1Department of Neurology, 2Department of Anatomy, and 3The Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53792
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ABSTRACT |
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Lynch, M.,
Ü. Sayin,
G. Golarai, and
T. Sutula.
NMDA Receptor-Dependent Plasticity of Granule Cell Spiking in the
Dentate Gyrus of Normal and Epileptic Rats.
J. Neurophysiol. 84: 2868-2879, 2000.
Because granule
cells in the dentate gyrus provide a major synaptic input to pyramidal
neurons in the CA3 region of the hippocampus, spike generation by
granule cells is likely to have a significant role in hippocampal
information processing. Granule cells normally fire in a single-spike
mode even when inhibition is blocked and provide single-spike output to
CA3 when afferent activity converging into the entorhinal cortex from
neocortex, brainstem, and other limbic regions increases. The effects
of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in -aminobutyric acid-A (GABAA)
receptor-dependent inhibition on spike generation were examined in
granule cells of the dentate gyrus. In contrast to the single-spike
mode observed in normal bathing conditions, perforant path stimulation
in Mg2+-free bathing conditions evoked graded burst
discharges in granule cells which increased in duration, amplitude, and
number of spikes as a function of stimulus intensity. After burst
discharges were evoked during transient exposure to bathing conditions
that relieve the Mg2+ block of the NMDA receptor, there was
a marked increase in the NMDA receptor-dependent component of the EPSP,
but no significant increase in the non-NMDA receptor-dependent
component of the EPSP in normal bathing medium. Supramaximal perforant
path stimulation still evoked only a single spike, but granule cell
spike generation was immediately converted from a single-spike firing
mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg2+-free
conditions and the expression of burst discharges evoked in normal
bathing medium with subsequent disinhibition were both blocked by
DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in
the perforant path, which is induced by NMDA receptors and is
also expressed by
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in
granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of
evoked synaptic response, and in the dentate gyrus reorganized by mossy
fiber sprouting in kindled and kainic acid-treated rats. NMDA
receptor-dependent plasticity of granule cell spike generation, which
can be distinguished from LTP and induces long-term susceptibility to
epileptic burst discharge under conditions of reduced inhibition, could
modify information processing in the hippocampus and promote epileptic
synchronization by increasing excitatory input into CA3.
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INTRODUCTION |
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The N-methyl-D-aspartate
(NMDA) subclass of glutamate receptors has been implicated in
activity-dependent plasticity of neural circuits in both the developing
and adult nervous system. In the developing brain, activation of the
NMDA receptor by neural activity regulates the formation of synaptic
connections and circuit organization (Ben-Ari et al.
1997; Constantine-Paton and Cline 1998
;
Hanse et al. 1997
). In the adult brain, the NMDA
receptor plays an important role in the induction of use-dependent
synaptic plasticity such as long-term potentiation and depression, and
in several forms of seizure-induced plasticity (Bear and Malenka
1994
; Meldrum et al. 1999
; Mody
1998
). For example, repeated seizures evoked by kindling
increase the NMDA receptor-dependent current in granule cells of the
dentate gyrus (Köhr et al. 1993
; Mody and
Heinemann 1987
; Mody et al. 1988
; Sayin
et al. 1999
) and induce progressive, permanent susceptibility
to additional seizures (Goddard et al. 1969
). The
induction of permanent seizure susceptibility by repeated kindled
seizures is NMDA-receptor dependent (Bengzon et al.
1999
; Sprengel et al. 1998
).
The initial seizure-induced increase of the NMDA receptor-dependent
current, while not permanent (Sayin et al. 1999),
nevertheless appears to play an important long-term role by initiating
structural and functional modifications which contribute to
long-lasting seizure susceptibility in hippocampal circuitry
(Sutula et al. 1996
). For example, repeated seizures
induce sprouting by the mossy fiber axons of granule cells, which
reorganize synaptic connectivity in the dentate gyrus and form
recurrent excitatory circuits that could contribute to increased
excitability and susceptibility to seizures (Buckmaster and
Dudek 1997
; Lynch and Sutula 2000
; Molnár and Nadler 1999
; Simmons et al.
1997
; Wuarin and Dudek 1996
). Both mossy fiber
sprouting (Sutula et al. 1996
) and the associated
progressive increase in seizure susceptibility induced by kindling can
be prevented by the NMDA receptor antagonist MK-801 administered at
doses that do not suppress evoked kindled seizures (Gilbert and
Mack 1990
; McNamara et al. 1988
; Sutula
et al. 1996
). Because activation of NMDA receptors only
transiently enhances synaptic transmission in granule cells but is
required for the progression of long-term seizure-induced alterations
in the structural organization and functional properties of hippocampal
circuitry, it was of interest to further examine how the initial
seizure-induced increase in the NMDA receptor-dependent component of
synaptic current and more slowly evolving NMDA receptor-dependent
cellular alterations may sequentially or independently contribute to
the generation of epileptic discharges in the dentate gyrus.
The effects of activation of NMDA receptors by transient exposure to Mg2+-free conditions and seizures on susceptibility to burst discharge and repetitive firing of granule cells were examined in the normal dentate gyrus, and in circuitry reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. The results demonstrated that activation of NMDA receptors by transient relief of Mg2+ block or seizures can induce alterations in granule cell firing patterns when inhibition is reduced, an action that potentially promotes epileptic synchronization and could modify information processing in hippocampal circuitry.
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METHODS |
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Surgical procedures
Adult male Sprague-Dawley rats (250-350 g) were anesthetized with a combination of ketamine (80 mg/kg im) and xylazine (10 mg/kg im) and were stereotaxically implanted with an insulated stainless steel bipolar electrode for stimulation and recording. The electrode was implanted in either the perforant path (8.1 mm posterior, 4.4 mm lateral, 3.5 ventral with respect to bregma) or the olfactory bulb (9.0 anterior, 1.2 lateral, 1.8 ventral with respect to bregma) and was fixed to the skull with acrylic.
Kindling procedures
After a 2-wk recovery period following electrode placement, the
unrestrained awake animals in the kindling group received twice-daily
kindling stimulation (5 days per wk) with a 1-s train of 62-Hz biphasic
constant current 1.0-ms-square wave pulses. The stimulation was
delivered at the lowest intensity that evoked an afterdischarge
according to standard procedures (Cavazos et al. 1991).
The electroencephalogram was recorded from the bipolar electrode, which
was switched to the stimulator for the delivery of kindling
stimulation. Evoked behavioral seizures were classified according to
standard criteria (Racine et al. 1975
; Sutula and Steward 1986
) and ranged from Class I (behavioral arrest) to
Class V (bilateral tonic-clonic motor activity with loss of postural tone, which are comparable to partial complex seizures with secondary generalization).
Administration of kainic acid
Adult male Sprague-Dawley rats (250-350 g) were injected with
kainic acid (9-12 mg/kg ip or sc) and were observed for signs of
behavioral seizure activity, which typically consisted of altered responsiveness to environmental stimuli, irregular tonic-clonic movements of the extremities, and alterations in postural tone. The
injected rats were observed for 2-3 h, when the most severe behavioral
alterations usually diminished. In previous studies, kainic acid
produced initially intense electrographic seizures, which gradually
diminished after 4-5 days (Sutula et al. 1992) and are
eventually followed by spontaneous recurrent seizures (Hellier
et al. 1998
). Rats that did not experience status epilepticus in response to the kainic acid injection were not used in this study.
Rats were killed 30-60 days after kainic acid treatment.
Preparation of hippocampal slices
Normal, kindled, and kainic acid-treated rats were decapitated
after induction of anesthesia by ether. The brains were rapidly removed
and placed into ice-cold artificial cerebrospinal fluid (ACSF) of the
following composition (in mM): 124 NaCl, 4.4 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 10 glucose, which was saturated with
95% O2-5% CO2 at pH 7.4. Transverse hippocampal slices were cut from the septal half of the
hippocampus with a vibratome or McIlwain tissue chopper at a thickness
of 400 µm. The slices were maintained for at least 1 h in ACSF
at 20-22°C before transfer to a submersion or interface recording
chamber containing oxygenated ACSF at 31-32°C. Granule cells were
impaled with tapered borosilicate glass micropipettes (100-150 M;
1.0/0.58 mm OD/ID) filled with 2 M potassium acetate, adjusted to pH
7.4. Some micropipettes also contained biocytin (2% wt/vol) for
intracellular staining and morphological analysis that has been
previously reported (Sutula et al. 1998
). Granule cells
were identified by morphological and physiological criteria such as
highly negative resting membrane potential, strong spike frequency
adaptation, and absence of a voltage "sag" in response to
hyperpolarizing current injection (Lübke et al.
1998
; Scharfman 1992
; Staley et al.
1992
). Granule cells were included in the study only when
stable impalements were obtained for at least 1 h, the resting
membrane potential was at least
60 mV, the input resistance was at
least 40 M
, and the action potential amplitude exceeded 50 mV. A
conventional bridge circuit was used for intracellular recording and
current injection; bridge balance was routinely monitored.
Extracellular recording electrodes (2-10 M
) containing 2 M NaCl
were positioned within 200 µm of the intracellular electrode.
Responses were amplified, digitized, and stored on optical disks for
off-line analysis.
Orthodromic synaptic responses were evoked in granule cells of the
dentate gyrus by monopolar constant-voltage stimuli (0.05 ms) delivered
by electrodes placed in the stratum moleculare of the dentate gyrus in
the region of the perforant path. Input-output curves were generated
using a sequence of increasing stimulation intensities. Based on
input-output curves, subsequent stimuli were delivered at the minimal
intensity that evoked the maximal response, unless otherwise indicated.
In an attempt to assess the contribution of other regions of
hippocampal circuitry to the burst responses recorded in the dentate
gyrus, transections were made by razor cuts in some slices to isolate
the dentate gyrus from the entorhinal cortex,
CA3a,b, and CA1 (illustrated in Fig.
3D). In some experiments, the NMDA component of the synaptic response was studied by replacing MgSO4 in the
ACSF with Na2SO4. We have
referred to ACSF with no added MgSO4 as
"Mg2+-free," but such solutions may still
contain trace concentrations of Mg2+. Kainic
acid, bicuculline methiodide(), picrotoxin, and
DL-2-amino-5-phosphonovaleric acid (APV) were obtained from Sigma.
Evoked responses were recorded, stored, and analyzed using software developed in the laboratory, pCLAMP 6.02, and Clampfit 6.02 (Axon Instruments).
Statistical methods
Group differences were evaluated for statistical significance by analysis of variance (ANOVA) or Student's t-test. When data were not normally distributed, analyses were performed using a nonparametric ANOVA by ranks, or the Mann-Whitney rank sum test. Averages are reported as mean ± SE.
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RESULTS |
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Passive membrane properties of granule cells from normal and epileptic rats
Data were collected from 32 control rats, 19 kainic acid-treated
rats killed 30-60 days after kainic acid-induced status epilepticus, and 41 kindled rats killed 1-90 days after kindling stimulation that
evoked a range of one afterdischarge to 120 Class V generalized tonic-clonic seizures. The average resting membrane potential of
granule cells from normal rats was 73.6 ± 1.4 mV (n = 61 cells) and did not differ in kainic acid-treated rats (
73.1 ± 1.2 mV, n = 48 cells) or kindled rats (
73.3 ± 0.8 mV, n = 96 cells). Average input resistance
calculated from the voltage deflection produced by 50-ms
hyperpolarizing and depolarizing current pulses was 59.1 ± 2.9, 61.0 ± 2.7, 62.7 ± 1.9 M
in granule cells from control,
kainic acid-treated, and kindled rats, respectively. There were no
significant differences in membrane properties as a function of
kindling site or number of evoked seizures, or in the threshold for
action potential generation in normal controls, kindled, or kainic acid
treated groups (Table 1).
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Synaptic responses and firing patterns of granule cells in normal ACSF
In hippocampal slices from normal rats, orthodromic stimulation of
the perforant path evoked an excitatory postsynaptic potential (EPSP)
and inhibitory postsynaptic potential (IPSP) in granule cells of the
dentate gyrus. The EPSP increased in amplitude with increasing stimulus
strength and was accompanied by an action potential when the EPSP
exceeded spike threshold, which was 53.3 ± 1.8 mV (Fig.
1A, Table 1). The EPSP was
followed by an IPSP, which was usually depolarizing at a typical
resting membrane potential in the range of
68 to
75 mV in the
recording conditions of these experiments (see Fig. 1A) and
became hyperpolarizing at less negative membrane potentials. In
agreement with previous reports (Fricke and Prince 1984
;
Lambert and Jones 1990
; Scharfman 1992
),
stimulation of the perforant path typically evoked a single-action
potential (Figs. 1A and 2A). Only 4 of 58 granule
cells from normal rats demonstrated two action potentials in normal
ACSF. Burst discharges, defined as three or more action potentials
evoked by an EPSP, were never evoked by perforant path stimulation of
granule cells from normal rats in standard ACSF (n = 58 cells). During bath application of the
-aminobutyric acid-A
(GABAA) receptor antagonist bicuculline (10 µM)
or picrotoxin (100 µM), supramaximal stimulation of the perforant
path in hippocampal slices from normal rats still evoked an EPSP and
only one or two action potentials (Fig. 1B; n = 28 of 28 granule cells).
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Synaptic responses and firing patterns of granule cells during and after transient exposure to Mg2+-free ACSF
The NMDA receptor channel in granule cells at resting membrane
potential is normally blocked by Mg2+ and plays a
relatively minor role in synaptic transmission (Lambert and
Jones 1990; Mody and Heinemann 1987
). Effects of
enhancing the NMDA receptor-dependent component of the synaptic current in granule cells from normal rats were assessed by comparing the perforant path-evoked synaptic response before, during, and after bath
application of Mg2+-free ACSF. In contrast to
granule cells in normal ACSF, perforant path stimulation of hippocampal
slices in Mg2+-free ACSF evoked a burst discharge
that increased in duration and amplitude as a function of stimulus
strength and was accompanied by multiple granule cell action potentials
(Fig. 2B; n = 34 of 38 cells). The evoked burst discharges in
Mg2+-free ACSF were field events (Fig.
3A) and were blocked by
application of the NMDA receptor antagonist APV (30 µM) (Fig.
3B; n = 5 of 6 cells).
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After transient exposure to Mg2+-free ACSF for 5-10 min followed by return to normal ACSF containing 1.3 mM Mg2+, supramaximal perforant path stimulation again evoked only a maximum of one or two action potentials (Fig. 2C). In striking contrast to normal granule cells that were not exposed to Mg2+-free ACSF, subsequent stimulation of the perforant path in ACSF containing 1.3 mM Mg2+ and bicuculline (10 µM) or picrotoxin (100 µM) evoked a prolonged burst with repetitive granule cell discharges (Fig. 2D; n = 27 of 30 cells, compare to Fig. 1B). The burst discharges were also field events (Fig. 3C) and increased in duration and amplitude as a function of stimulus intensity (Fig. 2D, n = 27 of 27 cells). The bursts evoked during reduction of GABAA receptor-mediated inhibition after transient exposure to Mg2+-free ACSF were APV sensitive (Fig. 2E; n = 19 of 20 cells).
Bursts evoked during transient exposure to Mg2+-free ACSF, and then during exposure to ACSF containing 1.3 mM Mg2+ and antagonists of GABAA receptor-mediated inhibition, were both suppressed by APV, which indicated that both the induction and the expression of granule cell bursts were NMDA receptor dependent. Burst discharges were also evoked in these conditions in transected hippocampal slices containing only a small sector of the granule cell layer, molecular layer, the subgranular hilus of the dentate gyrus, and the stratum lacunosum-moleculare of CA1, which suggested that the burst discharges were generated in local circuitry of the dentate gyrus (Fig. 3D; n = 6 of 6 slices). There was no difference in the action potential threshold during or after exposure to Mg2+-free ACSF (see Table 1), which indicates that the repetitive granule cell spiking was not caused by an alteration in the threshold for spike generation. These findings demonstrate that enhancement of the NMDA receptor-dependent component of synaptic transmission by transient exposure to Mg2+-free ACSF is sufficient to increase susceptibility to epileptic burst discharge when GABAA receptor-mediated inhibition is blocked.
The acquired susceptibility to burst discharge under conditions of reduced inhibition was not simply a consequence of exposure to Mg2+-free ACSF, as granule cells which did not demonstrate burst discharges in response to perforant path stimulation in Mg2+-free ACSF did not generate bursts when GABAA receptor-mediated inhibition was subsequently blocked (n = 4/4). This observation suggested that the induction of susceptibility to burst discharge under conditions of reduced inhibition was also a consequence of synaptic activity and was therefore activity-dependent.
The effects of evoked bursting during transient exposure to
Mg2+-free ACSF on the NMDA receptor-dependent and
non-NMDA receptor-dependent component of the evoked synaptic response
were evaluated in standard ACSF by comparing intracellular and
extracellular evoked responses before and after bath application of 30 µM APV. As reported previously (Lambert and Jones
1990; Mody and Heinemann 1987
), there was a small NMDA receptor-dependent component of the perforant path-evoked EPSP in granule cells examined in hippocampal slices from normal rats
(n = 4/5, Fig.
4A). After evoked bursting
during transient exposure to Mg2+-free ACSF,
there was a significant increase in the NMDA receptor-dependent component of the evoked EPSP (n = 3/4, Fig.
4A), which was also observed in the evoked extracellular
population EPSP (Fig. 4B, n = 4/4,
P < 0.01). Comparison of the non-NMDA
receptor-dependent component of the evoked intracellular EPSP before
and after exposure to Mg2+-free ACSF, obtained by
subtraction of the EPSPs evoked in 30 µM APV, revealed only a minor
increase in the non-NMDA receptor-dependent component (Fig.
4A). The extracellular population EPSP also demonstrated only a trend toward an increase in the non-NMDA receptor-dependent component, which did not reach statistical significance (Fig. 4B).
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Synaptic responses and firing patterns of granule cells in epileptic kainic acid-treated and kindled rats in normal ACSF
In hippocampal slices from kindled or kainic acid-treated rats in
normal bathing medium, orthodromic stimulation of the perforant path
also evoked an EPSP and a depolarizing IPSP at resting membrane potential, but did not evoke a burst response (Figs.
5A and
6, A1 and B1).
Previous studies in granule cells have demonstrated an increase in the
NMDA receptor-dependent synaptic current following status epilepticus
or repeated brief seizures (Köhr and Mody 1994;
Köhr et al. 1993
; Okazaki et al.
1999
; Patrylo and Dudek 1998
; Sayin et
al. 1999
), but stimulation of the perforant path with a single
pulse of supramaximal intensity still typically evoked only one action
potential in granule cells from epileptic kainic acid-treated or
kindled rats in normal bathing medium (Figs. 5A and 6,
A1 and B1; n = 122 of 132 cells).
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Effects of GABAA receptor blockade on synaptic responses and firing patterns of granule cells from epileptic kainic acid-treated rats
In contrast to normal rats, when GABAA
receptor-mediated inhibition was blocked with bicuculline or picrotoxin
in hippocampal slices from rats examined 30-60 days after kainic
acid-induced status epilepticus, stimulation of the perforant path
evoked granule cell burst discharges characterized by prolonged
depolarization and repetitive spikes (n = 38 of 39 cells; compare Fig. 5B to Fig. 1B). There was no
difference in the action potential threshold in normal and kainic
acid-treated rats (see Table 1). Simultaneous extracellular recording
demonstrated that the discharges were synchronous field events (Fig.
5B). Perforant path-evoked bursts were reliably elicited in
transected hippocampal slices (as in Fig. 3D,
n = 5 of 5 slices), which suggested that the burst
discharges were generated in local circuitry of the dentate gyrus.
Increasing the intensity of the perforant path pulse increased the
amplitude and duration of the EPSP, and the number of evoked granule
cell discharges (Fig. 5C). In contrast to the
"all-or-none" character of epileptiform events in other regions of
hippocampus or neocortex (Courtney and Prince 1977;
Schwartzkroin and Prince 1978
), the evoked epileptiform
discharges in the dentate gyrus were graded and increased in duration
with increasing stimulus strength. Spontaneous granule cell burst
discharges were not observed. The evoked repetitive granule cell
discharges were blocked by addition of APV (30 µM) and were therefore
dependent on the NMDA receptor (Fig. 5D; n = 31 of 33 cells).
Effects of GABAA receptor blockade on synaptic responses and firing patterns of granule cells from kindled rats
In hippocampal slices from kindled rats that had experienced 3-95 generalized Class V seizures, perforant path stimulation in normal bathing medium evoked an EPSP with a single-action potential (Fig. 6A1). Supramaximal intensity perforant path stimulation in hippocampal slices obtained from kindled rats and bathed in normal ACSF typically evoked only a single-spike discharge. Two action potentials were evoked in only 6 of 88 granule cells from kindled rats in normal ACSF; bursts and repetitive spiking were never observed. In ACSF-containing bicuculline or picrotoxin, perforant path stimulation evoked an APV-sensitive granule cell burst discharge (n = 46 of 49 cells, Fig. 6A). As in hippocampal slices from kainic-acid treated rats, the evoked bursts increased in amplitude and duration, and the number of spikes per burst also increased as a function of increasing stimulus intensity (data not shown). There were no differences in the resting membrane potential, input resistance, action potential threshold, or firing patterns as a function of kindling site or number of evoked seizures. In ACSF-containing bicuculline or picrotoxin, burst discharges were evoked in hippocampal slices obtained as long as 12 wk after the last kindled seizure.
The chronic epileptic state induced by kindling or treatment with
kainic acid is associated with the development of mossy fiber sprouting
in the dentate gyrus, which may contribute to generation of
epileptic burst discharges by formation of recurrent circuits
(Sutula et al. 1988; Tauck and Nadler
1985
). Histological evidence of mossy fiber sprouting induced
by kindling first becomes apparent by 7 days after the initial kindling
stimulation and increases with continued stimulation, but is not seen
within the first 3 days after initial stimulation (Cavazos et
al. 1991
). It was therefore of interest to assess effects of
the initial evoked kindled seizures on susceptibility to granule cell
burst discharge prior to the development of sprouting. In hippocampal slices prepared from rats 1 day after a single-evoked afterdischarge, perforant path stimulation evoked NMDA receptor-dependent granule cell
burst discharges in ACSF containing 10 µM bicuculline
(n = 9 cells from 4 rats), which were field events
(Fig. 6B1) and were blocked by APV (Fig. 6B2).
This observation demonstrates that mossy fiber sprouting is not
necessary for generation of epileptic burst discharges in the dentate
gyrus because bursts can be evoked in granule cells within 24 h
after an afterdischarge before mossy fiber sprouting has developed.
Perforant path stimulation also evoked NMDA receptor-dependent granule
cell burst discharges in ACSF containing 10 µM bicuculline in
hippocampal slices obtained after 1 wk of kindling stimulation
(n = 12 cells from 5 rats that experienced 5-8
afterdischarges; data not shown).
NMDA receptor-dependence of burst discharges in the chronic epileptic state
The increase in the NMDA receptor-dependent component of the
evoked synaptic current in kindled rats diminishes by 2.5-3 mo after
the last evoked seizure and becomes comparable to the NMDA receptor-dependent current in granule cells from age-matched normal rats (Sayin et al. 1999). The NMDA receptor-dependence
of evoked burst discharges was compared in hippocampal slices obtained
within 1 wk after 50-120 evoked Class V kindled seizures, and 3 mo
after the last of >100 Class V seizures, when the NMDA component of the synaptic current has diminished to normal. When
GABAA receptor-dependent inhibition was blocked
by 10 µM bicuculline in these groups, supramaximal perforant path
stimulation evoked epileptic burst discharges (Fig. 7, A and B;
n = 18 of 20 cells).
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It was of interest to compare the duration, number of action
potentials, and NMDA dependence of the burst discharges in these groups. For this analysis, the perforant path stimulus intensity was
standardized by using the lowest stimulus intensity required to evoke a
maximal response as defined by input-output curves. At this
standardized stimulus intensity, the burst duration, measured as the
interval from initial depolarizing deflection of the EPSP to the return
of the evoked depolarization to baseline resting membrane potential,
was shorter in granule cells from rats examined at 3 mo after the last
evoked seizure compared with rats examined within 1 wk after similar
numbers of kindled seizures; there were also fewer action potentials
per evoked burst compared with granule cells from acutely kindled rats
(Fig. 7, C and D). The duration of the evoked
bursts was strongly correlated with number of action potentials in all
groups (r = 0.77, P < 0.0001, Spearman
rank-order correlation). The number of action potentials per burst and
burst discharge duration in rats examined at 3 mo after the last evoked seizure were less sensitive to APV (Fig. 7), which is in agreement with
voltage-clamp recordings in granule cells studied at 2.5-3 mo after
the last evoked seizure (Sayin et al. 1999).
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DISCUSSION |
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Granule cells in the dentate gyrus, which normally generate a
single spike even when inhibition is reduced and thereby restrict the
flow of afferent activity into CA3, converted to a graded burst
discharge spike mode in conditions of reduced inhibition after burst
discharge was evoked during transient relief of the Mg2+ block of the NMDA in vitro, or after
preceding epileptic afterdischarges in vivo. We have referred to this
rapidly induced transformation in the pattern of granule cell spike
output to CA3 as "granule cell spike plasticity." Both the
induction and expression of this plasticity in granule cell spike
generation were prominently NMDA receptor dependent and could therefore
be distinguished from other forms of activity-dependent synaptic
plasticity in granule cells such as LTP, which depends on the NMDA
receptor for induction, but is also expressed through enhancement of
the AMPA receptor-dependent component of the synaptic response
(Maren et al. 1992; O'Connor et al.
1995
; Wang et al. 1996
; Xie et al.
1992
). This NMDA receptor-dependent transformation of granule
cell firing patterns was observed in normal granule cells and in the
epileptic dentate gyrus reorganized by mossy fiber sprouting. The
transformation of granule cell spike generation from a single-spike
firing mode to a graded burst discharge mode could modify information
processing and epileptogenesis in the hippocampus by increasing
susceptibility to epileptic synchronization and altering the
"filtering" properties of the dentate gyrus.
Features of granule cell spike plasticity
Granule cells acquired susceptibility to burst discharge when
inhibition was reduced in hippocampal slices examined: 1)
immediately after burst discharge evoked in
Mg2+-free ACSF, 2) at 24 h after
a single epileptic afterdischarge evoked in vivo, which induces
synaptic potentiation (Sutula and Steward 1986),
3) in kindled rats which have enhanced NMDA
receptor-dependent synaptic currents after repeated seizures
(Mody and Heinemann 1987
; Sayin et al.
1999
), and 4) in kainic acid-treated rats.
Because reduction of inhibition, which was required for expression of
the acquired capacity for burst discharge, occurs during use-dependent
fading of IPSPs (Deisz and Prince 1989; McCarren and Alger 1985
), in the initial period after status epilepticus induced by kainic acid or pilocarpine (Franck et al.
1988
; Hellier et al. 1999
; Michelson et
al. 1989
; Sloviter 1992
), and in kindled rats
experiencing spontaneous seizures (Rutecki et al. 1996
), the sequence of use-dependent increases in NMDA receptor-dependent currents followed by reduced strength of inhibition is likely to be
encountered in realistic settings of ongoing neural activity and
pathologies (Franck et al. 1995
; Isokawa
1996
; Isokawa et al. 1997
; Masukawa et
al. 1996
; Mathern et al. 1999
; Williamson et al. 1999
).
The NMDA receptor dependence and underlying mechanisms of granule cell spike plasticity in the dentate gyrus: contrasts to LTP
There were no differences in the threshold for spike generation between granule cells in normal controls and any of the experimental groups, which suggests that the observed effects were more likely to be caused by alterations in the functional properties of neural circuitry in the dentate gyrus rather than alterations in the intrinsic neuronal mechanisms of spike generation.
The induction of graded burst discharges in
Mg2+-free conditions, and the expression of burst
discharges evoked with subsequent disinhibition, were both blocked by
APV. While these observations implicate the NMDA receptor in the
induction and expression of alterations in granule cell spike
generation, the specific cellular mechanisms which underlie the
induction process, and the subsequent burst generation when inhibition
is reduced, remain uncertain. Enhancement of NMDA receptor-dependent
synaptic transmission has been observed to contribute to burst
discharges also in pyramidal cells of the hippocampus and cortex
(Anderson et al. 1987; Ben-Ari and Gho
1988
; Bush et al. 1999
; DeLorenzo et al.
1998
; Hoffman and Haberly 1989
; Telfeian
and Connors 1999
).
The requirement for synaptically evoked burst activity during exposure
to Mg2+-free conditions implies that cellular
events resulting from neural activity, not merely removal of
Mg2+ or relief of the Mg2+
block of the NMDA receptor, play a role in the induction of subsequent susceptibility to burst discharges when inhibition is reduced. The
cellular events induced by neural activity during
Mg2+-free conditions could include
Ca2+ influx, activation of other types of
receptors and second messenger systems, conformational changes related
to rapid activity-dependent processes such as phosphorylation or
dephosphorylation, or other unrecognized cellular alterations
(Meldrum et al. 1999; Mody 1998
).
Additional experiments will be required to understand how burst discharges during Mg2+-free conditions induce long-lasting susceptibility to burst discharge in granule cells. Whatever the specific mechanisms underlying the induction, however, the overall induction process is influenced by the NMDA receptor, as APV blocks the process. Furthermore, it is unlikely that gene expression is required for the induction of susceptibility to burst discharge, as the conversion of granule cell spike generation from a single-spike mode to the graded burst discharge mode is immediately apparent in normal bathing conditions when inhibition is reduced. If the process of induction required transcription and translation, a longer time interval would be expected for the conversion.
The mechanisms underlying the expression of the burst discharges in
disinhibited granule cells after burst discharges have been induced
during transient Mg2+-free conditions are also
NMDA receptor dependent, as the bursts are blocked by APV. Analysis of
the perforant path-evoked EPSP by both intracellular and extracellular
recordings in normal bathing conditions after the induction process
demonstrated a marked enhancement of the NMDA receptor-dependent
component of the synaptic response, but no significant increase in the
non-NMDA dependent component, which is likely to be mediated by AMPA
receptors. AMPA and kainate receptors, which contribute to synaptic
plasticity in other regions of the hippocampus and neocortex
(Bortolotto et al. 1999; Malenka and Nicoll
1999
), appear to play a minor role in the expression of
activity-dependent plasticity of spike generation in the dentate gyrus.
The prominent effects of the NMDA receptor on the induction and
expression of granule cell spike plasticity described in these experiments can be distinguished from other forms of activity-dependent synaptic plasticity such as long-term potentiation. In contrast to
long-term potentiation in granule cells, which depends on NMDA receptors for induction but also induces a prominent increase in
non-NMDA, AMPA receptor-dependent component of the evoked excitatory synaptic response (Maren et al. 1992; O'Connor
et al. 1995
; Wang et al. 1996
; Xie et al.
1992
), both the induction and the expression of the granule
cell spike plasticity observed in these experiments were NMDA receptor
dependent, but produced little alteration in the non-NMDA
receptor-dependent component of the evoked EPSP.
Functional effects of granule cell spike plasticity: implications for filtering properties of the dentate gyrus
The concept that the dentate gyrus acts as a "filter" for
input to the hippocampus is supported by 2-deoxyglucose autoradiography and electrophysiological observations demonstrating that seizures in
the normal rat do not propagate from the dentate gyrus to CA3, but do
so after seizures (Heinemann et al. 1992; Lothman
et al. 1992
). The cellular and circuit properties that are
responsible for the filtering function of the dentate gyrus are
uncertain, but this study provides some insights into how the filtering
property of the dentate gyrus could be dynamically impaired by
synchronous neural activity both in normal and epileptic rats. The
single-spike firing mode of granule cells in the normal dentate gyrus
serves to filter or restrict flow of afferent activity passing from the entorhinal cortex to CA3 by way of the dentate gyrus, as output to CA3
is "fixed" in the normal dentate gyrus even when
GABAA receptor-dependent inhibition is blocked.
In the burst discharge mode, granule cells fire in a pattern of
repetitive spikes that increase in number as a function of stimulus
strength, and the filtering of the spike output from the dentate gyrus
is thereby reduced or lost, with potential effects on the flow of
activity from the entorhinal cortex to CA3.
The activity-dependent transformation from the single-spike mode to the
graded burst discharge mode could potentially modify the balance of
excitation and inhibition in hippocampal pathways with consequences for
epileptogenesis. By enhancing propagation of neural activity through
the dentate gyrus into CA3, where pyramidal neurons demonstrate both
spontaneous burst discharges and high propensity for evoked epileptic
bursts (Johnston and Brown 1981; Schwartzkroin
and Prince 1978
), conversion to the graded burst discharge mode
could increase excitatory drive in CA3 and promote synchronization in
this highly epileptogenic region of hippocampal circuitry.
Mossy fiber sprouting is not necessary for burst discharge in the dentate gyrus
It has been suggested that the resistance of granule cells in the
dentate gyrus to burst discharge may be caused by absence of recurrent
excitatory circuits (Fricke and Prince 1984;
Golarai and Sutula 1996
; Lynch and Sutula
2000
; Patrylo and Dudek 1998
; Wuarin and
Dudek 1996
) which are normally found in regions such as CA3 and
CA1, where spontaneous and evoked bursts are observed when
GABAA receptor-dependent inhibition is blocked
(Dingledine et al. 1986
; Hablitz 1984
;
MacVicar and Dudek 1980
; Miles and Wong
1987
; Tancredi et al. 1990
; Traub et al.
1994
). In the present study, bursts were evoked in disinhibited
granule cells from normal rats immediately following enhancement of the
NMDA receptor-dependent component of the EPSP during transient exposure
to Mg2+-free ACSF, and at 24 h after a
single subconvulsive afterdischarge, when mossy fiber sprouting has not
yet developed. These findings demonstrate that recurrent circuitry
formed by mossy fiber sprouting is not necessary for
epileptic burst discharge in the dentate gyrus.
In epileptic kindled rats studied at 3 mo after the last evoked
seizure, when the dentate gyrus is reorganized by sprouting and other
seizure-induced alterations but the NMDA receptor-dependent synaptic
current is comparable to normal (Sayin et al. 1999), disinhibited granule cells continue to fire in the graded burst mode,
which is consistent with direct observation of enhanced propagation
from the entorhinal cortex to CA3 and reduced filtering by the dentate
gyrus in kindled rats (Behr et al. 1996
, 1998
). Because
the NMDA receptor-dependent component of the synaptic current in these
rats is comparable to controls at this time interval following a
seizure, seizure-induced cellular alterations other than enhancement of
the NMDA receptor-dependent synaptic response must be contributing to
the burst-firing mode. The long-term seizure-induced structural and
functional alterations in the reorganized dentate gyrus, which include
mossy fiber sprouting and other seizure-induced cellular alterations,
are sufficient to generate epileptic bursts when inhibition
is reduced. It should be noted, however, that burst duration is shorter
and spiking is reduced in epileptic rats examined at 3 mo after the
last seizure compared with granule cells from kindled rats that
recently experienced a seizure. This observation suggests that
enhancement of seizure-induced NMDA receptor-dependent synaptic
transmission may potentially have an additional proconvulsant effect in
the reorganized dentate gyrus, which has clinical implications in
regard to the importance of seizure control in people with epilepsy.
NMDA receptor-dependent plasticity: implications for relationships between synapse formation in development, information storage and memory, and epileptogenesis
NMDA receptors not only influence plasticity of granule cell spike
generation, but also play a role in several forms of activity-dependent plasticity that influence epileptogenesis. Repeated induction of LTP in
the dentate gyrus, which also is an NMDA receptor-dependent process,
increases the rate of kindling development in response to perforant
path stimulation (Sutula and Steward 1987). In addition, about 30 s of synchronous afterdischarge activates NMDA receptors, signal transduction, and gene expression, which eventually results in
permanent structural and functional alterations including mossy fiber
sprouting and increased susceptibility to evoked and spontaneous seizures (McNamara 1999
; Shin et al.
1990
). The importance of NMDA receptors in the induction of
mossy fiber sprouting and the associated progressive increase in
seizure susceptibility induced by kindling is indicated by the
observation that administration of the NMDA receptor antagonist MK-801
at doses that do not suppress evoked kindled seizures blocks both the
sprouting and the associated progression of kindling (Sutula et
al. 1996
).
NMDA receptor-dependent plasticity also regulates circuit formation in
normal development, and information storage in the adult nervous system
that plays a role in learning and memory (Bliss and Collingridge
1993; Scheetz and Constantine-Paton 1994
), but
also can be epileptogenic in the dentate gyrus. NMDA receptor-dependent mechanisms of plasticity confer an important capacity for
experience-dependent modification of synapses and circuits that
undoubtedly has significant adaptive advantages in the developing and
adult nervous systems, but with the potentially undesirable consequence
that this plasticity in the dentate gyrus also enhances susceptibility
to synchronous neural activity and paroxysmal behavioral dysfunction
during epileptic seizures.
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ACKNOWLEDGMENTS |
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The authors appreciate the helpful comments of L. Haberly and P. Rutecki.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-25020.
Present address of G. Golarai: Rm. 145, BMSB, Dept. of Neuroscience, University of New Mexico, Albuquerque, NM 87131.
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FOOTNOTES |
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Address for reprint requests: T. Sutula, Dept. of Neurology, H6/570, University of Wisconsin, Madison, WI 53792 (E-mail: sutula{at}neurology.wisc.edu).
Received 27 January 2000; accepted in final form 28 August 2000.
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REFERENCES |
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