1Department of Medicine (Neurology), 2Department of Neurobiology, and 3Department of Pharmacology and Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Bausch, Suzanne B. and James O. McNamara. Synaptic Connections From Multiple Subfields Contribute to Granule Cell Hyperexcitability in Hippocampal Slice Cultures. J. Neurophysiol. 84: 2918-2932, 2000. Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term (postnatal day 11; 40-60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABAA receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.
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INTRODUCTION |
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The hippocampus is a brain structure
that is important for learning and memory and is a common focus for
epileptic seizures. One of the primary afferent pathways into the
hippocampus arises from neurons in the entorhinal cortex. Propagation
of information from the entorhinal cortex through the hippocampal
formation, on a simplistic level, proceeds via a trisynaptic
feed-forward excitatory pathway. Information from the entorhinal cortex
is transmitted sequentially through a series of excitatory synapses; first to dentate granule cells, from there to CA3 pyramidal cells, and
last from CA3 to CA1 pyramidal cells (Amaral and Witter
1989). Thus as the first neurons in this series of excitatory
synapses through the hippocampal formation, the dentate granule cells
are in a position to limit transmission through the hippocampal network.
The hippocampal formation is an exquisitely seizure-prone structure
(Green 1964). Within this structure, the principal cells (i.e., CA1 and CA3 pyramidal cells of the hippocampus and the granule
cells of the dentate gyrus) display differing propensities for
epileptiform activity and seizures. The CA3 pyramidal cells are most
prone to epileptiform activity (Miles and Wong 1986
), which is thought to be due in part to recurrent excitatory synapses between CA3 pyramidal cells. In contrast, seizure-like activity is
difficult to induce in normal dentate granule cells, which is thought
to be due to the intrinsic properties of the granule cells, the lack of
recurrent excitatory synapses with their neighboring granule cells and
the presence of strong polysynaptic inhibitory synapses onto granule
cells. The difficulty in inducing seizures in dentate granule cells,
together with work by Lothman and colleagues (Collins et al.
1983
), have led to the hypothesis that the granule cells of the
dentate gyrus normally serve as a barrier to invasion of epileptiform
activity and seizures into the hippocampus. Collins et al.
(1983)
studied behavioral seizures and 2-deoxy-glucose (2DG)
activity during focal application of penicillin to the entorhinal cortex; as long as the 2DG increase was limited to the dentate gyrus
itself, little or no behavioral seizure activity occurred. Only after
2DG increases propagated beyond the dentate gyrus did behavioral
seizures emerge, leading to the idea that the granule cells served as a
barrier to seizure invasion of hippocampal circuitry. Field potential
recordings of seizures evoked in entorhinal-hippocampal slices provided
direct evidence in support of this idea. Behr et al.
(1996)
showed that low Mgo2+ induced seizures
in entorhinal cortex propagated through dentate gyrus to CA3 and CA1 in
4 of 18 slices from normal rats; propagation of these entorhinal cortex
seizures was blocked by a knife cut between the entorhinal cortex and
dentate gyrus (Behr et al. 1998
), implying that axons
connecting entorhinal cortex with dentate conveyed the seizures
to hippocampus. Moreover, limbic epileptogenesis is associated with
loss or attenuation of the barrier function (Behr et al.
1998
). Taken together, these findings suggest that elimination
of the filter function of the granule cells may be a pivotal event in
limbic epileptogenesis.
The single most common form of temporal lobe epilepsy in humans is
associated with selective neuronal loss termed Ammon's horn or
hippocampal sclerosis (Margerison and Corsellis 1966) and synaptic reorganization termed mossy fiber sprouting (de
Lanerolle et al. 1989
; Houser et al. 1990
;
Sutula et al. 1989
). Similarly, mossy fiber sprouting
also has been observed in numerous animal models of temporal lobe
epilepsy (Mello et al. 1992
; Sutula et al.
1988
; Tauck and Nadler 1985
). Mossy fiber
sprouting is the synaptic reorganization of the mossy fiber axons of
dentate granule cells into the inner molecular layer of the dentate
gyrus (Okazaki et al. 1995
; Represa et al.
1993
; Sutula et al. 1988
); a region almost
devoid of mossy fiber collaterals in normal animals (Mello et
al. 1992
; Ribak and Peterson 1991
; Seress
1992
; Sutula et al. 1988
; Tauck and
Nadler 1985
). The association between mossy fiber sprouting and
an epileptic phenotype has led to the popular hypothesis that one cause
of hyperexcitability in the sclerotic, epileptic hippocampus is that
the mossy fiber axons of dentate granule cells form synapses with
themselves and other granule cells, thus forming a recurrent excitatory
network. Indeed, anatomical studies have documented that sprouted mossy
fibers do form synapses onto granule cells (Okazaki et al.
1995
; Represa et al. 1993
; Sutula et al. 1988
, 1989
; Wenzel et al. 1995
),
suggestive of recurrent excitatory synapses between granule cells. Thus
the idea has emerged that recurrent excitatory synapses between granule
cells coincident with mossy fiber sprouting could compromise the
ability of the dentate gyrus to act as a barrier to invasion of
epileptiform activity into the hippocampus.
Ammon's horn sclerosis is often preceded by limbic status epilepticus
in animals and humans, which causes dramatic cell loss in regions such
as entorhinal cortex, septum, and hippocampus as well as dennervation
of hippocampal structures (Du et al. 1993; Green
et al. 1989
; Margerison and Corsellis 1966
;
Mathern et al. 1996
; but see Pennell et al.
1999
). Preparation of hippocampal slice cultures also produces
dramatic denervation. Although a substantial number of synapses are
maintained following slice preparation due to the laminar organization
of the hippocampus, profound denervation from extrahippocampal
structures occurs. This striking commonality between organotypic
hippocampal slice cultures and the sclerotic hippocampus in human
temporal lobe epilepsy prompted us to hypothesize that morphological
reorganizations and granule cell excitability in hippocampal slice
cultures should be similar to those observed in human limbic epilepsy
and animal models. To test this hypothesis, we performed
electrophysiological analyses of granule cell excitability and
morphological analyses of the synaptic organization in long-term
organotypic hippocampal slice cultures.
Portions of this manuscript were presented previously in abstract form
(Bausch and McNamara 1997; Bausch et al.
1998
).
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METHODS |
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Organotypic hippocampal slice cultures
Slice cultures were prepared using the method by Stoppini
et al. (1991) as described previously (Routbort et al.
1999
). Briefly, postnatal day 11 (P11)
Sprague-Dawley rat pups (Zivic-Miller, Zenople, PA) were anesthetized
with pentobarbital sodium and decapitated. The brains were removed;
hippocampi were dissected aseptically and placed onto an agarose
cushion. Hippocampi were then cut into 400-µm transverse sections
using a McIlwain tissue chopper and placed into Gey's balanced salt
solution (GBSS, GIBCO BRL) supplemented with 6.5 mg/ml glucose.
Sections were separated with a Teflon spatula, and the middle four to
six slices of each hippocampus (with the entorhinal cortex removed)
were placed onto tissue culture membrane inserts (Millipore) in a
tissue culture dish containing medium consisting of 50% minimum
essential medium, 25% Hank's buffered salt solution, 25%
heat-inactivated horse serum, 0.5% GlutaMax II, 10 mM HEPES (all from
GIBCO BRL), and 6.5 mg/ml glucose (pH 7.2). Medium was changed two to
four times per week. Cultures were maintained at 37°C under room air
+5% CO2. Physiological recordings and anatomical
labeling were performed at 40-60 days in vitro (DIV).
All treatment of animals was according to National Institutes of Health and institutional guidelines.
Acute hippocampal slices
Acute hippocampal slices were isolated from young adult (5-6 wk) male Sprague-Dawley rats (Zivic-Miller, Zenople, PA); the age of the rat approximating the age of slice cultures at time of recording. Rats were anesthetized with halothane and decapitated, and brains were immediately removed and placed in ice-cold buffer. The brain was blocked and attached to a wax block using cyanoacrylate glue, and 400-µm transverse slices were cut using a vibratome. The middle four to six slices of each hippocampus were placed in a submerged holding chamber containing buffer composed of (in mM): 120 NaCl, 3.5 KCl, 1.3 MgSO4, 2.5 CaCl2, 1.24 NaH2PO4, 25.6 NaHCO3, and 10 glucose equilibrated with 95% O2-5% CO2 for at least 1 h before recording.
Electrophysiological recording
For acute hippocampal slices, slices were placed into a
recording chamber mounted to a Zeiss Axioskop microscope and held down
with a harp made of platinum wire and nylon strings. For hippocampal
slice cultures, a portion of the tissue culture insert membrane
containing a single slice culture was cut, and the membrane and slice
culture were placed into the recording chamber. The membrane was held
down with platinum wires. Both acute slices and slice cultures were
superfused at room temperature with a recording buffer composed of (in
mM) 120 NaCl, 3.5 KCl, 1.3 MgSO4, 2.5 CaCl2, 1.24 NaH2PO4, 25.6 NaHCO3, and 10 glucose, equilibrated with 95%
O2-5% CO2. Bicuculline
methiodide (BMI, 10 µM; Sigma), D()-2-amino-5-phosphonopentanoic acid (D-APV,
50 µM; Tocris Cookson), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX,
10 µM; Tocris Cookson), and
6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX, 10 µM;
Tocris Cookson) were diluted immediately before use in recording buffer
and applied by bath superfusion. Recording pipettes (whole cell, 2-5
M
; extracellular, 1-3 M
) were pulled on a Flaming-Brown puller
and filled with 3 M NaCl for extracellular recordings or with (in mM)
100 K-gluconate, 30 KCl, 10 HEPES, 10 EGTA, 3 MgCl2, and 2 Na2ATP (pH 7.2 with KOH) for whole cell current-clamp recordings. Whole cell
recordings were obtained using visual identification and were excluded
if series resistance was >10 M
. Evoked responses were elicited by
stimulation (0.3-ms square pulse, 0.03 Hz, 20-700 µA) of the hilar
mossy fiber pathway using a concentric bipolar electrode (MCE-100,
Rhodes Medical Supply) and a Grass stimulator.
Current-clamp data were collected within 2-5 min of establishing whole cell configuration. The resting membrane potential (RMP) was read from the amplifier. Input resistance (Rin) was calculated with pCLAMP software using points from the linear portion of a current-voltage plot of the change in membrane voltage in response to a series of 450-ms, 25- to 50-pA steps. Spike properties were determined by generating a series of 450-ms, 25- to 50-pA steps. Spike threshold was determined as the first current step that elicited an action potential. The number of action potential spikes was counted at 1) threshold for spike generation and 2) following a 200-pA current step.
Dentate granule cell layer field potential recordings were deemed
acceptable if hilar stimulation yielded an action potential spike
(spike) that immediately followed the stimulus artifact with a response
threshold 100 µA (e.g., Fig. 4). The basis for concluding that this
waveform was an action potential spike was that the waveform could be
abolished with tetrodotoxin (TTX; 1 µM; Calbiochem; data not shown).
Given the very short latency and lack of an underlying field excitatory
postsynaptic potential (EPSP), this action potential spike was most
likely due to the antidromic stimulation and the subsequent synchronous
firing of a population of dentate granule cells. The spike immediately
following the stimulus artifact in field potential recordings was
therefore called an antidromic population spike. Neither the amplitude
of the antidromic population spike nor the shape of the waveform was
used as criterion for acceptable recordings. Evoked responses were
measured at a stimulus intensity sufficient to evoke a maximal response
(200-700 µA) in both acute slices and slice cultures. Short-latency
EPSP amplitudes (Fig. 8, C-E, right) were
measured from baseline to peak positivity (see Fig. 8B,
right). Short-latency EPSP durations (Fig. 8, C-E,
left) were measured from the point immediately following the
antidromic population spike to a point at 50% of the peak amplitude
(excluding any spikes) during the decay phase of the EPSP (see Fig.
8B, left). Seizures were defined as a burst of rhythmic
activity
3 s in duration that evolved over time and exhibited an
abrupt onset and an abrupt termination (see Figs.
1 and 2).
Recordings from slice culture experiments investigating seizure
activity were analyzed independently by two investigators, and the
percentages of cultures showing seizures were averaged; investigators
agreed on 15 of 17 traces. Epileptiform bursts were defined as bursts
of rhythmic spikes or spikes superimposed on positive field potential
shifts that were
80 ms in duration, but that did not fit the criteria
for seizures (see Fig. 3A,
BMI). Spontaneous postsynaptic potential (PSP) data were collected as two to six runs of 30 s duration per run (Fig. 3A).
Spontaneous activity in acute slice experiments was monitored on an
oscilloscope; no spontaneous activity was noted. PSPs were defined as
waveforms that were blocked by antagonists of fast synaptic
transmission (BMI + APV + CNQX or NBQX). Data were collected using an
Axopatch 1D amplifier (2-kHz analog filter) and pCLAMP or Axotape
(Figs. 1 and 2; 3.33- to 10-kHz acquisition rate) software.
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Knife cuts between the dentate gyrus and different regions of the hippocampal formation were performed with a scalpel blade under a Reichert Stereo Zoom dissecting microscope 45 min to 1 h prior to electrophysiological experiments as depicted in Fig. 8A. To separate the CA1/CA2 regions of the hippocampus from the dentate gyrus (CA1 cut), two knife cuts were placed in a plane roughly parallel to the hippocampal fissure at the approximate border between stratum radiatum and stratum lacunosum-moleculare; knife cuts extended into stratum radiatum of CA3 and thus severed the Schaeffer collateral projection from CA3 to CA1. To separate the CA3 a/b region of the hippocampus from the dentate gyrus (CA3 cut) one knife cut was placed at the open end of the dentate hilus in a straight line between the two blades of the granule cell layer. This CA3 cut extended through the CA1/CA2 pyramidal cell layer. CA3 cuts did not isolate the CA3c region from the remainder of the dentate gyrus. Combined cuts of CA1 and CA3 were used to isolate the dentate gyrus/CA3c region from the rest of the hippocampal formation (DG cut). Data from uncut and CA1, CA3, and the combination CA1/CA3 cut (DG cut) cultures were obtained from different slice cultures because recordings before and after knife cuts in the same slice culture were not technically feasible for the following reasons. Knife cuts disrupted the integrity of the recordings when performed in the recording chamber with electrodes in place due to slight movement of the culture (data not shown). Removing the electrodes and repositioning them after cuts was not attempted; data obtained following repositioning of electrodes is difficult to interpret since field potential waveforms can be altered drastically by slight changes in the location of the recording electrode.
Histology
NEUROBIOTIN.
Individual neurons were visualized and filled with neurobiotin using
whole cell recording techniques. Recording buffer and methods were
identical to those described for electrophysiological recording except
that neurobiotin (0.4 or 0.5%; Vector) was added to the K-gluconate
pipette solution immediately prior to use to permit subsequent
visualization of the recorded neuron. The neurobiotin-containing solution was allowed to diffuse into the neuron for 20-45 min after
establishing whole cell configuration (following collection of
current-clamp data, the membrane was voltage clamped at 70 mV) before
gently pulling away the pipette to reseal the membrane. Cultures were
then either 1) fixed immediately or 2) placed
back into tissue culture media in the incubator for 45 min following recording before fixation. Cultures were fixed overnight with 4%
paraformaldehyde in 0.1 M phosphate buffer (PB [pH 7.4]), rinsed, removed from the tissue culture insert membrane, sunk in 30% sucrose in 0.1 M PBS (PB containing 0.15 M NaCl and 2.7 mM KCl [pH 7.4]) and
stored frozen at
70°C. Cultures were then thawed, rinsed, and
processed using the avidin-biotin complex (ABC) method. Briefly, cultures were treated with PBS containing 10% methanol and 0.6% H2O2 for 30 min, rinsed,
treated with a blocking buffer consisting of PBS with 2% bovine serum
albumin (BSA) and 0.75% Triton X-100 for 1 h, and incubated in
ABC elite (Vector) diluted in PBS containing 2% BSA and 0.1% Triton
X-100 according to kit instructions overnight at 4°C. Cultures were
then rinsed, incubated in 0.05% 3,3'-diaminobenzidine (DAB, Sigma),
0.028% CoCl2, and 0.020% nickel ammonium
sulfate in PBS for 15 min, and treated with 0.05% DAB, 0.028%
CoCl2, 0.02% nickel ammonium sulfate, and
0.00075% H2O2 in PBS until
staining was evident under an Axiovert 135 microscope at ×100
magnification. Cultures were then rinsed, mounted onto subbed glass
slides, dehydrated, cleared in xylenes, and coverslipped. The CA3c
region was defined as the CA3 pyramidal cell layer located between the
blades of the dentate granule cell layer. The CA3a/b region was defined as the CA3 pyramidal cell layer excluding the CA3c region. No attempt
was made to differentiate CA1 from CA2. Camera lucida reconstructions
were drawn using a Zeiss Axioskop microscope at ×250 magnification.
Statistical analysis
Numbers and error bars represent means ± SE in the stated
number of slice cultures except where otherwise stated. All statistical analysis was performed with Sigma Stat software. Data fitting a
nonparametric distribution were tested for significance using the
Kruskal-Wallis ANOVA by ranks test with Dunn's post hoc comparison when comparing multiple groups, Mann-Whitney rank sum test when comparing two experimental groups, or a z-test when
comparing proportions. Data fitting a normal parametric distribution
were tested for significance using a two-way ANOVA with least
significance difference (LSD) post hoc comparison when comparing
multiple groups or a t-test when comparing two experimental
groups. Significance was defined as P 0.05.
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RESULTS |
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Hyperexcitability in slice cultures
DENTATE GRANULE CELLS IN HIPPOCAMPAL SLICE CULTURES WERE HYPEREXCITABLE. Spontaneous seizures. In the first series of experiments, field potential recordings from the dentate granule cell layer were conducted in an effort to detect the occurrence of spontaneous seizures.
Field potentials were recorded in the dentate granule cell layer for approximately 45 min (48 ± 2 min, mean ± SE) in physiological buffer. Despite the brevity of the recording period, electrographic seizures (Fig. 1) composed of negative spikes superimposed on a relatively flat baseline were observed in 22% of cultures (2 of 9 cultures). The average duration of seizures in these two cultures with seizures was 32 s. The common occurrence of spontaneous seizures detected in physiological buffer in a relatively brief recording period was consistent with a striking increase of excitability of these cultures. To determine whether the propensity of granule cells to express seizures was under (
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GLUTAMATERGIC TRANSMISSION WAS REQUIRED FOR HYPEREXCITABILITY. Positive PSPs following the hilar-evoked population spike raised the possibility that excitatory glutamatergic synaptic transmission may contribute to granule cell hyperexcitability in slice cultures. Thus the contribution of N-methyl-D-aspartate (NMDA) and AMPA/KA receptor responses to evoked granule cell hyperexcitability in slice cultures was investigated.
NMDA receptors. The NMDA receptor antagonist D-APV was used to investigate the contribution of NMDA receptors to granule cell hyperexcitability in slice cultures. As described above, in physiological recording buffer, hilar stimulation elicited a short-latency EPSP immediately following the antidromic population spike. In the presence of BMI, hilar stimulation yielded a short-latency EPSP and longer-latency epileptiform bursts of EPSPs with superimposed spikes. D-APV, when applied concurrently with BMI, significantly decreased the duration of the short-latency evoked EPSP (Fig. 5A, compare Fig. 8, D and E, left, no cut), altered the underlying waveform (Fig. 5A) and decreased the number of longer-latency hilar-evoked EPSPs with superimposed spikes by 47 ± 10% (Fig. 5A). The short- and longer-latency hilar-evoked EPSPs remaining after NMDA receptor blockade were dependent on AMPA/KA receptor activation as shown by blockade of these components by further addition of the AMPA/kainate receptor antagonist, CNQX (Fig. 5A). D-APV had similar effects on spontaneous interictal bursts but did not significantly affect their frequency (data not shown). Thus NMDA receptor activation contributes to granule cell hyperexcitability in slice cultures. AMPA/KA receptors. The AMPA/KA receptor antagonist, NBQX, was used to investigate the contribution of AMPA/KA glutamate receptors to granule cell hyperexcitability in slice cultures. NBQX was chosen over other AMPA/KA receptor antagonists because of its high affinity for AMPA/KA receptors and low affinity for the glycine site on the NMDA receptor (Randle et al. 1992Possible mechanisms underlying granule cell hyperexcitability
Collectively, our data provide evidence for granule cell
hyperexcitability in hippocampal slice cultures in comparison to slices
acutely isolated from normal rats. Neither spontaneous seizures nor
interictal bursting have been reported in slices acutely isolated from
normal rats, even in the presence of BMI (Cronin et al.
1992; Patrylo and Dudek 1998
; Wuarin and
Dudek 1996
). Similarly, hilar-evoked PSPs and multiple
population spikes have not been reported in acute slices from normal
rats, even following GABAA receptor blockade
(Cronin et al. 1992
; Patrylo and Dudek
1998
; Tauck and Nadler 1985
; Wuarin and
Dudek 1996
). Given the dependence of hilar-evoked EPSPs on
glutamatergic transmission in slice cultures, we hypothesized that
newly formed excitatory glutamatergic synaptic inputs to dentate
granule cells may contribute to granule cell hyperexcitability.
However, granule excitability also could be influenced by small
differences in ion concentration, oxygenation, osmolality, etc., which
may be different in our recording conditions compared with conditions
used by investigators performing similar experiments in acute slices.
Another possibility is that the membrane properties of granule cells
may be different in slice cultures and acute slices. To begin to assess
the possible mechanisms contributing to increased granule cell
excitability in long-term slice cultures, we investigated each of these possibilities.
RECORDING CONDITIONS DID NOT PRODUCE GRANULE CELL
HYPEREXCITABILITY.
To investigate the possibility that our recording conditions promote
granule cell hyperexcitability, granule cell responses were measured in
acute hippocampal slices under conditions identical to those used for
slice culture recordings. Similar to previous reports (Cronin et
al. 1992; Patrylo and Dudek 1998
; Tauck
and Nadler 1985
; Wuarin and Dudek 1996
), in both
physiological buffer and in the presence of BMI, no seizures or
interictal spikes were observed (data not shown), and hilar stimulation
elicited a sole antidromic population spike with no PSPs (Fig.
6). Furthermore, glutamate receptor
antagonists had no effect on hilar-evoked responses (Fig. 6). Thus our
recording conditions were not sufficient to account for granule cell
hyperexcitability in slice cultures.
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ALTERED MEMBRANE PROPERTIES CANNOT ACCOUNT FOR GRANULE CELL
HYPEREXCITABILITY.
To investigate the possibility that the membrane properties of dentate
granule cells may be different in slice cultures and acute slices,
whole cell current-clamp recordings were performed. Average RMP of
dentate granule cells in hippocampal slice cultures was 66 ± 8 mV (n = 10), consistent with the RMP of
67 mV
measured under similar recording conditions in acutely prepared
hippocampal slices (Edwards et al. 1989
). Input
resistance (Rin) of granule cells in
slice cultures was 135 ± 11 M
(n = 11), a
result not significantly different (t-test;
P > 0.05) from the 186 ± 23 M
reported by
Staley and colleagues (1992)
when a calcium chelator was
included in the patch pipette solution (our pipette solution contained
the calcium chelator EGTA). Action potential spike threshold for
granule cells in slice cultures was
44 ± 1.2 mV
(n = 9), which is significantly higher than the
49 ± 0.3 (t-test; P < 0.05)
previously reported for granule cells in acute hippocampal slices
(Staley et al. 1992
). However, a higher spike threshold should serve to make granule cells in hippocampal slice cultures less
excitable than granule cells in acute hippocampal slices. Thus
alterations in dentate granule cell membrane properties cannot account
for granule cell hyperexcitability in hippocampal slice cultures.
ANATOMICAL REARRANGEMENTS IN SLICE CULTURES CONTRIBUTE TO GRANULE CELL HYPEREXCITABILITY. Morphology. To investigate the possibility that newly formed excitatory glutamatergic synaptic inputs to dentate granule cells may contribute to granule cell hyperexcitability, single CA1 pyramidal cells, CA3 pyramidal cells, and dentate granule cells were filled with neurobiotin, and axonal projections of individual neurons were traced.
Axons from most CA1 pyramidal cells (7/9) projected toward the subiculum and/or CA3a/b but remained within the CA1 region (Fig. 7A). However, in 22% of CA1 pyramidal cells (2/9), axon collaterals were detected within the CA3c pyramidal cell layer and the dentate gyrus (Fig. 7B) including the hilus, granule cell, and dentate molecular layers, suggesting that a subpopulation of CA1 pyramidal cells may synapse onto granule cells, CA3c pyramidal cells, and/or hilar neurons. CA1 pyramidal cell dendrites were confined mainly to the CA1 region (Fig. 7A); however, in 22% of CA1 pyramidal cells, dendrites were seen in the dentate molecular layer (not shown). Thus any neurons that project axons to the molecular layer could synapse onto CA1 pyramidal cell dendrites. These data are suggestive of synapses between CA1 pyramidal cell and neurons in the dentate gyrus.
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DISCUSSION |
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Field potential recordings of dentate granule cells were performed
in organotypic hippocampal slices isolated at P11 and
maintained long term (40-60 days) in vitro. The principal findings are
the following. Relatively brief recordings conducted in physiological buffer disclosed spontaneous seizures recruiting granule cells in 22%
of cultures; recordings conducted in the presence of the GABAA receptor antagonist, BMI, documented
seizures in 75% of cultures. Hilar stimulation evoked PSPs and
multiple population spikes in both physiological recording buffer and
in the presence of BMI. PSPs were eliminated by a combination of NMDA
and AMPA/KA receptor antagonists, demonstrating the requirement for
excitatory synaptic transmission. These findings stand in sharp
contrast to the lack of seizures and to the isolated hilar-evoked
population spike without PSPs observed in acute hippocampal slices
isolated from normal rats under identical recording conditions. A
combination of anatomical and physiological approaches provided strong
evidence that newly formed excitatory synaptic connections to the
dentate gyrus contribute to granule cell hyperexcitability in slice
cultures. Anatomical labeling of individual pyramidal cells in CA3 and
CA1 demonstrated axonal projections to the dendritic regions of the dentate granule cells; likewise labeling of individual dentate granule
cells disclosed axonal projections to both CA3 and CA1 as well as to
dendritic regions of the granule cells themselves. Physiological
granule cell responses to hilar stimulation recorded in the absence and
presence of knife cuts disconnecting CA1 and/or CA3 regions from the
dentate gyrus suggested that these newly formed synapses were
functional. Knife cuts severing connections between CA1 and the dentate
gyrus greatly reduced measures of synaptic transmission with smaller
reductions evident after severing connections between CA3a/b and
dentate gyrus. Remarkably, some residual enhanced response of the
granule cells to hilar stimulation persisted even after isolation of
the dentate gyrus from both CA1 and CA3 a/b. Taken together, these data
are consistent with the proposal that reciprocal excitatory synaptic
connections among dentate granule cells on the one hand and between
dentate granule cells and pyramidal cells in CA3 and CA1 on the other
contribute to the striking hyperexcitability of the dentate granule
cells in long-term organotypic hippocampal slice cultures. In support of this interpretation, a paper (Gutierrez and Heinemann
1999) appeared during preparation of this manuscript that also
provided evidence for functional synaptic pathways between CA1
pyramidal cells and granule cells as well as between granule cells
themselves in organotypic hippocampal slice cultures.
Dentate granule cell seizures
The dentate granule cells normally limit invasion of seizure
activity into the hippocampus, a function that may be pivotal in
preventing epileptic activity in limbic circuitry. Our findings demonstrate that long-term culture of organotypic hippocampal slices
reliably produces seizures that recruit dentate granule cells. A
diversity of in vitro models of temporal lobe seizures have been
described in the past 10-15 yr (Anderson et al. 1986; Avoli et al. 1996
; Benedikz et al.
1993
; Bragdon et al. 1992
; Bruckner et
al. 1999
; Gutierrez et al. 1999
; Jensen
and Yaari 1988
; Konnerth et al. 1986
;
McBain et al. 1989
; Merlin 1999
;
Muller 1993
; Ogata 1978
;
Richardson and O'Reilly 1995
; Routbort et al. 1999
; Schwartzkroin and Prince 1977
;
Sombati and DeLorenzo 1995
; Swann et al.
1993
; Tancredi and Avoli 1987
; Traynelis
and Dingledine 1988
). In most instances, seizures are triggered
by subjecting a hippocampal slice acutely isolated from a normal animal
to some intervention (e.g., ionic manipulation,
GABAA receptor antagonist, etc). Most often these
seizures involve CA3 and/or CA1 pyramidal cells. Only two reports
disclose seizures recruiting the seizure-resistant dentate granule
cells in acute hippocampal slices; following 4-aminopyridine induced
seizures in entorhinal cortex (Avoli et al. 1996
) and during low-calcium perfusion combined with exposure to electric fields
(Richardson and O'Reilly 1995
). Moreover, perfusion of 7-8 DIV hippocampal slice cultures with solutions containing low magnesium evoked seizures involving the dentate granule cells (Gutierrez et al. 1999
). By contrast, we report the
occurrence of isolated seizures arising spontaneously in brief
recordings (45 min) conducted in physiological buffer (e.g., without
ionic manipulations or convulsant agents). The occurrence of
spontaneous seizures in the present study is all the more surprising
because the recordings were performed at 27-29°C, and reduction of
temperature from 33 to 28°C has been shown to exert powerful
anti-seizure effects in hippocampal slices (Traynelis and
Dingledine 1988
). The onset of seizures in 75% of cultures
within minutes of addition of BMI underscores the powerful effects of
GABAA-mediated inhibition in control of dentate
granule cell excitability in these slice cultures. Why addition of BMI
to hippocampal slice cultures elicited an isolated seizure rather than
recurrent seizures is unclear. However, this finding is consistent with
similar data obtained in CA1 following BMI application to hippocampal
slice cultures (Scanziani et al. 1994
).
Comparison of hippocampal slice cultures with in vivo models and human temporal lobe epilepsy
A critical issue raised by this study is whether abnormalities evident in long-term hippocampal slice cultures are similar to abnormalities in animal models or humans with limbic epilepsy. Indeed, striking parallels emerge.
DENTATE GRANULE CELL SEIZURES AND HYPEREXCITABILITY.
However, one notable difference between hippocampal slice cultures and
acute slices isolated from animal models or humans with temporal lobe
epilepsy is the incidence of seizures involving the granule cells. In
our study, relatively brief recordings conducted in physiological
buffer disclosed spontaneous seizures including granule cells in 22%
of cultures; recordings conducted in the presence of the
GABAA receptor antagonist, BMI, documented
seizures in 75% of cultures. These findings contrast sharply with
previous reports from numerous laboratories in which investigators
recorded extracellular granule cell layer field potentials in acute
slices isolated from rat or resected human epileptic hippocampus
(Cronin et al. 1992; Franck et al. 1995
;
Isokawa and Fried 1996
; Isokawa et al.
1991
, 1997
; Masukawa et al. 1991
;
Molnar and Nadler 1999
; Okazaki et al.
1999
; Patrylo and Dudek 1998
; Tauck and
Nadler 1985
; Urban et al. 1990
;
Williamson et al. 1995
). Surprisingly, none of these
investigators reported seizures involving granule cells in either
physiological buffer or physiological buffer containing GABAA receptor antagonists. Several factors could
explain this discrepancy including the following: recording duration,
maintenance of neuronal network connections, different gap junction
coupling, and/or altered ionic homeostasis. The contributions of all
factors in a subset of slice culture may be sufficient for seizure
initiation; whereas, in acute slices from epileptic hippocampus, these
parameters may need to be manipulated. In validation of this idea,
Dudek and colleagues (Patrylo and Dudek 1998
;
Wuarin and Dudek 1996
) have reported spontaneous
seizures in a subset of acute slices prepared from KA-treated epileptic
rats following perfusion with a GABAA receptor
antagonist and elevated extracellular K+ (6-9
mM). We are unaware of any similar studies using slices isolated from
humans with limbic epilepsy or, indeed, of any reports of spontaneous
seizures recruiting granule cells in slices isolated from humans with
limbic epilepsy. Such a multifactorial mechanism would likely yield a
large degree of variability and heterogeneity in physiological
responses and may explain why seizures were observed in only a subset
of slice cultures in our study. Once the various mechanisms
contributing to dentate granule cell seizures in hippocampal slice
cultures are elucidated, this information can guide subsequent investigations into the operative factors contributing to temporal lobe
epilepsy in humans and animal models.
MORPHOLOGICAL ABNORMALITIES.
Some of the morphological abnormalities reported here also have been
described in animal models and humans with limbic epilepsy. Despite
deafferentation, hippocampal slice cultures have been reported to
retain relatively normal cytoarchitecture and synaptic connectivity
(Daily et al. 1994; Frotscher and Heimrich
1993, 1995
; Gahwiler et al. 1997
;
Li et al. 1993
, 1994
; Robain et
al. 1994
; Stoppini et al. 1993
,
1997
; Zimmer and Gahwiler 1987
). However, recent studies (Gahwiler et al. 1997
; Pavlidis
and Madison 1999
) estimate that these normal synaptic
connections are increased 10 times relative to acute slices. Similar
expansions of normal synapses have been reported in epileptic rat and
human hippocampus. Indeed, the most extensively studied synaptic
rearrangement in the epileptic rat and human hippocampus is mossy fiber
sprouting, an expansion of a small normal projection (de
Lanerolle et al. 1989
; Houser et al. 1990
;
Laurberg and Zimmer 1981
; Mello et al. 1992
; Molnar and Nadler 1999
; Okazaki et
al. 1995
; Represa et al. 1993
; Ribak and
Peterson 1991
; Seress 1992
; Sutula et al. 1988
, 1989
; Tauck and Nadler
1985
). A slight mossy fiber expansion is also present in
long-term hippocampal slice cultures (Routbort et al.
1999
). By analogy, our findings in slice culture suggest that
the small CA3 pyramidal cell to dentate granule cell projection found
in normal rat (Ishizuka et al. 1990
; Li et al.
1994
; Scharfman 1993
, 1994a
,b
)
may be expanded in the epileptic hippocampus. This hypothesis remains
to be addressed in rat and human.
Hippocampal slice cultures as a model to investigate mechanisms underlying synaptic rearrangements and epileptogenesis
The in vitro long-term hippocampal slice culture model provides a simple system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis. Currently, the only avenue available to investigate these mechanisms is in vivo because acute in vitro slices are short-lived and slice preparation severs many axonal projections. However, in vivo studies are hampered by slow data collection, the inability to control reagent concentration in the brain and unwanted side effects caused by systemically administered reagents. In contrast to in vivo studies, the external environment of slice cultures can be altered easily without unwanted systemic side effects and, unlike acute in vitro slices, hippocampal slice cultures can be maintained for weeks to months. Thus the hippocampal slice culture preparation is an attractive alternative to in vivo and acute in vitro preparations.
Despite the prominent advantages offered by slice cultures, potential disadvantages are also evident. Hippocampal slice cultures are subjected to trauma, cell death, and deafferentation from extrahippocampal regions during slice preparation, are bathed in culture media rather than cerebral spinal fluid, and are covered in a prominent glial layer. Such differences from in vivo conditions may alter neuronal activity/excitability, dendritic and/or axonal morphology, synapse formation, synaptic properties, and the molecular bases for these functional and structural modifications as well as protein expression and/or protein-protein interactions. These disadvantages notwithstanding, slice cultures provide a relatively simple chronic in vitro model of limbic epileptogenesis; it seems likely that some commonalities exist between limbic epileptogenesis in vivo and in this in vitro preparation. Thus while the long-term in vitro hippocampal slice culture model does not obviate the need for in vivo animal models, slice cultures can provide a relatively simple first step in investigating the cellular and molecular mechanisms underlying epileptogenesis and synaptic rearrangements.
In summary, long-term organotypic hippocampal slice cultures provide a chronic in vitro model of limbic epilepsy with striking electrophysiological and morphological similarities to both human and in vivo models. This in vitro model provides a simple, useful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis, which will bring us one step closer toward developing novel new therapies for temporal lobe epilepsy.
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ACKNOWLEDGMENTS |
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We thank Dr. Nell Cant for use of a microscope for camera lucida reconstructions and Dr. George Augustine for helpful discussions.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-17771, NS-32334 (J. O. McNamara), NS-07370, and NS-10387 (S. B. Bausch) and the American Epilepsy Society with support from the Milken Family Foundation (S. B. Bausch).
Present address of S. B. Bausch: Uniformed Services University of the Health Sciences, Dept. of Pharmacology, Rm. C2007, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799.
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FOOTNOTES |
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Address for reprint requests: J. O. McNamara, Duke University Medical Center, 401 Bryan Research Bldg., Research Dr., Box 3676, Durham, NC 27710 (E-mail: jmc{at}neuro.duke.edu).
Received 3 February 2000; accepted in final form 30 August 2000.
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REFERENCES |
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