Departments of Neurology and Anatomy and The Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53792
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ABSTRACT |
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Lynch, Michael and
Thomas Sutula.
Recurrent Excitatory Connectivity in the Dentate Gyrus of Kindled
and Kainic Acid-Treated Rats.
J. Neurophysiol. 83: 693-704, 2000.
Repeated seizures induce mossy fiber
axon sprouting, which reorganizes synaptic connectivity in the dentate
gyrus. To examine the possibility that sprouted mossy fiber axons may
form recurrent excitatory circuits, connectivity between granule cells
in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by
kindling and kainic acid. The experiments were designed to functionally
assess seizure-induced development of recurrent circuitry by exploiting
information available about the time course of seizure-induced synaptic
reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When
recurrent inhibitory circuits were blocked by the GABAA
receptor antagonist bicuculline, focal application of glutamate
microdrops at locations in the granule cell layer remote from the
recorded granule cell evoked trains of excitatory postsynaptic
potentials (EPSPs) and population burst discharges in epileptic rats,
which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 µM
tetrodotoxin and were therefore dependent on network-driven synaptic
events. Excitatory connections were detected between blades of the
dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after 1
wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting.
Excitatory connectivity between blades of the dentate gyrus was also
assessed in slices deafferented by transection of the perforant path,
and bathed in artificial cerebrospinal fluid (ACSF) containing
bicuculline to block GABAA receptor-dependent recurrent
inhibitory circuits and 10 mM [Ca2+]o to
suppress polysynaptic activity. Low-intensity electrical stimulation of
the infrapyramidal blade under these conditions failed to evoke a
response in suprapyramidal granule cells from normal rats
(n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 ± 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation
of monosynaptic excitatory connections between blades of the dentate
gyrus. Recurrent excitatory circuits developed in the dentate gyrus of
epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy
fiber pathway.
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INTRODUCTION |
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Mossy fiber axons in the dentate gyrus undergo
sprouting and reorganization of their terminal field in experimental
models of epilepsy and in the human epileptic temporal lobe (de
Lanerolle et al. 1989; Houser et al. 1990
;
Mikkonen et al. 1998
; Represa et al.
1989
; Sutula et al. 1988
, 1989
;
Tauck and Nadler 1985
). In epileptic rats, sprouted axon
collaterals of granule cells project from the hilus of the dentate
gyrus into the supragranular layer in both transverse and longitudinal
directions along the septotemporal axis (Buckmaster and Dudek
1999
; Sutula et al. 1998
). Mossy fiber axons
from granule cells in the infrapyramidal blade of the dentate gyrus
cross the hilus and form synapses in the supragranular layer of the
suprapyramidal blade (Sutula et al. 1998
).
Reorganization of the mossy fiber pathway also occurs in the hilus of
the dentate gyrus of epileptic rats, where mossy fibers form more small
terminal boutons, and have fewer giant boutons and greater axon length
compared with normal rats (Buckmaster and Dudek 1999
;
Sutula et al. 1998
). Axonal remodeling and synaptic reorganization of mossy fiber axons that alters connectivity and local
circuits within the hilus and supragranular layer in the dentate gyrus
may modify functional properties of the dentate gyrus and influence
epileptogenesis, seizure propagation, and memory. Current source
density analysis has demonstrated that the terminal field of the
sprouted mossy fiber pathway in the supragranular layer of the dentate
gyrus is the site of an abnormal inward current that is consistent with
synaptic transmission in the sprouted pathway (Golarai and
Sutula 1996
), but the functional significance of this
seizure-induced plasticity is uncertain.
Recurrent excitatory connections are absent or minimal in the normal
dentate gyrus, but are prominent in other regions of the hippocampus or
in the neocortex that are susceptible to epileptic synchronization
(Claiborne et al. 1986; Deuchars and Thomson
1996
; Deuchars et al. 1994
; MacVicar and
Dudek 1980
; Miles and Wong 1986
).
Seizure-induced formation of recurrent excitatory circuits by sprouted
mossy fibers in the dentate gyrus could potentially contribute to
epileptogenesis by increasing excitatory drive. Because strong systems
of inhibition in the dentate gyrus and formation of recurrent
inhibitory circuits by sprouted mossy fibers may mask or suppress
activity in excitatory circuits, it is not surprising that
seizure-induced recurrent excitation may not be detected unless
GABAA receptor-mediated inhibition is blocked, or in conditions such as use-dependent fading of inhibitory
postsynaptic potentials/inhibitory postsynaptic currents (IPSPs/IPSCs)
(Deisz and Prince 1989
; McCarren and Alger
1985
). Because sprouting increases with repeated seizures
(Cavazos et al. 1991
), it would also be anticipated that
evidence for recurrent excitatory circuits would be more easily
detectable after many repeated seizures.
In previous studies, focal application of glutamate to the granule cell
layer evoked long-latency trains of excitatory postsynaptic potentials
(EPSPs) in granule cells from rats that had experienced status
epilepticus induced by treatment with kainic acid or pilocarpine, but
not in granule cells from normal rats (Molnar and Nadler
1997; Wuarin and Dudek 1996
,
1997
). Variable latency burst discharges and excitatory
postsynaptic currents (EPSCs) are more frequently evoked in granule
cells by antidromic activation of the mossy fiber pathway in
hippocampal slices from rats that experienced status epilepticus than
in controls, which suggests that mossy fiber sprouting could be
contributing to recurrent excitation (Cronin et al.
1992
; Okazaki et al. 1999
; Patrylo and
Dudek 1998
; Tauck and Nadler 1985
). These
results are consistent with the development of recurrent excitatory
circuitry in the dentate gyrus of rats after status epilepticus, but do
not address the critical question of whether sprouted mossy fiber axons
form functionally significant monosynaptic recurrent excitatory
circuits with other granule cells. Furthermore, it is uncertain if the
functional consequences of seizure-induced synaptic reorganization are
similar in kindled rats that experience brief repeated seizures and
rats that have experienced status epilepticus. In this study, we have used glutamate microstimulation methods and focal electrical
microstimulation to exploit the opportunity provided by the kindling
model, which induces mossy fiber sprouting with a predictable time
course and in a gradually progressive manner, and detailed knowledge
now available about the anatomic features of sprouted mossy fiber axon
collaterals, to make new inferences about formation of recurrent circuits in dentate gyrus of epileptic rats.
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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 mm anterior, 1.2 mm lateral, 1.8 mm ventral with respect to bregma) and was fixed to the skull with acrylic. Methods of animal handling and all experimental procedures were approved by the Research Animal Care Committee of the University of Wisconsin.
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 week) 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
could be switched to the stimulator by a digital circuit for the
delivery of the kindling stimulation. The evoked behavioral seizures
were classified according to standard criteria (Sutula and
Steward 1986
) and ranged from class I (behavioral arrest with
electrographic afterdischarge) to class V (bilateral tonic-clonic motor
activity with loss of postural tone with prolonged afterdischarges,
which are comparable to partial complex seizures with secondary
generalization). Kindled rats were killed within 24 h after the
final kindling stimulation.
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 and returned to their cages after 2-3 h,
when the most severe behavioral alterations usually diminished. Previous studies have documented that kainic acid produces initially intense electrographic seizures that gradually diminish and usually cease by 4-5 days (Ben-Ari et al. 1981; Lothman
et al. 1981
; Sutula et al. 1992
) and that are
eventually followed by spontaneous recurrent seizures
(Cavalheiro et al. 1982
; Cronin and Dudek
1988
; Pisa et al. 1980
). Rats that did not
experience status epilepticus in response to the kainic acid injection
were not used in this study. Rats were killed 4-8 wk after kainic acid
treatment. Kainic acid was obtained from Sigma.
Preparation of hippocampal slices
Rats were decapitated after induction of anesthesia by ether,
and the brains were rapidly removed and placed into ice-cold artificial
cerebrospinal fluid (ACSF) with 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 (Technical Products International) at a
thickness of 400-450 µm, perpendicular to the septotemporal axis.
The slices were allowed to equilibrate for at least 1 h in
oxygenated ACSF at 20-22°C, before being transferred to a submersion
recording chamber and bathed in ACSF at 31-32°C. In the recording
chamber, slices were perfused with ACSF containing 6.2 mM
K+ and GABAA
receptor-mediated inhibition was blocked with 10 µM ()-bicuculline
methiodide (Sigma). In some experiments, the voltage-dependent block of
N-methyl-D-aspartate (NMDA) receptor channels by
Mg2+ was relieved by replacing
MgSO4 in the ACSF with
Na2SO4 (Mayer et al.
1984
; Nowak et al. 1984
). We have referred to
ACSF with no added MgSO4 as
"Mg2+-free", but such solutions may still
contain trace concentrations of Mg2+.
Recording and stimulation methods
Granule cells were impaled with tapered glass micropipettes
(100-150 M) filled with 2 M potassium acetate, adjusted to pH 7.4. Granule cells were identified by distinctive 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 (Fricke and Prince 1984
; Lübke et al. 1998
; Scharfman
1992
; Staley et al. 1992
). Granule cells were
included in this study when stable impalements were obtained, 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. Bridge balance was routinely monitored by a conventional bridge
circuit used for intracellular recording and current injection.
Extracellular recording electrodes (2-10 M
) containing 2 M NaCl
were positioned within 200 µm of the intracellular electrode.
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. Responses were amplified, digitized,
and stored on optical disks for off-line analysis.
Assessment of connectivity in the dentate gyrus by glutamate microstimulation
Excitatory connectivity between granule cells was investigated
by determining whether focal application of glutamate in the molecular
layer of the dentate gyrus evoked an EPSP in a recorded granule cell
during bath application of 10 µM bicuculline to block GABAA receptor-dependent IPSPs. In previous
studies in the hippocampus and dentate gyrus, application of glutamate
microdrops to dendrites and cell bodies under these conditions
activated local excitatory synaptic circuits, but not axons,
presynaptic terminals, or GABAA receptor-dependent inhibitory circuits (Christian and Dudek
1988a,b
). Glutamate was applied to the molecular layer of the
dentate gyrus through a glass micropipette with a tip of ~20 µm,
which was filled with L-glutamic acid (20 mM) in ACSF.
Delivery of the glutamate was controlled by a picospritzer (General
Valve) that generated positive-pressure pulses (50 ms, 40 psi) at the
blunt end of the micropipette. The glutamate solutions were routinely
mixed with trace amounts of India ink to monitor micropipette placement
and glutamate solution flow. In preliminary experiments, direct
application of India ink or ink-stained ACSF did not elicit responses
from recorded cells (n = 5), and the distribution of
the ink after pressure injection typically indicated diffusion for
~100 µm from the pipette tip. After obtaining a stable granule cell
impalement, the micropipette containing glutamate was lowered beneath
the surface of the slice at the most distal site in the dentate gyrus (usually either at the blade crest or tip) from the impaled cell, and a
single pulse of glutamate was applied to the molecular layer. Subsequent pulses were applied closer to the impaled cell, in steps of
~100 µm. In some slices, glutamate pulses were also applied in the
molecular layer of the opposite blade of the dentate gyrus, and in the
hilus. After systematic stimulation at locations remote from the
recorded cell, glutamate was directly applied adjacent to the impaled
cell to verify the integrity of the stimulation and recording
procedure. This usually resulted in a large, long-lasting depolarization with repetitive discharges, followed by a refractory period.
Assessment of connectivity in the dentate gyrus by electrical microstimulation
Connectivity between blades of the dentate gyrus was also
assessed by electrical microstimulation applied in transverse
hippocampal slices transected by razor cuts that removed the
CA3a,b regions of the hippocampus, the subiculum, and the
entorhinal cortex. Perforant path connections between blades were
transected by removing the crest of the dentate gyrus (see Fig. 6). The
stimulation pulses consisted of constant-voltage 100 µs pulses
delivered by a bipolar electrode (100 µm tip separation; World
Precision Instruments) to the molecular layer of the infrapyramidal
blade while recording from a granule cell in the opposite
suprapyramidal blade. Electrical microstimulation experiments were
conducted at low stimulus intensities (<10 V), in ACSF with 10 µM
()-bicuculline methiodide to block GABAA
receptor-mediated inhibition and 10 mM Ca2+ to
suppress polysynaptic recurrent excitatory and inhibitory circuits
(Berry and Pentreath 1976
; Crepel et al.
1997
; Miles and Wong 1987
; Williams and
Johnston 1991
).
Histological procedures
After recording, hippocampal slices were immersed in an aqueous solution of 10% (vol/vol) Formalin in 0.9% (wt/vol) NaCl, and stored for at least 24 h at 4°C. After cryoprotection and freezing on dry ice, tissue sections of 60 µm thickness were cut on a freezing microtome in the same plane as the transverse hippocampal slice. All sections were mounted on slides, stained with cresyl violet, and juxtaposed with detailed drawings made during recording of sites of stimulation and recording.
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RESULTS |
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Passive membrane properties of granule cells from normal and epileptic rats
Data were collected from 23 control rats, 30 rats that experienced
kainic acid-induced status epilepticus, and 21 kindled rats. To assess
evolving alterations in connectivity in the dentate gyrus induced by
kindling, kindled rats were killed at one of three time points:
1) within 24 h of the first evoked afterdischarge (n = 5), before the development of mossy fiber
sprouting (Cavazos et al. 1991); 2) at ~ 1 wk after onset of kindling stimulation (n = 5),
when mossy fiber sprouting first becomes detectable by histological
methods (Cavazos et al. 1991
); or 3) after
extensive kindling (30-180 generalized class V seizures;
n = 11), when mossy fiber sprouting is well developed
(Cavazos et al. 1991
; Sutula et al.
1988
). The average resting membrane potential of dentate gyrus
granule cells from control rats was
72.3 ± 1.5 mV (mean ± SE, n = 46 cells), from kainic
acid-treated rats was
72.6 ± 1.3 mV (n = 71 cells), and from kindled rats was
72.7 ± 1.2 mV (n = 50 cells; P = 1.0, ANOVA).
Granule cell input resistances were 58.5 ± 3.3 M
, 60.4 ± 3.3 M
, and 65.5 ± 3.7 M
for control, kainic acid-treated,
and kindled rats (P = 0.4, ANOVA).
Glutamate microstimulation in the dentate gyrus of normal rats
Application of glutamate to the molecular layer of the dentate
gyrus in ACSF containing 10 µM bicuculline failed to evoke EPSPs in
11 granule cells from 7 normal rats (Fig.
1), which confirmed previous observations
(Wuarin and Dudek 1996). In these 11 granule cells,
which included 4 infrapyramidal granule cells and 7 suprapyramidal cells, glutamate application to the molecular layer of the same blade
at distances of 200-800 µm from the impaled cell (n = 11) or to the opposite blade (n = 5) did not evoke an
EPSP or any apparent change in ongoing activity recorded by
intracellular or extracellular methods.
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Because lowering the Mg2+ content of the
bathing medium in the presence of GABAA receptor
blockade has been shown to enhance excitatory polysynaptic activity in
other regions of the hippocampus by relieving
Mg2+ block of the NMDA receptor (Heinemann
et al. 1992; Tancredi et al. 1990
; Traub
et al. 1994
), we also tested for glutamate-evoked responses in
ACSF in which Mg2+ was omitted. In
Mg2+-free ACSF with 10 µM bicuculline,
glutamate application also failed to evoke EPSPs in 14 of 14 granule
cells from 8 normal rats. Glutamate microdrops were applied at
~100-µm intervals along the molecular layer of the dentate gyrus at
an average of six sites per slice, usually in the same blade as the
impaled cell, but also in the hilus (n = 7) and
opposite blade (n = 6). EPSPs were not observed in
response to glutamate microapplication at any of these sites in granule
cells from normal rats (Fig. 2). These
results suggest that enhancement of excitatory transmission by relief
of the Mg2+ block of the NMDA receptor is not
sufficient to increase excitatory connectivity between granule cells in
the dentate gyrus of normal rats.
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Glutamate microstimulation in the dentate gyrus of kainic acid-treated rats
In contrast, application of glutamate microdrops to the molecular
layer of the dentate gyrus in hippocampal slices obtained from 14 rats
at 4-8 wk after kainic acid-induced status epilepticus evoked trains
of EPSPs in 6 of 30 granule cells in ACSF with 10 µM bicuculline
(Fig. 1 and Table 1), confirming previous
reports (Wuarin and Dudek 1996). Trains of EPSPs were
evoked by glutamate application in the same blade as the impaled
granule cell at sites from 200-900 µm from the recording site (Fig.
3). These results were observed in
granule cells in both the suprapyramidal (n = 4), and
infrapyramidal (n = 2) blades. The stimulation sites
that evoked EPSPs appeared to be distributed in an irregular patchy distribution relative to the recorded granule cell (Fig. 3). The patchy
distribution of the evoked responses is consistent with the irregular
distribution of sprouted mossy fiber synaptic terminals in the
supragranular layer described in detailed anatomic studies of granule
cells filled with biocytin in hippocampal slices and in vivo in kainic
acid-treated rats (Buckmaster and Dudek 1999
; Sutula et al. 1998
). In these studies, sprouted mossy
fiber synaptic terminals projected into irregular and randomly
distributed regions of the supragranular layer, rather than in the
diffuse pattern implied by the distribution of labeled terminals
examined by Timm histochemistry at the light microscopic level. The
patchy pattern of evoked responses is also consistent with the patchy
distribution of labeled terminals examined at the ultrastructural level
(Okazaki et al. 1995
; Ribak et al. 1998
;
Sutula et al. 1988
; Zhang and Houser
1999
).
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When Mg2+ was removed from the ACSF, glutamate
microdrops evoked trains of EPSPs in 8 of 13 granule cells from kainic
acid-treated rats (P < 0.02 vs.
Mg2+-free conditions in control rats, Fisher's
exact test; Table 1), which suggests that synaptic transmission in
circuits that contribute to recurrent excitatory connectivity in the
dentate gyrus may involve NMDA receptors. The evoked EPSP trains
occurred at variable latency (5-1,000 ms) after glutamate application,
and lasted for 250 ms to 15 s, which suggested that the trains
were generated by buildup of reverberating network activity
(Ayala et al. 1973; Christian and Dudek
1988a
; Hablitz 1984
; Miles and Wong
1983
). The distribution of stimulation sites that evoked EPSPs
in Mg2+-free ACSF was also irregular and patchy
relative to the recorded cells (not shown). In 4 of 13 granule cells
from kainic acid-treated rats in Mg2+-free ACSF
with 10 µM bicuculline, glutamate microdrops evoked EPSP trains that
were accompanied by action potentials or granule cell burst discharges
(Fig. 4). Granule cell EPSP trains
elicited by glutamate microapplication were detected in extracellular
electrodes and, in three of three cases, were blocked by 1 µM
tetrodotoxin, indicating that they were network-driven synaptic events
and were not due to effects of glutamate on presynaptic terminals (Fig. 4).
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Because recent anatomic studies have demonstrated that seizure-induced
mossy fiber sprouting from infrapyramidal granule cells can cross the
hilus and form terminal boutons in the supragranular layer of the
suprapyramidal blade (Sutula et al. 1998), glutamate microdrop application was also used to test for connectivity between blades. In two of five hippocampal slices from kainic acid-treated rats, application of glutamate in the molecular layer of the
infrapyramidal blade evoked trains of EPSPs in suprapyramidal granule
cells (see Fig. 3). Application of glutamate to the molecular layer of
suprapyramidal blade did not evoke a response in three of three
infrapyramidal granule cells from kainic acid-treated rats.
Application of glutamate to the hilus of hippocampal slices from kainic
acid-treated rats did not evoke responses in granule cells in the
infrapyramidal blade (n = 3) or the suprapyramidal
blade (n = 5) in Mg2+-free ACSF
with 10 µM bicuculline, in agreement with previous reports
(Wuarin and Dudek 1996
).
Glutamate microstimulation in the dentate gyrus of kindled rats
Evidence for recurrent excitatory circuitry was also assessed in
hippocampal slices from rats that experienced brief repeated seizures
evoked by kindling. The time course of development of EPSPs evoked by
glutamate microstimulation was evaluated at progressive stages of
kindling to assess whether functional connectivity developed in the
dentate gyrus with a time course corresponding to the time course of
development of mossy fiber sprouting, which has been characterized in
detail in previous studies (Cavazos et al. 1991; Sutula et al. 1988
). In 4 of 20 granule cells in
hippocampal slices from kindled rats that experienced 30-180
generalized class V seizures, application of glutamate to the molecular
layer of the dentate gyrus evoked trains of EPSPs. As with
glutamate-evoked EPSP trains in hippocampal slices from kainic
acid-treated rats, granule cell EPSP trains in hippocampal slices from
kindled rats were evoked at a variable latency (5-800 ms) and varied
in duration (200 ms to 5 s). In Mg2+-free
ACSF with 10 µM bicuculline, EPSP trains were evoked in 8 of 12 granule cells from kindled rats (Fig. 5,
Table 1). Glutamate application to the suprapyramidal blade evoked
EPSPs in five of seven suprapyramidal granule cells, and application to
the infrapyramidal blade evoked EPSPs in three of five infrapyramidal
granule cells at distances ranging from 200 to 700 µm from the
impaled cell. As in kainic acid-treated rats, systematic application
of glutamate pulses in ~100-µm steps along the blade revealed that
locations along the blade that evoked EPSPs were distributed in an
irregular patchy pattern relative to the recorded granule cell (not
shown).
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Because of the possibility that sprouted mossy fibers may form
recurrent excitatory circuits with granule cells, it was of interest to
determine whether there was a correlation between the development of
excitatory connectivity detected by glutamate microstimulation and the
development of mossy fiber sprouting induced by seizures in kindled
rats. Kindling is particularly advantageous to address this question,
because mossy fiber sprouting develops gradually in response to
repeated seizures, and both the time course and the extent of sprouting
have been characterized as a function of repeated seizures
(Cavazos et al. 1991). Sprouting is not apparent in
kindled rats at 24 h after a single afterdischarge, but first
becomes apparent at 4-7 days after the initial stimulations that evoke
synchronous afterdischarges (Cavazos et al. 1991
). In
seven granule cells from five kindled rats killed 24 h after a
single evoked afterdischarge, when sprouting has not yet developed (Cavazos et al. 1991
), glutamate application failed to
evoke a response in Mg2+-free bathing medium with
10 µM bicuculline (Fig. 5; P = 0.02 vs. extensively
kindled rats, Fisher's exact test). In contrast, glutamate application
in disinhibited Mg2+-free ACSF to the dentate
gyrus of kindled rats killed at 1 wk after five to eight evoked
afterdischarges, when histological evidence of sprouting first becomes
apparent (Cavazos et al. 1991
), evoked trains of EPSPs
in four of nine granule cells from five rats (Fig. 5). This observation
is consistent with the possibility that sprouted mossy fibers may be
contributing to increased excitatory connectivity by forming recurrent
excitatory circuits between granule cells in the dentate gyrus.
Electrical microstimulation in the dentate gyrus of normal and epileptic rats
The ability to evoke EPSPs in granule cells by focal
microinjection of glutamate into regions of the dentate gyrus depends not only on the presence of axonal connections between the cells, but
is also influenced by factors such as the rate of diffusion of
glutamate after pressure injection, the peak local concentration at
sites of glutamate receptors, and rate of glutamate removal from the
extracellular space (Goodchild et al. 1982). Because these technical factors influence the latency of EPSPs evoked by focal
microinjection of glutamate, it is not possible to determine whether
the evoked EPSPs are generated by activation of polysynaptic or
monosynaptic circuits. Moreover, observations that the trains of EPSPs
and burst discharges evoked by glutamate microstimulation occurred at a
variable latency after injection (see Fig. 3) and were blocked by bath
application of tetrodotoxin suggests that these population events may
have been generated by buildup of excitatory activity spreading in a
network of recurrent circuits (Johnston and Brown 1984
;
Wong et al. 1986
).
Previous studies have not allowed the assessment of the latency of
responses evoked in the reorganized circuitry of the dentate gyrus
(Okazaki et al. 1999; Wuarin and Dudek
1996
). Electrical microstimulation methods were therefore used
to obtain a more reliable estimate of latency of EPSPs evoked by focal
stimulation of the dentate gyrus in hippocampal slices from epileptic
rats. If sprouted mossy fibers that arise from granule cells in the infrapyramidal blade of the dentate gyrus form excitatory connections with granule cells in the suprapyramidal blade as suggested by both in
vitro and in vivo labeling of sprouted mossy fiber axons (Sutula
et al. 1998
), activation of granule cells in the infrapyramidal blade should evoke an EPSP in granule cells of the suprapyramidal blade
with a latency compatible with mossy fiber monosynaptic connections
(Doller and Weight 1982
; Jonas et al.
1993
; Williams and Johnston 1991
). This
prediction was tested by stimulating the infrapyramidal blade while
recording from granule cells in the suprapyramidal blade in transected
hippocampal slices from normal and kainic acid-treated rats in which
perforant path connections to granule cells in each blade were cut
(Fig. 6) and polysynaptic activity was
suppressed by 10 mM
[Ca2+]o (Berry
and Pentreath 1976
; Crepel et al. 1997
;
Miles and Wong 1987
).
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In transected slices of the dentate gyrus from kainic acid-treated rats containing only the infrapyramidal and suprapyramidal blades, the intervening hilus, and a small sector of CA3c (Fig. 6), which were bathed in ACSF with 10 µM bicuculline to suppress IPSPs and 10 mM Ca2+ to suppress polysynaptic activity, stimulation of the molecular layer of the infrapyramidal blade with a 100-µs constant voltage pulse (<10 V) evoked EPSPs in 5 of 18 suprapyramidal granule cells. EPSP latency (measured from the center of the stimulation artifact to EPSP onset) was 2.59 ± 0.36 ms (Fig. 6). In contrast, stimulation of the infrapyramidal blade in hippocampal slices from normal rats failed to evoke EPSPs in 15 suprapyramidal granule cells (P < 0.05, Fisher's exact test). These observations are evidence that seizure-induced granule cell axonal reorganization results in the formation of monosynaptic excitatory connections between blades of the dentate gyrus.
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DISCUSSION |
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There has been considerable interest in determining whether
sprouted mossy fibers induced by repeated seizures form recurrent circuits in the dentate gyrus (McNamara 1999). Paired
intracellular recording could provide definitive evidence about
formation of recurrent excitatory circuits by demonstrating that
current injection that evokes spikes in one granule cell evokes an EPSP
or EPSC in another granule cell. Paired recordings in hippocampal
regions such as CA3 that are known to have extensive recurrent
connections, however, have demonstrated that not more than 5% of
neurons form recurrent excitatory circuits (MacVicar and Dudek
1980
; Miles and Wong 1986
). As a preliminary
alternative to laborious paired recording experiments that would be
required to conclusively resolve this question, we employed the methods
of glutamate microstimulation, which has been used previously in
studies of the hippocampus and dentate gyrus (Christian and
Dudek 1988a
,b
; Wuarin and Dudek 1996
), and a
focal electrical microstimulation technique in transected hippocampal
slices, to assess development of excitatory connectivity in dentate
gyrus of rats that had experienced seizures. The design of the
experiments exploited information about the time course of development
of recurrent mossy fiber circuitry induced by kindled seizures
(Cavazos et al. 1991
), and the detailed information now available about the anatomic features of sprouted mossy fiber axon
collaterals in kainic acid-treated rats (Buckmaster and Dudek 1999
; Sutula et al. 1998
).
Confirming previous anatomic and physiological studies, the experiments
did not detect evidence for excitatory connections between regions of
the dentate gyrus in normal rats when GABAA receptor-dependent recurrent inhibitory circuitry was blocked and
excitatory synaptic transmission was enhanced by increasing the NMDA
receptor-dependent synaptic current. The experiments provided new
evidence that excitatory connections develop in the dentate gyrus of
kindled rats in a time course that paralleled the development of mossy
fiber sprouting (Cavazos et al. 1991). In
addition, the spatial patterns of intrablade and interblade connectivity in kainic acid-treated rats corresponded to patterns of
arborization of sprouted mossy fiber axons described in anatomic studies (Sutula et al. 1998
), and focal electrical
microstimulation experiments furthermore demonstrated interblade
excitatory connections at a latency consistent with monosynaptic
excitatory pathways.
EPSPs evoked by glutamate microinjection and electrical microstimulation as evidence for recurrent excitatory connections
The observation that glutamate application in the granule
cell and molecular layer can evoke EPSPs in other granule cells of
epileptic kindled and kainic acid-treated rats is consistent with the
development of recurrent excitatory circuits in response to repeated
seizures, but these results are not sufficient to conclude that
recurrent excitatory circuits are formed by synapses of sprouted mossy
fibers on other granule cells. In the initial description of EPSPs
evoked by glutamate microstimulation in kainic acid-treated rats
(Wuarin and Dudek 1996), and in our experiments in
kindled and kainic acid-treated rats, there was a long and variable
latency between glutamate application and the onset of EPSPs. This
delay (as long as 1,000 ms after glutamate application in our
experiments) may be at least partially explained by technical factors
such as the glutamate diffusion, peak local concentration at sites of
glutamate receptors, and glutamate removal, but could also be caused by
a gradual buildup of polysynaptic excitatory activity spreading in a
network of recurrent circuits (Johnston and Brown 1984
;
Wong et al. 1986
). If recurrent circuits formed by
sprouted mossy fibers on other granule cells contribute to generation
of the trains of EPSPs evoked by glutamate microstimulation, it would
be expected that 1) the development of EPSPs should
correlate with the time course of mossy fiber sprouting, 2)
the spatial distribution of the sites of glutamate application that
evoke trains of EPSPs should correspond to projection patterns of
sprouted mossy fiber axons, and 3) electrical
microstimulation of regions of the dentate gyrus that contain cells of
origin of sprouted mossy fibers should evoke EPSPs in granule cells
with a latency consistent with a monosynaptic pathway.
Spatial features of seizure-induced excitatory connectivity in epileptic rats
When GABAA receptor-dependent IPSPs were
blocked by bicuculline, EPSPs were evoked in granule cells of the
epileptic rats by application of glutamate to sites in the molecular
layer of the same blade as well as the opposite blade of the dentate
gyrus relative to the recorded granule cell. Although the EPSPs were evoked at distances from ~200-900 µm from the recorded cell within the same blade, the distribution of sites that evoked EPSPs was patchy.
This anatomic feature of the responses and the pattern of interblade
projection from infrapyramidal to suprapyramidal blade are consistent
with the projection patterns of biocytin-filled sprouted mossy fiber
axons observed in anatomic studies (Sutula et al. 1998).
Time course of induction of excitatory connectivity in epileptic rats: relationship to development of mossy fiber sprouting
In previous studies, spontaneous burst discharges were not
observed 2-4 days after kainic acid-induced status epilepticus, but
were observed months later after the development of sprouting (Cronin et al. 1992; Patrylo and Dudek
1998
; Wuarin and Dudek 1996
). In the present
study we have further explored the relationship between development of
increased excitatory connectivity as assessed by glutamate microdrops
in the kindling model, which induces mossy fiber sprouting with a
predictable time course and in a gradually progressive manner. Trains
of EPSPs were not evoked in dentate gyrus from rats examined 24 h
after a single kindled afterdischarge, when sprouting has not yet
developed, but were evoked by glutamate application in hippocampal
slices from rats examined at ~1 wk after initial kindling stimulation
when histological evidence of sprouting first becomes apparent
(Cavazos et al. 1991
). At 24 h after a single
afterdischarge, the NMDA receptor-dependent component of the evoked
population EPSP and the evoked population spike have been enhanced by
kindling stimulation (Sutula and Steward 1986
;
unpublished observations), but this seizure-induced enhancement of
granule cell synaptic transmission, which precedes development of
sprouting, was not accompanied by development of excitatory connectivity in the dentate gyrus. These observations were confirmed by
the inability to evoke EPSPs by glutamate microstimulation in
hippocampal slices from normal rats exposed to
Mg2+-free ACSF, which enhances excitatory
transmission by increasing the NMDA receptor-mediated component of
synaptic transmission (Herron et al. 1985
;
Schneiderman and MacDonald 1987
; Traub et al.
1994
). These previous studies and the present results together suggest that acute seizure-induced increases in synaptic efficacy, which are sufficient to alter susceptibility to burst discharges (unpublished observations), are not sufficient to alter excitatory connectivity in the dentate gyrus. More slowly developing chronic cellular alterations, possibly mossy fiber sprouting, are required.
Mossy fiber sprouting was not directly assessed by histological
analysis in our experiments, but the time course of development of
excitatory connectivity in kindled rats was comparable to the time
course of mossy fiber sprouting in previous studies (Cavazos et
al. 1991). Furthermore, previous studies in kainic
acid-treated rats with extensive sprouting have not uniformly
demonstrated burst discharges when GABAA
receptor-mediated inhibition is blocked (Cronin et al.
1992
; Patrylo and Dudek 1998
), and as in our
study, glutamate microstimulation does not uniformly evoke EPSPs in the presence of sprouting (Molnar and Nadler 1997
;
Wuarin and Dudek 1996
, 1997
). In slices
demonstrating evoked EPSPs, the patchy distribution of sites where
glutamate evoked responses suggests dependence on underlying local circuitry.
Available anatomic studies of the sprouted pathway demonstrate that
hippocampal slices, regardless of orientation, transect the mossy fiber
pathway and processes of other neurons in the dentate gyrus and
therefore invariably limit attempts to comprehensively assess the
structural and functional features of the sprouted pathway
(Amaral and Witter 1989; Buckmaster and Dudek
1999
; Buckmaster et al. 1996
; Sutula et
al. 1998
). The experiments reported here, although still
preliminary, have nevertheless provided new spatial and temporal
details about the development of recurrent excitatory circuits in the
epileptic dentate gyrus. A complete analysis of the spatial
relationship between the pattern of evoked EPSPs and sprouted mossy
fiber terminals would likely require in vivo paired recording and
detailed anatomic analysis of axonal projections from the site of stimulation.
Recurrent excitatory circuits that could contribute to increased excitatory connectivity in the epileptic dentate gyrus
Although the induction of glutamate-evoked EPSPs in granule cells
is strong evidence that recurrent excitatory connections develop in the
dentate gyrus of epileptic rats, the identity of circuits that
contribute to this seizure-induced connectivity is uncertain. Granule
cells have reciprocal connections with polymorphic neurons in the hilus
in the dentate gyrus, and also form distinctive giant mossy fiber
synapses with "thorny excresences" or complex spines of mossy
cells, which provide excitatory feedback to granule cells
(Amaral 1978; Buckmaster et al. 1996
;
Claiborne et al. 1986
; Jackson and Scharfman
1996
; Scharfman et al. 1990
; Seress and Pokorny 1981
). Seizure-induced enhancement of excitatory
transmission in this pathway, or in the polysynaptic circuit from the
dentate gyrus or entorhinal cortex to CA3 with direct projection from CA3 back to granule cells, could play a role in the generation of the
glutamate-evoked EPSPs observed in the present study (Scharfman 1996
). However, we were unable to elicit EPSPs in hippocampal slices from normal rats, even in disinhibited
Mg2+-free ACSF, which enhances
polysynaptic excitatory circuitry in other regions of the hippocampus
(Crepel et al. 1997
; Tancredi et al.
1990
; Traub et al. 1994
). Furthermore, because
mossy cells, pyramidal neurons in CA3, and other polymorphic neurons in
the hilus are especially susceptible to seizure-induced cell death (Ben-Ari 1985
; Cavazos et al. 1994
;
Meldrum et al. 1973
; Nadler and Cuthbertson
1980
; Sloviter 1983
), it seems improbable that enhancement of transmission in these excitatory circuits would generate
glutamate-evoked EPSPs in the epileptic hippocampus, which undergoes
substantial seizure-induced loss in these regions.
Other possible sources of increased excitatory connectivity include
seizure-induced sprouting by surviving mossy cell axons, which might
increase polysynaptic innervation to granule cells, and sprouting by
mossy fiber axons in the hilus, which has been demonstrated in
biocytin-filled granule cells of kainic acid-treated rats
(Buckmaster and Dudek 1999; Sutula et al.
1998
). Because sprouting by mossy fibers in the hilus increases
the number of small synapses (Sutula et al. 1998
), which
almost exclusively terminate on GABAergic interneurons in normal rats
(Acsády et al. 1998
), it seems unlikely that
reorganization in these polysynaptic circuits would increase excitatory
innervation to granule cells (Kotti et al. 1997
).
Monosynaptic excitatory circuits in the dentate gyrus of epileptic rats
In a further attempt to identify the specific circuit or circuits
that contribute to recurrent excitatory connectivity induced by
seizures in the dentate gyrus, electrical microstimulation of the
molecular layer of the infrapyramidal blade was used to more accurately
measure the latency of the recurrent connections that form between
blades of the dentate gyrus. These measurements were performed in
transected slices of the hippocampus that contained only the
infrapyramidal and suprapyramidal blades, the intervening hilus, and a
small sector of CA3c. In these slices (see Fig.
6), severing the perforant path connection between blades by removing the crest of the dentate gyrus eliminated the possibility of antidromic back-propagation from the infrapyramidal to suprapyramidal blade, and
the small remaining sector of CA3c after
transection reduced the possibility of activation of polysynaptic
circuits through the CA3 network (Scharfman 1994-1996
).
In addition, polysynaptic activity was further reduced by bath
application of ACSF with 10 µM bicuculline to block IPSPs and 10 mM
[Ca2+]o, which suppresses
polysynaptic activity (Berry and Pentreath 1976
;
Crepel et al. 1997
; Miles and Wong 1987
).
Under these recording conditions in kainic acid-treated rats,
low-intensity electrical stimulation of the molecular layer of the
infrapyramidal blade evoked EPSPs in granule cells of suprapyramidal
blade at a latency of 2.6 ms, which were not observed in normal rats.
This latency indicates monosynaptic transmission and is consistent with
the possibility that sprouted mossy fibers form functional recurrent excitatory circuits between blades of the dentate gyrus in epileptic rats.
Net functional effects of seizure-induced recurrent neuronal circuits
Synapses formed by sprouted mossy fibers on dendrites of granule
cells would result in recurrent excitatory circuits that could increase
excitatory drive and promote epileptogenesis. Conversely, recurrent
inhibitory circuits formed by mossy fiber synapses on inhibitory
interneurons would be expected to enhance inhibition. Ultrastructural
analysis of asymmetric synapses formed by sprouted mossy fibers has
provided evidence that both types of synapses are formed, but the
relative abundance of these functionally distinct circuits has not been
formally assessed (Franck et al. 1995; Kotti et
al. 1997
; Okazaki et al. 1995
; Ribak and
Peterson 1991
; Ribak et al. 1998
; Zhang
and Houser 1999
). Under normal physiological conditions,
activity in recurrent excitatory circuits is balanced by activity in
inhibitory circuits and may be masked or suppressed by recurrent
inhibition (Crepel et al. 1997
; Miles and Wong
1987
; Scharfman 1996
), except for brief periods
when dynamic alterations in afferent activity or use-dependent fading
of inhibition may disrupt the balance. Synchronous excitatory activity
that overwhelms inhibition and generates a seizure is a relatively
uncommon event in the presence of strong systems of recurrent of
inhibition. More complete understanding of the contribution of
seizure-induced circuit reorganization to epileptogenesis will need to
resolve two critical questions: 1) what is the relative
abundance of seizure-induced inhibitory and excitatory circuits in
regions of the brain that are sites of seizure initiation, and
2) what are the dynamic processes that result in reduction
of inhibition and are permissive for epileptic synchronization?
Conclusions
The time course of development, patterns of excitatory
connectivity, and the latency of excitatory connections demonstrated in
these experiments support the possibility that sprouted mossy fibers
form monosynaptic recurrent circuits with other granule cells that
contribute to recurrent excitation in the epileptic dentate gyrus.
Paired intracellular recordings will be necessary to provide
unequivocal physiological evidence for seizure-induced formation of
functional granule cell-to-granule cell connections. Furthermore,
because anatomic evidence has demonstrated that seizure-induced sprouting produces marked septotemporal divergence in the normally lamellar projection of the mossy fiber terminal field (Sutula et
al. 1998) and preparation of hippocampal slices invariably alters the circuitry of the hippocampus, in vivo recordings of pairs of
granule cells may be required to fully assess the extent and functional
significance of mossy fiber sprouting. Despite these limitations, the
data presented here provide evidence that seizure-induced mossy fiber
sprouting is associated with the development of aberrant monosynaptic
recurrent excitatory circuitry that may be more extensive than
previously appreciated.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-25020.
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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.
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 30 June 1999; accepted in final form 6 October 1999.
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REFERENCES |
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