamp
recording, we found that <2% (1/53) of the granule cells tested from
saline-injected animals showed an increase in EPSCs in response to
flash photolysis of the granule cell layer. In contrast, within 2 wk
after kainate treatment, the proportion of granule cells showing
multiple EPSCs in response to photostimulation was 10%. This
proportion increased to 32% by 4 wk and reached 66% in the animals
10-51 wk after kainate treatment. These data suggest that functional
excitatory synapses between granule cells are formed relatively early
after a kainate-induced injury and that the number of these connections
(i.e., the number of granule cells connected together through
excitatory synapses) increases progressively over time.
Temporal change in seizure frequency after injury
A central concept in temporal lobe epilepsy is that 1)
a latent period (i.e., a period without chronic seizures) occurs after an injury before the onset of recurrent spontaneous seizures and 2) the frequency of seizures tends to increase with time
after the precipitating insult. These characteristics of temporal lobe epilepsy and post-traumatic epilepsy suggest that the injury triggers a
time-dependent process that increases the probability that epileptic seizures will occur. We hypothesize that after the kainate-induced injury, the granule cell layer becomes progressively more capable of
generating epileptiform bursts as the number of newly formed excitatory
connections between granule cells increases. Since inhibition has been
shown to mask multisynaptic excitatory circuits (Christian and
Dudek 1988a,b
; Dichter and Spencer 1969a
,b
;
Miles and Wong 1983
, 1986
,
1987
; Miles et al. 1984
; Patrylo
and Dudek 1998
), we tested this hypothesis in conditions of
reduced inhibition. We used photostimulation of the granule cells to
evoke activity in a few granule cells, and we then determined whether
this localized firing could propagate synaptically through the granule
cell layer and generate epileptiform bursts. Photostimulation in
conditions of reduced inhibition did not evoke bursts of action
potentials in granule cells from control rats or in granule cells from
rats tested within the first 4 wk after kainate treatment. In contrast, photostimulation of the granule cell layer evoked epileptiform bursts
in all the granule cells tested from rats 10-51 wk after kainate
treatment. Although Timm stain in the IML was already present in slices
from the animals 1-4 wk after kainate treatment, it was generally more
robust in slices from the animals 10-51 wk after treatment. This
result is consistent with the hypothesis that with increased mossy
fiber sprouting and synaptic connectivity, the granule cell layer
becomes more susceptible to ictal-like events when inhibition is depressed.
Evidence for new recurrent excitatory circuits versus alternative mechanisms
When considered separately, part of the data in this study can be interpreted as resulting from a number of changes other than formation of excitatory circuits between granule cells. For example, increased amplitude and frequency of EPSCs support only indirectly the notion of increased excitatory synaptic input. Postsynaptic mechanisms, including changes in cell membrane properties and/or changes in glutamate receptor number and/or function, could explain both the increase in frequency and amplitude of spontaneous EPSCs. Based only on this set of data, the argument can be made that the apparent increase in synaptic input may result not only from new excitatory connections between granule cells, but also from changes in synaptic projections from other areas including entorhinal cortex, hilus, and CA3 (Fig. 12). Furthermore, the data obtained with flash photolysis do not prove the presence of excitatory connections between granule cells. Dual intracellular or whole cell patch-clamp recordings are needed to provide the unequivocal demonstration of functional synaptic contacts between granule cells. However, postsynaptic changes and increased inputs from other areas are not consistent with the results from the photostimulation experiments. The most plausible interpretation, however, for the repetitive EPSCs observed in granule cells from kainate-treated rats in response to focal stimulation of the granule cell layer is probably the presence of new excitatory synapses between granule cells. The increased susceptibility to generate seizure-like activity in slices from animals of the long-term group could result, for example, from increased K+ sensitivity, decreased levels of inhibition or an increased number of gap junctions between granule cells. In our experiments with high [K+]o and bicuculline, epileptiform bursting was observed only weeks to months after treatment. There is little or no evidence to suggest or a priori reason to believe that changes in K+ sensitivity, formation of new gap junctions, loss of inhibition, or other related hypothesized mechanisms would occur progressively over time after kainate-induced status epilepticus; however, several lines of evidence suggest that mossy fiber sprouting and new recurrent excitatory circuits would develop progressively over weeks and months.
|
The strongest support of the hypothesis that kainate treatment induces
an epileptogenic synaptic reorganization comes from the combination of
the three main lines of results. The association of a progressively
more robust degree of Timm staining in the IML with 1)
spontaneous EPSCs of large amplitude and high frequency, 2)
repetitive EPSCs in response to photostimulation of the granule cells
layer, and 3) epileptiform bursting in conditions of reduced inhibition, strongly support the notion of a time-dependent synaptic reorganization between granule cells. The data obtained in bicuculline and high [K+]o confirm
that inhibition can suppress these potential excitatory circuits
because epileptiform activity was detected only when bicuculline was
present and [K+]o was
elevated. The results from the flash photolysis experiments support and
expand on the work of Molnar and Nadler (1999) in the
pilocarpine model by showing that the number of excitatory connections
from granule cells to granule cells probably increases over a
relatively long period of time. This preliminary description of changes
at the level of a neuronal network at different times after kainate
treatment should contribute to a better understanding of the mechanisms
underlying the latent period between insult and the appearance of
seizures, and the progressive increase in seizure frequency with time
after kainate treatment.
Conclusions
These results suggest that the kainate-induced injury triggers a
process leading to increased seizure susceptibility of the dentate
gyrus. A number of factors, including formation of new gap junctions
between granule cells, altered inhibitory input to granule cells, and
changes in glutamate and GABA receptor properties may contribute to the
increased seizure susceptibility. However, our data indicate that
synaptic reorganization plays a central role in the time-dependent
changes in seizure susceptibility that follow an injury, such as the
one produced by kainate-induced status epilepticus. Recent work has
shown that kainate and pilocarpine treatments evoke axonal sprouting in
CA1 pyramidal neurons cells (Esclapez et al. 1999;
Perez et al. 1996
), an increase in the spontaneous
glutamatergic input to pyramidal cells (Esclapez et al.
1999
), and an increased propensity to generate all-or-none, network bursts when inhibition is depressed (Meier and Dudek
1996
; Smith and Dudek 2001
). This suggests that
injury-induced synaptic reorganization can produce epileptiform
bursting not only in the dentate gyrus but also in other brain areas.
The general hypothesis can be proposed that injury-induced synaptic
reorganization includes the progressive formation of new recurrent
excitatory synapses resulting in an increased seizure probability.
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ACKNOWLEDGMENTS |
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We thank P. Dou for technical assistance and E. A. Swiss for word processing.
This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-16683 (F. E. Dudek) and NS-32662 (J.-P. Wuarin).
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FOOTNOTES |
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Address for reprint requests: F. E. Dudek (E-mail: edudek{at}cvmbs.colostate.edu).
Received 12 June 2000; accepted in final form 20 November 2000.
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REFERENCES |
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