Department of Pharmacology and Cancer Biology and Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Okazaki, Maxine M., Péter Molnár, and J. Victor Nadler. Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth. A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in ~74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.
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INTRODUCTION |
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Temporal lobe epilepsy is the most common form of epilepsy in the
adult population, afflicting 800,000 Americans. Despite years of
study, the etiology of this condition is poorly understood. Roughly
two-thirds of persons with temporal lobe epilepsy have lesions in one
or both hippocampi (Meldrum and Bruton 1992
). The most
consistent pathological finding in these brains is a dramatic loss of
interneurons from the hilus of the dentate gyrus, a condition referred
to as "endfolium sclerosis" (Margerison and Corsellis 1966
). This lesion appears to precede the seizures in many
cases and is postulated to facilitate seizure generation and
propagation (Sloviter 1994
). There are a number of ways
in which endfolium sclerosis could lead to seizures. One of these is
the lesion-induced growth of excitatory axon collaterals, with
subsequent formation of novel feedback circuitry.
The mossy fibers are axons of the hippocampal dentate granule cells.
The mossy fiber pathway normally projects to the pyramidal cells of
hippocampal area CA3 and to interneurons of the CA3 area and dentate
hilus; it is thought to make few, if any, recurrent synapses onto
granule cells. A common feature of temporal lobe epilepsy (Babb
et al. 1991; Franck et al. 1995
; Sutula
et al. 1989
) and of animal models of temporal lobe epilepsy
(Okazaki et al. 1995
; Represa et al.
1993
; Sutula et al. 1988
) is the development of
numerous mossy fiber-granule cell synapses. These synapses mediate
monosynaptic recurrent excitation that is normally weak or absent
(Cronin et al. 1992
; Golarai and Sutula
1996
; Masukawa et al. 1992
; Patrylo and
Dudek 1998
; Tauck and Nadler 1985
; Wuarin and Dudek 1996
). Novel innervation of this type is expected to have profound consequences for hippocampal function. Dentate granule cells have been shown to resist the propagation of seizures through the
limbic circuit (Collins et al. 1983
; Lothman et
al. 1992
). Several properties of granule cells and their
connectivity appear to contribute to this high resistance, including
the lack of intrinsic capacity for burst discharge, strong tonic
inhibition from GABA interneurons, and absence of a synaptic mechanism
for synchronization of burst discharge. Granule cell discharge can be
synchronized by nonsynaptic mechanisms (Schweitzer et al.
1992
), but this occurs only during very strong afferent
bombardment that is likely to compromise synaptic inhibition
(Lothman et al. 1992
). Synaptic interconnections serve
as the anatomic substrate for synchronization of CA3 pyramidal cell
discharge (Miles et al. 1984
). Area CA3 is one of the
major sites of epileptiform bursting and seizure initiation in the
limbic system. By analogy, the recurrent mossy fiber pathway, if
sufficiently powerful, could facilitate the synchronized firing of
granule cells, thereby enhancing their participation in seizures.
However, the recurrent mossy fibers also may drive GABA
inhibition. Some of these fibers appear to contact GABA interneurons (Kotti et al. 1997; Sloviter 1992
). They
have been suggested to provide a critical excitatory drive needed to
activate these neurons that normally is provided by the
associational-commissural pathway (which degenerates in the lesioned
hippocampal formation) (Sloviter 1991
). It also has been
suggested that mossy fiber terminals in the epileptic brain release
GABA in addition to glutamate, thus becoming at once both excitatory
and inhibitory (Sloviter et al. 1996
). Furthermore, GABA
interneurons may form additional synapses on granule cells
(Davenport et al. 1990
; Mathern et al.
1995
). Under the right conditions, strong recurrent inhibition
mediated by the granule cell-GABA interneuron-granule cell circuit
could facilitate seizures by assuring that granule cells fire
synchronously (Freund and Buzsáki 1996
). At other
times, enhanced recurrent inhibition may suppress the emergence of seizures.
This study was undertaken to examine the cellular electrophysiological
effects of activating the recurrent mossy fiber pathway. We recorded
the excitatory postsynaptic current (EPSC) and inhibitory postsynaptic
current (IPSC) evoked in granule cells by antidromic stimulation of the
mossy fibers at two survival times after pilocarpine-induced status
epilepticus: 10 wk, a time when mossy fiber growth is essentially
complete, and 4-6 days, a time when Timm histochemistry reveals no
evidence of mossy fiber growth (Okazaki et al. 1995
). For comparison, similar studies were performed with stimulation of the
perforant path.
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METHODS |
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Pilocarpine-induced status epilepticus
Male Sprague-Dawley rats (150-200 g; Zivic-Miller Laboratories,
Allison Park, PA) received a single injection of pilocarpine hydrochloride (330-360 mg/kg ip). The animals were pretreated 30 min
earlier with scopolamine methyl bromide and terbutaline hemisulfate (2 mg/kg ip, each) to block peripheral side effects and maintain
respiration. Not all pilocarpine-treated rats developed status
epilepticus. If status epilepticus, defined as a continuous limbic
motor seizure of stage 2 or higher (Racine 1972),
developed, it was terminated 3-4 h after onset with a single injection
of phenobarbital sodium (50 mg/kg ip). Pilocarpine-treated rats that exhibited only a few brief seizures, but did not develop status epilepticus, were used as drug-treated controls. Studies of this treatment group accounted for any possible effects of pilocarpine not
associated with status epilepticus. Age-matched rats, either untreated
or injected with saline, were used as untreated controls.
Preparation and incubation of hippocampal slices
The rat was decapitated under ether anesthesia, the brain was removed, and transverse 400-µm-thick slices of the caudal hippocampal formation were cut with a vibratome. Most of the slices were trimmed and transferred to a holding chamber that contained artificial cerebrospinal fluid [ACSF, which contained (in mM) 122 NaCl, 25 NaHCO3, 3.1 KCl, 1.8 CaCl2, 1.2 MgSO4, 0.4 KH2PO4, and 10 D-glucose, pH 7.4] continuously gassed with 95% O2-5% CO2 at room temperature. Adjacent slices were immersed in 0.1% (wt/vol) Na2S, 0.1 M sodium phosphate buffer, pH 7.3, and set aside for Timm and Nissl staining.
Stimulation and recording
Slices used for electrophysiological recording corresponded to
horizontal plates 98-100 of Paxinos and Watson (1986).
Beginning 1.5 h after preparation, individual slices were
transferred to a small experimental chamber, placed on a nylon net and
barely submerged in the superfusion medium. The superfusion rate was 1.5 ml/min. The monopolar stimulating electrode was a 25-µm-diam nichrome wire insulated to the tip with a polymerized polyvinyl resin
(Formvar). It was placed in stratum lucidum of area CA3b >100 µm
from the opening of the dentate hilus. Then an extracellular electrode
fashioned from borosilicate glass (filled with 1 M NaCl; resistance of
2-6 M
) was used to probe for the location in the granule cell body
layer where the antidromic population spike was of maximal amplitude.
With the extracellular recording electrode positioned at this location,
the stimulating electrode was moved perpendicular to the pyramidal cell
body layer until stimulation evoked an antidromic population spike of
the greatest possible amplitude. The antidromically evoked response
declined dramatically when the stimulating electrode was moved even
slightly away from stratum lucidum. The final optimization of the
antidromic population spike amplitude was achieved by adjusting the
depth of both the stimulating and recording electrodes. The stimulus
current was set to a near-maximal value (490 µA) and rectangular
pulses of 100-µs duration were applied every 30 s.
Whole cell patch-clamp recordings were made from dentate granule cells
located close to the extracellular recording electrode. The patch
electrode was fashioned from Sutter (Novato, CA) borosilicate glass
pulled to a narrowly tapered tip with a resistance of 5-8 M. The
tip was filled by vacuum with a solution that contained (in mM) 140 cesium gluconate, 15.5 HEPES, and 3.1 MgCl2, pH 7.2 and
276-277 mosm. The electrode then was backfilled with the internal solution, which consisted of (in mM) 120 cesium gluconate, 10 HEPES, 2 MgATP, and 10 QX-314 (N-ethyl lidocaine) chloride, pH 7.2 and 276-277 mosm. Seals were formed by the "blind" approach (Blanton et al. 1989
), and whole cell access was
obtained in current clamp mode. Only cells with
Vm more than
60 mV on break-in were accepted
for study. The liquid junction potential was determined to be 10 mV
with use of the method described by Neher (1992)
. This
value was subtracted from all membrane potentials. Whole cell
recordings were made with an Axon Instruments (Foster City, CA)
Axopatch 200A patch clamp amplifier beginning 15-20 min after break-in. Membrane resistance was determined in current clamp mode by
injecting 50-pA hyperpolarizing rectangular pulses of 200-ms duration
just before the start of recording. Series resistances ranged from 8 to
20 M
and were compensated
75%. The series resistance did not
change by >20% during the experiment. Signals were filtered <2 kHz,
digitized at 10 kHz, and stored to disk with use of a Digidata board
and PClamp 6 running on a Micron P-90 or P-100 microcomputer. Normally,
10 sequentially obtained traces were averaged, and the averaged
waveforms were analyzed off-line with functions incorporated in PClamp
6. Unless stated otherwise, all traces presented in the figures are
averaged records.
The antidromically evoked EPSC was studied at a holding potential of
80 mV in the presence of 30-100 µM bicuculline methiodide or 50 µM picrotoxin and was defined as the inward current abolished by 50 µM D-2-amino-5-phosphonopentanoate (D-AP5)
and either 20 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 5 µM
2,3-dihydroxy-6-nitro-7-sulfamyl-benzo(F)quinoxaline (NBQX). The
antidromically evoked (feedback or mossy fiber-driven) GABAA IPSC was studied at a holding potential of 0 mV and
was defined as the outward current abolished by bicuculline methiodide or picrotoxin. Activation of postsynaptic GABAB receptors
was prevented by the use of a cesium-based internal solution that included QX-314 but not GTP.
Responses to perforant path stimulation were studied in a similar
fashion with the following modifications. The stimulating electrode was
placed in the subiculum just across the hippocampal fissure from the
dentate molecular layer. An extracellular recording electrode then was
positioned in that portion of the granule cell body layer where the
amplitude of the postsynaptic response was greatest. After optimizing
the depth of both electrodes, the stimulus current was adjusted to a
value just below that which evoked a population spike. Finally, a whole
cell patch-clamp recording was established from a granule cell in close
proximity to the extracellular recording electrode. The feed forward
(or perforant path-driven) GABAA IPSC was defined as
described above for the feedback GABAA IPSC. In these
experiments, the AMPA/kainate-receptor- and NMDA-receptor-mediated
components of the perforant path EPSC routinely were discriminated and
analyzed separately. The NMDA component was studied at a holding
potential of 20 mV and was defined as the inward current recorded in
the presence of 30 µM bicuculline methiodide that was abolished by
addition of 50 µM D-AP5 to the superfusion medium. The
AMPA/kainate component was studied at a holding potential of
80 mV
and was defined as the inward current recorded in the presence of 30 µM bicuculline methiodide and 50 µM D-AP5 that was
abolished by addition of 20 µM DNQX to the superfusion medium.
Histology
Slices set aside for Timm and Nissl staining remained in
the Na2S solution for 90 min and then were fixed in
phosphate-buffered 0.9% (wt/vol) saline that contained 10% formalin.
After storage in 10% formalin at 4°C for 1-2 days, the slices were
embedded in albumin-gelatin and cut into 30-µm-thick sections with a
Vibratome. Alternate sections were mounted on slides coated with chrome
alum-gelatin, stained for the presence of heavy metals as described by
Danscher (1981), and lightly counterstained with cresyl
violet. The remaining sections were stained with cresyl violet.
Sections used for histological analysis were those that corresponded to
horizontal plates 101-103 of Paxinos and Watson (1986)
,
the region of the hippocampal formation immediately rostral to the
slices used for electrophysiological recording.
Histological analyses were performed without knowledge of the
experimental treatment or electrophysiological data. Hilar neurons were
counted in two cresyl violet-stained sections, and the results were
averaged. The hilar area related to the dentate gyrus (corresponding to
area CA4 of Lorente de Nó 1934) was outlined with
the aid of a camera lucida. Only cells within this area with a diameter that exceeded 10 µm were included in the count, thus excluding glia
and displaced granule cells. Neurons at the lower edge of the granule
cell body layer (e.g., basket cells) also were excluded. The
cross-sectional area in which neurons were counted was measured with
use of NIH Image software. Hilar neuron density was calculated by
dividing the number of neurons counted by the cross-sectional area of
the counting region.
A separate group of 23 rats that had experienced 3-4 h of
pilocarpine-induced status epilepticus and 9 pilocarpine-treated control rats was used to determine the extent of neuronal degeneration in the hippocampal formation. The animals were anesthetized deeply with
pentobarbital sodium 1 day after pilocarpine treatment and perfused
transcardially with phosphate-buffered 0.9% (wt/vol) saline for 1 min
followed by 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate
buffer, pH 7.4. After 3-20 days of postfixation at 4°C, the brains
were cut into 40-µm-thick frozen coronal or horizontal sections.
Alternate sections were stained with cresyl violet or impregnated with
silver to visualize degenerating neuronal somata and terminals
(Nadler and Evenson 1989).
Materials
D-Gluconic acid lactone, HEPES, phenobarbital
sodium, pilocarpine hydrochloride, ()scopolamine methyl bromide, and
terbutaline hemisulfate were purchased from Sigma Chemical (St. Louis,
MO). D-AP5 and DNQX were purchased from Tocris Cookson
(Bristol, UK), bicuculline methiodide from Research Biochemicals
(Natick, MA), and cesium hydroxide (99.9%; 50 wt%) from Aldrich
(Milwaukee, WI). QX-314 chloride was obtained from Astra USA
(Westborough, MA) and Alomone Labs (Jerusalem, Israel). NBQX was a gift
from Novo Nordisk (Måløv, Denmark).
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RESULTS |
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Antidromically evoked EPSCs were recorded more frequently and were sometimes larger in slices from rats that had developed status epilepticus
The antidromically evoked inward current recorded at a holding
potential of 80 mV consisted of bicuculline-sensitive and -insensitive components (Fig. 1,
top). In each of 49 cells, the bicuculline-insensitive
component was abolished or nearly abolished by addition of 20 µM DNQX
and 50 µM D-AP5 to the superfusion medium (Fig. 1,
bottom). It thus was taken to represent the antidromically evoked EPSC. The EPSC typically exhibited slow response kinetics that
could not be described adequately by a monoexponential rise and decay.
In some instances, multiple components clearly were present. The
bicuculline-sensitive inward current represented the
GABAA-receptor-mediated feedback IPSC. The IPSC was
directed inwardly at a holding potential of
80 mV because
ECl (calculated as described by Staley and Mody
1992
) was about
54 mV under the conditions of our
experiments.
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A small, brief biphasic current recorded just before the EPSC coincided with the simultaneously recorded antidromic population spike, and its amplitude varied with the antidromic spike amplitude (Fig. 1). This "inflection" of the antidromic spike overlapped the onset of the EPSC (and sometimes the IPSC), and it could not be adequately removed by electronic subtraction of traces in the presence and absence of bicuculline or in the presence and absence of DNQX/D-AP5. Thus the latency to onset of the antidromically evoked EPSC could not be accurately determined.
Antidromically evoked EPSCs were observed most frequently in dentate granule cells from long-term survivors of pilocarpine-induced status epilepticus. The success rate in these animals was ~74% (Fig. 2, Table 1). However, antidromic stimulation at this survival time also evoked an EPSC in ~38% of granule cells from pilocarpine-treated control rats and in ~26% of granule cells from age-matched untreated or saline-treated control rats. The peak amplitudes of 9 of the 25 antidromically evoked EPSCs from the status epilepticus group were greater than the peak amplitude of the largest EPSC recorded from any control granule cell; 4 were >100 pA. Because of this cohort of unusually large responses, the mean peak amplitude of the antidromically evoked EPSC was about three times as large as control in granule cells from the status epilepticus group. The largest EPSCs were not always associated with especially robust mossy fiber growth, as indicated by Timm histochemistry. Among the long-term survivors, there were no significant between-group differences in either the membrane potential on break-in, input resistance or amplitude of the antidromic population spike (Table 1). There was also no correlation between any of these measures and the peak amplitude of the EPSC (P > 0.1, Spearman-rho rank correlation).
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No such effects of pilocarpine-induced status epilepticus on the antidromically evoked EPSC were found 4-6 days after treatment (Table 1). Antidromic stimulation of the mossy fibers evoked an EPSC in only 1 of 19 granule cells from rats that had developed status epilepticus. In contrast, antidromic stimulation evoked an EPSC in roughly the same percentages of control granule cells in short-term survivors (40% of the treated control group and 36% of the untreated control group) as in long-term survivors. Again there were no significant between-group differences in either the membrane potential on break-in or the amplitude of the antidromic population spike. However, input resistance was higher in granule cells from rats that had developed status epilepticus.
Delayed inward currents sometimes were observed in granule cells from both status epilepticus and control groups
Although antidromic stimulation normally evoked at most a single
short-latency inward current, in some granule cells it also evoked a
delayed inward current during superfusion with bicuculline or
picrotoxin (Fig. 3). Delayed currents
ranged from large (as large as 700 pA) and complex to small and
unimodal. They appeared at varying latencies and were present after
some stimulus pulses and not others. In some instances, the delayed
inward current appeared during wash-in of the GABAA
antagonist and then disappeared after the antagonist had equilibrated.
Persistent delayed inward currents most commonly were observed in the
long-term status epilepticus group (6 cells) but also appeared in four
cells from the long-term treated control group and in three cells each
from the short-term treated control and short-term untreated control
groups. Each granule cell in which this response was recorded came from
a different rat. In each treatment group, the peak amplitude of at
least one of these responses was 100 pA. Delayed inward currents were
abolished by superfusion with DNQX/D-AP5, suggesting that
they were polysynaptic EPSCs.
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NMDA receptors contributed to the antidromically evoked EPSC
To determine whether antidromic stimulation activated NMDA
receptors, we studied the pharmacology of the postsynaptic response recorded at holding potentials of 80 and
30 mV (Fig.
4). Only rats that had survived
10 wk
after pilocarpine administration were used in this study. At a holding
potential of
80 mV, addition of the selective AMPA/kainate receptor
antagonist NBQX (5 µM) to the superfusion medium left only a small
antidromically evoked inward current. The small residual current
disappeared on the addition of 50 µM D-AP5. However, NBQX
unmasked a much larger component when the holding potential was changed
to
30 mV. The bicuculline- and NBQX-insensitive inward current
exhibited slow response kinetics and was abolished by addition of 50 µM D-AP5 to the superfusion medium. It thus represented
the NMDA component of the antidromically evoked EPSC. Because the
antidromically evoked EPSC essentially was eliminated with a
combination of NBQX and D-AP5 (or DNQX and
D-AP5), we conclude that this response is glutamatergic and
depends predominantly on activation of AMPA/kainate and NMDA receptors.
Similar results were obtained in each of four granule cells from
pilocarpine-treated control rats and in each of six granule cells from
rats that had developed status epilepticus.
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I-V curves were generated in three experiments each from the
status epilepticus and pilocarpine-treated control groups. Each of
these curves exhibited a region of negative slope conductance with the
peak NMDA current between 20 and
40 mV (Fig.
5). However, the NMDA component of the
response from granule cells in the status epilepticus group was
generally larger. Table 2 compares the total charge transfer through NMDA receptors at
30 mV with the total
charge transfer through AMPA/kainate receptors at
80 mV. By this
measure, the NMDA component was a small fraction of the AMPA/kainate
component in three of the four granule cells from control rats. In
contrast, the NMDA component was 1.4-2.2 times as large as the
AMPA/kainate component in five of the six granule cells from rats that
had developed status epilepticus.
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Amplitude of antidromically evoked IPSCs depended on age and the prior development of status epilepticus
At a holding potential of 0 mV, antidromic stimulation of the mossy fibers evoked a GABAA-receptor-mediated feedback IPSC (Fig. 6) in all granule cells studied (Fig. 7). The peak amplitude of this response differed among treatment groups in two respects. First, the GABAA feedback IPSC was only about one-third as large in the older control rats compared with the younger controls (Table 3). Second, the amplitude of the feedback GABAA IPSC was depressed significantly 4-6 days after pilocarpine-induced status epilepticus when compared with the control groups. There were no significant between-group differences at the longer survival time and the results from the status epilepticus group did not differ significantly with survival time (P > 0.05).
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Antidromic stimulation evoked synaptically mediated field responses and delayed bursts in the dentate gyrus of some slices from both status epilepticus and control groups
We and others have recorded complex antidromically evoked field
responses from the granule cell body layer in some hippocampal slices
from epileptic brain (Cronin et al. 1992;
Masukawa et al. 1992
; Patrylo and Dudek
1998
; Tauck and Nadler 1985
). These responses, consisting of repetitive population spikes, were not observed in the
present study whether or not a GABA antagonist was present. In many
instances, the field response consisted simply of an antidromic population spike, and GABA and glutamate antagonists were without effect (Fig. 8, right). In
other instances, most commonly in slices from the long-term status
epilepticus group, a small negative deflection followed the antidromic
spike (Fig. 8, left). The peak of this negative wave
corresponded in time to the peak of the EPSC, when an antidromically
evoked EPSC was present in the recorded cell. GABA antagonists had
variable effects, if any, on this component of the response, but
DNQX/D-AP5 or NBQX either abolished it or markedly reduced
its amplitude. In addition, when antidromic stimulation evoked a
delayed inward current in the recorded cell, it usually also evoked a
delayed burst in the extracellular record. The burst consisted of a
biphasic shift in extracellular potential (negative-positive) on which
small negative deflections were superimposed. The peak negativity of
the burst occurred ~100 ms after the stimulus. A delayed field burst
was observed in all six slices from the long-term status epilepticus
group in which a delayed inward current was recorded and in all but one
such slice from each of the long-term treated control, short-term
treated control and short-term untreated control groups. Like the
delayed inward current, delayed field bursts were abolished by
superfusion with DNQX/D-AP5.
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Responses to perforant path stimulation were unaffected by the prior development of status epilepticus
Electrical stimulation of the perforant path where it crosses the subiculum consistently evoked an EPSC with a latency to onset of ~4-5 ms and a feed forward GABAA IPSC with a latency to onset of ~7-12 ms (Fig. 9). Pilocarpine-induced status epilepticus did not significantly change the mean peak amplitude, half-width, or latency to onset of the AMPA-kainate component of the EPSC, the NMDA component of the EPSC, or the feed forward GABAA IPSC (Fig. 10). However, the peak amplitude of the feed forward IPSC, like that of the feedback IPSC, declined significantly with age.
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Pilocarpine-induced status epilepticus killed about half the hilar neurons in the caudal hippocampal formation
Silver impregnation performed 1 day after
pilocarpine-induced status epilepticus revealed widespread neuronal
degeneration in many brain regions, including layers 2 and 3 of the
somatosensory neocortex, layers 5 and 6 of the cingulate cortex, the
entire pyriform cortex, the lateral septum, the claustrum-insula
region, and several nuclei of the amygdala and thalamus. This
distribution of damage is consistent with previous reports
(Clifford et al. 1987; Turski et al.
1983
). Within the hippocampal formation, neuronal degeneration
was less extensive than in most other vulnerable brain regions.
Argyrophilic (degenerating) granule and pyramidal cells were found
scattered and in small clusters throughout the cell body layers (Fig.
11). Degenerating granule cells were
most numerous at the apex of the granule cell arch. Numerous
argyrophilic neurons were present in the dentate hilus. Silver
granules, indicative of terminal degeneration, densely filled the inner
third of the dentate molecular layer, but there was little evidence of
terminal degeneration in the perforant path zone. We found no evidence of somatic or terminal argyrophilia in pilocarpine-treated control rats.
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When examined with a Nissl stain 10 wk after pilocarpine-induced
status epilepticus, all sections cut from slices adjacent to those used
for electrophysiological recording exhibited relatively normal
histology (Fig. 12). Preservation of
the normal lamination in area CA3 facilitated accurate placement of the
antidromic stimulating electrode. The only obvious histological
abnormality was a substantial loss of hilar neurons. Cell counts
revealed about a 50% reduction in their density both 4-6 days and
10 wk after treatment (Fig. 13).
There was also a significant age-related reduction of hilar neuron
density irrespective of treatment. The latter effect could be accounted
for by a corresponding increase in the cross-sectional area of the
hilus with age.
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Pilocarpine-induced status epilepticus increased the density of supragranular mossy fiber-like Timm stain
As reported previously (Okazaki et al. 1995),
pilocarpine-induced status epilepticus led to robust recurrent mossy
fiber growth as demonstrated by Timm histochemistry. Except in some
animals with the most extensive growth (e.g., Fig.
14, D-F), Timm staining of
the supragranular zone appeared denser in the infrapyramidal blade of
the dentate gyrus than in the suprapyramidal blade. Recurrent mossy
fiber growth was evident only in long-term survivors; none could be
seen 4-6 days after pilocarpine-induced status epilepticus.
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In most sections from treated and untreated control rats, scattered
clusters of mossy fiber-like Timm stain were present in the
supragranular zone (Fig. 14, A-C). These clusters
consistently were observed in sections cut from slices caudal and
immediately rostral to the slices used for electrophysiological
recording. They were always more numerous in the infrapyramidal blade
of the dentate gyrus and at the apex of the granule cell arch than in
the suprapyramidal blade. Supragranular mossy fiber-like Timm stain was
greatest at the caudal pole of the dentate gyrus and was virtually
absent from sections more than ~400 µm rostral to the level at
which electrophysiological recordings were made. These observations are
consistent with previous descriptions of Timm histochemistry in normal
rats (Gaarskjaer 1978; Haug 1974
). Timm
staining of the supragranular zone in treated control rats was
indistinguishable from Timm staining in untreated controls.
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DISCUSSION |
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Whole cell patch-clamp recordings in pilocarpine-treated rats revealed the following. 1) Antidromic stimulation of the mossy fibers evoked an EPSC in many dentate granule cells from control rats. 2) The same stimulus evoked an EPSC in a much higher percentage of granule cells from rats that exhibited recurrent mossy fiber growth and, in these instances, the peak amplitude of the response tended to be greater. 3) The antidromically evoked EPSC was glutamatergic and had both AMPA-kainate and NMDA components. The NMDA component of the response tended to be considerably greater in rats with recurrent mossy fiber growth. 4) The peak amplitude of the antidromically evoked IPSC was 40% less than control 4-6 days after status epilepticus and not significantly different from control in long-term survivors. However, control values declined with age. 5) In contrast to the antidromically evoked EPSC, neither the AMPA/kainate nor the NMDA component of the perforant path EPSC appeared to be affected by pilocarpine-induced status epilepticus. However, the size of the feed forward IPSC, like that of the feedback IPSC, declined with age.
Antidromically evoked EPSCs in rats with recurrent mossy fiber growth
These responses differed from controls in three ways: they were detected more frequently, the peak amplitude of 36% of them was greater than the peak amplitude of the largest EPSC recorded from any control granule cell, and the NMDA component was generally much larger. A key finding was that neither the probability of evoking an EPSC nor the peak amplitude of the EPSC was enhanced 4-6 days after pilocarpine administration. At this survival time, Timm histochemistry revealed no evidence of recurrent mossy fiber growth. Another important finding was that the effects were specific to the antidromically evoked EPSC; the perforant path EPSC remained unchanged.
Our results are consistent with previous reports on the functional
consequences of recurrent mossy fiber growth. Dentate granule cells
become hyperexcitable in response to afferent stimulation both in vivo
(Buckmaster and Dudek 1997a) and in slice preparations (Cronin et al. 1992
; Franck et al. 1995
;
Masukawa et al. 1992
; Patrylo and Dudek
1998
; Tauck and Nadler 1985
; Williamson
et al. 1995
). Field recordings in vivo revealed development of
a novel current sink in the inner portion of the molecular layer, the major site of recurrent mossy fiber synapses, in kindled rats with
mossy fiber growth (Golarai and Sutula 1996
). A
-opioid receptor agonist, which inhibits the release of glutamate
from mossy fiber terminals, reduced the amplitude of granule cell
population spikes in hippocampal slices with supragranular mossy fibers
(Simmons et al. 1997
). Wuarin and Dudek
(1996)
reported that microdrop application of glutamate to the
granule cell body layer increased the frequency of spontaneous EPSPs in
granule cells. Similarly, we reported that laser-evoked photolysis of
caged glutamate in the granule cell body layer evoked an apparently
unitary EPSC in granule cells (Molnár and Nadler
1997
). The basal frequency of spontaneous EPSCs was reported to
be substantially greater in granule cells from hippocampal slices with
supragranular mossy fibers (Simmons et al. 1997
). In the
presence of bicuculline, EPSCs of unusually high-amplitude and long
duration ("giant" EPSCs) were observed both spontaneously and
during stimulation of the perforant path. It was suggested that the
giant perforant path-evoked EPSPs resulted from disynaptic activation
of mossy fiber-granule cell synapses.
Thus present and past findings can be explained by formation of mossy
fiber-granule cell synapses after prolonged or repeated seizures. Lack
of correlation between the size of the antidromically evoked EPSC and
the extent of recurrent mossy fiber growth, as indicated by Timm
histochemistry, does not argue against this conclusion. Timm stain
density should correlate with measures of population activity, but not
necessarily with responses of an individual granule cell. The only
other excitatory input to the dentate granule cells reported to be
activated by stimulation of the CA3 area is the dentate associational
pathway (Scharfman 1994a, 1996
). Activation of granule
cell EPSCs through this disynaptic pathway requires stimulus-evoked
action potentials in hilar mossy cells. Thus one reason to discount the
possibility that an expanded associational projection contributed to
the antidromically evoked EPSC is that limbic status epilepticus is
believed to kill the majority of hilar mossy cells (Scharfman
and Schwartzkroin 1990
; Sloviter 1987
).
Consistent with this idea, degeneration of hilar neurons in the present
study was associated with dense terminal argyrophilia in the inner
third of the dentate molecular layer, the region to which the
associational pathway projects.
The wide variation in peak amplitudes of the antidromically evoked EPSC
may arise at least partly from similar variability in mossy fiber
terminal morphology. The largest of these terminals in the inner third
of the dentate molecular layer is ~2 µm in diameter (Okazaki
et al. 1995). Three or four of these large terminals form a
collar around the granule cell dendrite and each terminal forms
multiple synaptic contacts. Activation of such synaptic complexes may
have generated the large amplitude antidromically evoked EPSCs observed
in this study, as well as the giant spontaneous EPSCs observed by
others (Simmons et al. 1997
). However, many recurrent
mossy fiber boutons are much smaller than 2 µm and appear to form
synaptic contact with only a single spine. Unitary synaptic currents
are expected to vary with the number of release sites. The often
complex morphology of the antidromically evoked EPSC suggests that
polysynaptic activity in the recurrent mossy fiber pathway also
contributes. Antidromic activation of granule cells would be expected
to provoke reverberating excitation. Thus many of the EPSCs we recorded
probably included both a monosynaptic component from direct activation
of granule cells presynaptic to the recorded cell and polysynaptic
components from granule cells that were activated synaptically by the
stimulus. The variable amplitude, slow time course and complex
morphology of the antidromically evoked EPSC also may reflect the
asynchronous discharge and broad dispersion of the mossy fiber-granule
cell synapses. Three pieces of evidence support this possibility.
First, granule cells appear to become innervated by other granule cells
located both nearby and some distance away (Okazaki et al.
1995
; Sutula et al. 1998
). Furthermore, the
recurrent mossy fibers often branch from the parent axon deep within
the hilus, several hundred micrometers from the target cells. Thus
conduction time would be expected to differ considerably for the
different mossy fibers activated. Second, Langdon et al.
(1993)
suggested that action potentials typically invade mossy
fiber boutons after a variable delay due to the variable impedance
mismatch between the thin mossy fiber axon and the morphologically
diverse boutons. Even if action potentials arrive at two recurrent
mossy fiber boutons simultaneously, they may release transmitter
asynchronously. Third, mossy fiber-granule cell synapses can be located
on a rather large percentage of the dendritic tree particularly in
cases of robust recurrent growth. Unitary EPSCs generated distally will
be distorted by filtering through the membrane time constant to a
greater extent than those generated proximally. The greater the
contribution of distal synapses, the slower the time course and the
smaller the peak amplitude. A final consideration is variation in the
percentage of stimulated mossy fibers that remain within the plane of
the slice until they reach the fascia dentata. One might predict that
the higher the percentage of these fibers activated, as reflected by
the size of the antidromic population spike, the larger the
antidromically evoked EPSC would be. However, we found no correlation
between the peak amplitudes of the antidromic population spike and
antidromically evoked EPSC. Hence differences in mossy fiber activation
appear to be weakly contributory at most.
Our findings that NMDA receptors contribute to the
antidromically evoked EPSC and that pilocarpine-induced status
epilepticus augments the size of the NMDA component are of particular
interest considering the well-established role of NMDA receptors in
epileptic phenomena (Dingledine et al. 1990;
Traub and Jeffreys 1994
). Because NMDA-receptor-mediated
EPSCs could be evoked in the presence of an AMPA/kainate receptor
antagonist, these responses were presumably monosynaptic and resulted
from the activation of recurrent mossy fibers. We have confirmed that
the unitary mossy fiber-granule cell EPSC has an NMDA component
(Molnár and Nadler 1997
). NMDA receptors also
contribute to the mossy fiber EPSC recorded in CA3 pyramidal cells
(Weisskopf and Nicoll 1995
) and dentate basket cells
(Kneisler and Dingledine 1995
). The synaptic NMDA
current measured at a holding potential of
30 mV was ordinarily a
small fraction of the synaptic AMPA/kainate current measured at
80 mV. However, the relative size of the NMDA current increased
several-fold in most instances after pilocarpine-induced status
epilepticus. This change probably did not reflect upregulation of NMDA
receptors throughout the granule cell because the NMDA and AMPA/kainate components of the perforant path EPSC appeared to be unaffected. In
fact, in five of six cells, NMDA receptors came to play as prominent a
role in antidromically evoked excitatory transmission as they do in
perforant path transmission. Interestingly, Patrylo and Dudek
(1998)
reported that D-AP5 either blocked
antidromically evoked bursting in slices from rats with kainate-induced
recurrent mossy fiber growth or reduced its probability of occurrence.
Recurrent NMDA receptor activation may play a significant role in
seizure propagation under the depolarizing conditions that exist when granule cells are bombarded by high-frequency activity of the perforant path.
Pilocarpine-induced status epilepticus did not alter the
current-voltage relationship of the NMDA component, suggesting that there was no marked change in the ability of Mg2+ to block
the NMDA channel. In this respect, the pilocarpine model differs from
the kindling model, in which Mg2+ has reduced ability to
block NMDA channels in dentate granule cells (Köhr et al.
1993).
Antidromically evoked EPSCs in control rats
There is currently no published anatomic evidence for the presence
of mossy fiber-granule cell synapses in normal rat hippocampal formation. Thus we were surprised to record an antidromically evoked
EPSC in many granule cells from control rats. In a few instances, the
EPSC was followed by a delayed inward current and field burst. When
using antidromic stimulation to study the recurrent mossy fiber
pathway, one is concerned about the possibility of activating other
afferent pathways. As stated before, the only other excitatory input to
the dentate granule cells reported to be activated in this manner is
the associational pathway. Electrically evoked firing of CA3 pyramidal
cells drives hilar mossy cells (Scharfman 1994b), which
innervate dentate granule cells (Scharfman 1995
).
Antidromic stimulation of the mossy fiber pathway in area CA3b
inevitably activates some CA3 pyramidal cells, and for technical reasons, we could not differentiate a monosynaptic recurrent mossy fiber EPSC from a polysynaptic (CA3 pyramidal cell-hilar mossy cell-dentate granule cell) associational EPSC on the basis of onset
latency. For several reasons, however, we do not think that the
associational input contributed in a major way to the antidromically evoked EPSC. First, every antidromically evoked EPSC tested had an NMDA
component. Again, because these responses were evoked in the presence
of an AMPA/kainate receptor antagonist, they must have been activated
monosynaptically and thus could not have originated from the
polysynaptic associational pathway. Scharfman (1994a)
confirmed that blockade of AMPA/kainate receptors prevents the activation of mossy cells by input from area CA3. Second, we could evoke apparently unitary EPSCs in some granule cells from both pilocarpine-treated control rats (Molnár and Nadler
1997
) and age-matched untreated controls (Molnár and
Nadler, unpublished observations) by laser-evoked photolysis of caged
glutamate in the granule cell body layer. Finally, we have not been
able to evoke an EPSC in granule cells from control rats by uncaging
glutamate within the dentate hilus in the presence of bicuculline. At
least half the hilar interneurons are mossy cells (Amaral
1978
), and these neurons are highly excitable due to their low
spike threshold (~7-8 mV from resting Vm),
small IPSPs, and high-input resistance (Scharfman and
Schwartzkroin 1988
). Nevertheless, we did not activate a mossy
cell-granule cell connection in ~600 attempts; the probability of
evoking an EPSC by photolysis in the granule cell body layer with use
of the same laser power and pulse duration was much higher. Although
these considerations do not eliminate the possibility that the
associational pathway contributed to the antidromically evoked EPSC in
some instances, they suggest that recurrent mossy fibers played the
major role.
Previous electrophysiological tests for mossy fiber-granule cell
synapses in hippocampal slices from control rats have produced negative
results. In contrast to our work, Wuarin and Dudek
(1996) reported that neither antidromic stimulation of the
mossy fibers nor glutamate microstimulation in the granule cell body
layer evoked an excitatory synaptic response in dentate granule cells. There is no obvious explanation for these discrepant findings. However,
Timm histochemistry suggests that the location in the hippocampal
formation from which the slice was taken may be a crucial variable. In
our study, slices were prepared from a portion of the caudal
hippocampal formation in which clusters of mossy fiber-like Timm
staining normally were present in the supragranular zone. These
clusters were most numerous at the caudal pole of the dentate gyrus and
virtually disappeared at a level just rostral to that at which our
recordings were made. They presumably indicate the presence of closely
spaced recurrent mossy fiber boutons at those locations. Although we do
not know whether the postsynaptic targets of these presumptive
recurrent mossy fibers are, in fact, granule cells, our
electrophysiological data support the hypothesis that at least some of
them are. Previous studies may have utilized hippocampal slices from
more rostral levels of the hippocampal formation, where supragranular
mossy fibers appear to be scarce.
Thus we suggest that the recurrent mossy fiber pathway, previously thought unique to the epileptic brain, represents expansion of a minimal pathway already present in the caudal dentate gyrus. The putative existence of mossy fiber-granule cell synapses in normal rats requires confirmation by electron microscopy.
Antidromically evoked field responses
Previous reports have associated recurrent mossy fiber growth with
antidromically evoked repetitive population spikes, delayed field
bursts, and spontaneous bursting (Cronin et al. 1992;
Masukawa et al. 1992
; Patrylo and Dudek
1998
; Tauck and Nadler 1985
; Wuarin and
Dudek 1996
). These responses were postulated to have resulted in large part from the establishment of reverberating excitation in the
dentate gyrus. Field recordings provided evidence of enhanced population activity in the present study as well. These responses consisted of a glutamate-mediated field EPSP with or without a delayed
field burst. Both components of the field response coincided in time
with an antidromically evoked glutamatergic synaptic current. The
delayed burst, like the delayed inward current, appeared at variable
latencies and after some stimulus pulses but not others. These
long-latency responses have been attributed to the unmasking by
GABAA receptor antagonists of a polysynaptic recurrent
excitatory circuit (Cronin et al. 1992
). Antidromically
evoked synaptic field responses were observed in rats from both status
epilepticus and control groups, but more commonly from the long-term
status epilepticus group. This observation supports the view that the
extracellularly recorded antidromically evoked synaptic responses
depended on the presence of mossy fiber-granule cell synapses. However,
synaptically evoked field responses were observed in only a minority of
slices, even in those with robust mossy fiber growth. We attribute this relatively meager evidence of reverberating excitation to recording at
room temperature, which would be expected to reduce neuronal excitability. When recordings were made at a temperature of 32°C, we
often recorded complex antidromically evoked field responses in slices
from pilocarpine-treated rats (Okazaki and Nadler 1994
). Previously published recordings of similar antidromically driven waveforms from slices of epileptic hippocampus also were made at
32-35°C.
Feedback and feed forward IPSCs
The GABAA-mediated feedback IPSC was depressed
substantially 4-6 days after pilocarpine-induced status epilepticus.
This result was expected because 40% of hilar GABA neurons were
reported to degenerate under these conditions (Obenaus et al.
1993). Silver impregnation and cell counts also indicated
degeneration of about half the hilar neurons, although we do not know
what proportion of these were GABA neurons as opposed to mossy cells.
Conversely, the GABAA-mediated feed forward (perforant
path-activated) IPSC was unaffected. These results are consistent with
the limited information we have about the vulnerability of different
GABA neuronal subtypes to prolonged seizures. The "HIPP" cells
(cell body located in the hilus, axonal projection to the perforant path terminal zone), a type of GABA interneuron that also contains somatostatin and neuropeptide Y, are highly vulnerable to seizures (Buckmaster and Dudek 1997b
; Lurton and
Cavalheiro 1997
; Mathern et al. 1995
;
Sloviter 1987
). Because the dendrites of the HIPP cell
remain confined predominantly to the hilus (Freund and
Buzsáki 1996
), these cells are thought to mediate
feedback, but not feed forward, inhibition. The dentate basket cells
and "MOPP" cells (cell body located in the molecular layer,
axonal projection to the perforant path terminal zone) remain intact
after status epilepticus (Buckmaster and Dudek 1997b
;
Obenaus et al. 1993
; Sloviter 1987
). The
dendrites of both cell types receive direct innervation from the
perforant path and participate in feed forward inhibition (although
basket cells also mediate feedback inhibition).
The between-group difference in peak amplitude of the feedback IPSC had
disappeared within 10 wk after pilocarpine administration. However,
this change with survival time resulted not from restoration of
feedback inhibition in the status epilepticus group but rather from a
marked reduction of peak IPSC amplitude in the control groups. The size
of the antidromic population spike tended to be smaller in the slices
from older rats. Thus the antidromic stimulus may have provided a
weaker drive for activation of GABA neurons in the feedback circuit.
However, for the treated controls, the threefold reduction in peak IPSC
amplitude with survival time appears disproportionate to the 20%
reduction in peak antidromic population spike amplitude. Furthermore
the peak amplitude of the feed forward IPSC also was reduced
substantially. This loss of GABA synaptic function in granule cells may
have either a biological or technical explanation. There is some
evidence for a loss of GABA neurons with aging in the rat hippocampal
formation (Shetty and Turner 1998). Some of the GABA
neurons that die off during the aging process may be of the same types
that are killed by pilocarpine-induced status epilepticus.
Alternatively, GABA neurons of the dentate gyrus may survive slice
preparation less well as animals grow older.
Several lines of evidence suggest that some form of granule cell
inhibition is enhanced in rats after pilocarpine-induced or kainic
acid-induced status epilepticus. Buckmaster and Dudek (1997a,b
) found enhanced paired-pulse inhibition and an
increased threshold for maximal dentate activation with stimulation of
the perforant path in vivo. In addition, GABAA receptor
current density in acutely dissociated granule cells was 78% greater
than control, indicating enhanced expression of those receptors
(Gibbs et al. 1997
). Conversely, Isokawa
(1996)
found no difference from control in the peak amplitude
of the feed forward IPSC during low-frequency stimulation of the
perforant path in hippocampal slices. We confirm the latter result and
further report no lasting between-group difference in the feedback IPSC
either. These findings do not necessarily contradict reports of
enhanced inhibition in vivo, however. Peak amplitude of the IPSC evoked
at one standard stimulus intensity serves only as a crude measure of
synaptic inhibition. Subtle changes easily could have been missed. For
example, high-frequency stimulation of the perforant path at a holding
potential of
30 mV (to maximize NMDA current) was reported to reduce
the size of the GABAA-mediated IPSC specifically in granule
cells from rats that had been made epileptic with pilocarpine
(Isokawa 1996
). Furthermore, slice preparation removes
much of the normal inhibitory circuitry of the dentate gyrus. Thus our
stimuli activated only a portion of the granule cell inhibition that is
present in the intact animal. Finally the additional GABAA
receptors reported to be expressed by dentate granule cells from
epileptic brain may not have been activated by the stimulation
protocols we used. One possibility is that they are present mainly at
extrasynaptic sites and are activated only by stimulus trains.
Although our results support the hypothesis that seizure-induced mossy
fiber growth mediates recurrent excitation, they reveal little about
its effect on GABA inhibition. Relative to the controls, feedback
inhibition increased with survival time after status epilepticus. At
first glance, this finding might be regarded as evidence of
compensation for the seizure-induced loss of hilar GABA neurons,
perhaps through the formation of new mossy fiber-GABA neuron and/or
GABA neuron-granule cell synapses. This interpretation would be
unjustified, however, because the change resulted not from enhancement
of the IPSC in the status epilepticus group but rather from a
diminished IPSC in the control groups. Our results neither support nor
refute the possibility that seizure-induced mossy fiber growth drives
GABA inhibition, as others have suggested (Kotti et al.
1997; Sloviter 1992
). Further studies of this
issue need to consider the possibility of time-dependent changes in the controls.
Short-term effects of pilocarpine-induced status epilepticus
Antidromic stimulation evoked only a single small EPSC in 19 granule cells tested 4-6 days after pilocarpine-induced status epilepticus. This frequency of success was significantly less than in
either control group, suggesting a reversible loss of connectivity.
Mossy fiber synapses may have become anatomically or functionally
disconnected from postsynaptic granule cells during the period of mossy
fiber growth. There is ample precedent for a reversible loss of
excitatory afferent synapses on peripheral neurons during regrowth of
their axons after axotomy (e.g., Mendell et al. 1976;
Purves 1975
). In addition, 30% of perforant path synapses were lost transiently after status epilepticus induced by
intracerebroventricular administration of kainic acid (Nadler et
al. 1980
). The apparent disconnection of synapses occurred in
the absence of overt terminal degeneration. A similar process may
account for transient loss of the antidromically evoked EPSC in the
present study. However, our results do not suggest any disconnection of
perforant path synapses in this instance. Silver impregnation revealed
degeneration of some dentate granule cells within a day after status
epilepticus. Therefore loss of the antidromically evoked EPSC may be
explained to some degree by degeneration of presynaptic granule cells.
To the extent that the associational pathway contributes to the
antidromically evoked EPSC, seizure-induced degeneration of hilar mossy
cells also may play a role.
The input resistance of granule cells increased reversibly after pilocarpine-induced status epilepticus. The molecular basis of this change requires further investigation. It probably cannot be explained by reduced resting K+ conductance, because K+ channels were blocked with intracellular Cs+. Along with degeneration of presynaptic GABA neurons, increased membrane resistance may contribute to hyperexcitability of the dentate gyrus during the first few days after status epilepticus. However, this result requires confirmation under recording conditions that do not alter the intracellular milieu.
Relation to epileptic phenomena
Rats that develop status epilepticus after administration of
pilocarpine invariably become epileptic, that is, they exhibit unremitting spontaneous seizures (Lemos and Cavalheiro
1996; Mello et al. 1993
). The density of
supragranular mossy fiber-like Timm staining has been correlated with
the development of spontaneous seizures although not with seizure
frequency. Our results support the hypothesis that expansion of the
recurrent mossy fiber pathway increases the excitatory drive to dentate
granule cells and thus facilitates repetitive synchronous discharge.
Given that dentate granule cells are normally difficult to recruit into
epileptiform activity and thus present a barrier to seizure
propagation, recurrent mossy fiber growth may contribute to
epileptogenesis in the pilocarpine-treated rat. Although the
pilocarpine model differs from human temporal lobe epilepsy in some
respects (e.g., extent of cell death in area CA1 and in neocortical
layer 2), both are characterized by extensive loss of hilar neurons and
recurrent mossy fiber growth. Some studies have reported hyperexcitable
electrophysiological responses in the dentate gyrus of hippocampi
resected for pharmacologically intractable temporal lobe epilepsy that
could be explained by expanded recurrent excitatory circuitry
(Franck et al. 1995
; Masukawa et al.
1992
). However, recurrent mossy fiber growth is probably neither necessary nor sufficient for epileptogenesis. Timm-positive supragranular mossy fibers are present in aged, nonepileptic human hippocampi (Cassell and Brown 1984
), but not in all
persons with temporal lobe epilepsy (Franck et al.
1995
). In addition, administration of cycloheximide before
pilocarpine appears to suppress mossy fiber growth without effect on
the development of recurrent spontaneous seizures (Longo and
Mello 1997
). The circumstances under which recurrent mossy
fiber growth contributes to epileptogenesis remain to be defined.
The existence of mossy fiber-granule cell synapses in the normal brain,
if confirmed anatomically, also must be considered with regard to
seizure propagation. By analogy with research on area CA3, these
connections, though sparse, would be expected to promote the
synchronization of granule cell discharge when inhibition is
compromised. Thus mossy fiber-granule cell synapses may play some role
in epileptic events even in the absence of mossy fiber growth, for
example when seizures originate from a mass lesion or vascular
malformation outside the hippocampal formation. It may not be
coincidental that supragranular mossy fibers are most numerous at the
caudal end of the hippocampal formation (homologous to the human
anterior hippocampus), which also has a particularly low threshold for
epileptiform discharge (Bragdon et al. 1986; Gilbert et al. 1985
; Racine et al. 1977
).
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ACKNOWLEDGMENTS |
---|
We thank Drs. G. Oxford, J. Kauer, and N. Lambert for assistance with the whole cell recording technique, B. Gordon and L. Watson for performing some of the histological studies, and K. Gorham for secretarial assistance.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-17771.
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FOOTNOTES |
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Address for reprint requests: J. V. Nadler, Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710.
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 21 August 1998; accepted in final form 2 December 1998.
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REFERENCES |
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