Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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DeFazio, R. Anthony and John J. Hablitz. Alterations in NMDA Receptors in a Rat Model of Cortical Dysplasia. J. Neurophysiol. 83: 315-321, 2000. Recent studies have demonstrated an important role for the N-methyl-D-aspartate receptor (NMDAR) in epilepsy. NMDARs have also been shown to play a critical role in hyperexcitability associated with several animal models of human epilepsy. Using whole-cell voltage clamp recordings in brain slices, we studied evoked paroxysmal discharges in the freeze-lesion model of neocortical microgyria. The voltage dependence of epileptiform discharges indicated that these paroxysmal events were produced by a complex pattern of excitatory and inhibitory inputs. We examined the effect of the NMDAR antagonist D-2-amino-5-phosphopentanoic acid (APV) and the NMDA receptor subunit type 2B (NR2B)-selective antagonist ifenprodil on the threshold, peak amplitude, and area of evoked epileptiform discharges in brain slices from lesioned animals. Both compounds consistently raised the threshold for evoking the discharge but had modest effects on the discharge peak and amplitude. For comparison with nonlesioned cortex, we examined the effects of ifenprodil on the epileptiform discharge evoked in the presence of 2 µM bicuculline (partial disinhibition). In slices from nonlesioned cortex, 10 µM ifenprodil had little effect on the threshold whereas 71% of the recordings in bicuculline-treated lesioned cortex showed a >25% increase in threshold. These results suggest that NR2B-containing receptors are functionally enhanced in freeze-lesioned cortex and may contribute to the abnormal hyperexcitability observed in this model of neocortical microgyria.
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INTRODUCTION |
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Neuronal migration disorders resulting in cortical
dysplasia, microgyria, and heterotopia are associated with intractable seizure disorders in humans (Palmini et al. 1994).
Several animal models have been developed to examine neural mechanisms
underlying hyperexcitability in dysplastic cortex (Baraban and
Schwartzkroin 1995
; Dvorak et al. 1978
;
Roper 1998
). The rat neonatal freeze-lesion model
reproduces many of the anatomic findings found in human microgyria
(Dvorak et al. 1978
). In this model, a freezing probe is
briefly placed on the skull of a neonatal rat pup. This procedure destroys the deep layer neurons present near the surface of the cortical plate but preserves progenitor cells and radial glia, which
ultimately give rise to superficial neuronal layers in the area of the
lesion. The local loss of deep layer cells results in a microsulcus in
the otherwise lissencephalic rat brain. Neocortical brain slices from
rats >2 wk old containing the microsulcus are hyperexcitable.
Epileptiform discharges, which propagate over large distances, are
evoked by weak intracortical stimulation (Hablitz and DeFazio
1998
; Jacobs et al. 1996
, 1999
; Luhmann
and Raabe 1996
; Luhmann et al. 1998
;
Prince et al. 1997
). Electrophysiological studies have
indicated changes in intrinsic membrane properties (Luhmann et
al. 1998
), alterations in GABAergic inhibition (DeFazio and Hablitz 1999
; Jacobs et al. 1996
; Qu
et al. 1998
), and modification of glutamate receptors
(Qu et al. 1998
). It is likely that these factors
interact to produce hyperexcitability.
Chronic changes in
N-methyl-D-aspartate receptors
(NMDARs) are involved in human temporal lobe epilepsy (Mathern
et al. 1998) and the kindling model of temporal lobe epilepsy
(Mody and Heinemann 1987
). In addition, acute models of
interictal spiking show sensitivity to NMDAR antagonists (e.g.,
Hablitz and Lee 1992
; Lee and Hablitz 1990
). Variable results with epileptiform activity in slices
from freeze-lesioned cortex have been reported. The NMDAR antagonist D-2-amino-5-phosphonopentanoic acid (APV, 50-100
µM) has been reported to completely block hyperexcitability in slices
from freeze-lesioned animals (Jacobs et al. 1996
, 1999
).
In other studies, APV had no effect or only reduced late recurrent
epileptiform activity; blockade of
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors
was necessary to completely abolish hyperexcitability (Luhmann
and Raabe 1996
; Luhmann et al. 1998
). However,
in both cases an important role for NMDARs in the initiation and/or
propagation of the epileptiform discharge was apparent. It remains to
be determined if NMDAR participation reflects pathological function of
normal receptors (e.g., caused by diminished inhibition), alterations in the receptors themselves (e.g., expression of receptors with enhanced current flow), and/or abnormal excitatory interconnectivity (e.g., sprouting of horizontal excitatory collaterals).
Native NMDARs are thought to contain subunits from two different
classes termed NR1 and NR2 (for review see Monaghan et al. 1998). Each
class has a number of subtypes that can alter the pharmacological and
kinetic properties of the receptor. In heterologous expression systems,
NR1 homomers form poorly conducting NMDAR channel complexes compared
with NR1-NR2 heteromers. The primary candidates for native NMDAR in rat
somatosensory cortex are NR1 (and its splice variants) and NR2A, B,
and/or C. During neocortical development, there is a shift from
predominately NR2B to both NR2A and NR2B receptor subunit expression
(Sheng et al. 1994
), with a concomitant decrease in the
decay time constant of excitatory postsynaptic currents (Flint
et al. 1997
; Roberts and Ramoa 1999
; Stocca and Vicini 1998
). In human cortical dysplasia,
evidence exists for an increased expression of NR2 (possibly NR2B)
subunits within the region of the dysplasia, but not in surrounding or "nonspiking" cortex (Ying et al. 1998
). The absence
of NR2 immunoreactivity in nonspiking cortex compared with the
abundance of NR2 immunoreactivity in actively epileptic cortex has led
to the suggestion that increases in NR2 expression alone could explain
hyperexcitability in human cortical dysplasia (Ying et al.
1998
).
In the present study, we tested the hypothesis that functional
alterations in NMDA receptor subunit composition are responsible for
the hyperexcitability observed in brain slices from freeze-lesioned rats. NR2B-containing receptors are more sensitive than NR2A-containing receptors to the polyamine-site antagonist ifenprodil (Williams 1993). We therefore compared the effects of APV and ifenprodil in lesioned and nonlesioned animals. A preliminary account of some of
these results appeared in DeFazio and Hablitz (1998)
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METHODS |
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Animals were housed and handled according to approved
guidelines. Timed pregnant Sprague-Dawley dams arrived on embryonic day
15. Freeze lesions were produced using modifications of the technique
of Dvorak and Feit (1977). On postnatal day (PN) 2, rat pups
were anesthetized by hypothermia (5 min on ice). After making a midline
incision, the cold probe was placed on the surface of the skull near
the midline for 3-5 s. The cold probe consisted of a 2 mm diameter
copper rod extending from a 60 ml methanol-filled centrifuge tube
cooled to about
50°C with dry ice. After the incision was sutured,
the animals were placed under a heat lamp and returned to their home
cage after 30 min.
Brain slices were prepared from PN 17-27 animals. Rats were anesthetized with ketamine (100 mg/kg) before decapitation. The brain was rapidly removed and submerged in oxygenated (95% O2/5% CO2), ice-cold, low-calcium saline (containing, in mM: 125 NaCl, 3.5 KCl, 26 NaHCO3, 10 glucose, 4 MgCl2). Coronal sections (300 µm) containing somatosensory cortex were cut using a Vibratome (TPI). Slices were stored in saline consisting of (in mM) 125 NaCl, 5 KCl, 26 NaHCO3, 10 glucose, 2 CaCl2, and 2 MgCl2 bubbled with 95% O2/5% CO2.
Whole cell voltage clamp recordings were made 1-2 mm lateral to
the lesion and at least 1 mm lateral to the stimulation site (Fig.
1A). All records were
obtained from visually identified neocortical pyramidal cells in layer
II/III. Cells were identified by their location below the pial surface,
pyramidal shape, and presence of a prominent apical dendrite (Fig.
1B). Recordings were made at room temperature and at a
holding potential of 60 mV. Series resistance was regularly monitored
and cells with series resistance >20 M
or significant changes in
series resistance during the experiment were discarded. The internal
solution consisted of 140 K-isethionate or K-methyl sulfate, 10 HEPES,
5 EGTA, 0.1 CaCl2, 4 MgATP, 0.4 NaGTP, and 5 QX-314 (all
concentrations in mM). Slices were continuously perfused with the
storage saline listed above. Whole cell recordings were made with an
Axopatch-1B amplifier. No series resistance compensation was employed.
Recordings were digitized at 5-10 kHz using a Digidata 1200 data
acquisition system and Clampex 7 software (Axon Instruments). Data
analysis was performed using custom scripts written for Origin 5 (Microcal Software). Stimulating electrodes were made from a twisted
pair of nichrome wires (0.002 in. diameter) and placed in deep cortical layers ~0.5 mm lateral to the lesion. An example of electrode positioning is shown in Fig. 1B. In slices from control
animals, the recording site was ~2-3 mm lateral to the medial edge
of cortex (corresponding to the longitudinal cerebral fissure) and at
least 1 mm lateral to the stimulation site. Threshold stimulus currents ranged from 30-500 µA at 80 µsec and were not statistically
significant between the control and lesion groups. A Weco SC-100
constant current source and a WPI pulse generator were employed to
deliver current pulses through the stimulating electrode. The threshold for epileptiform discharges was determined by varying the stimulation current (80 µsec duration). To generate stimulus intensity curves, the stimulus duration was varied, under digital control, from 20 to 300 µsec in 10-50 µsec steps. The interstimulus interval (30 s) was
kept constant throughout the experiment. To quantify the effects of
NMDAR antagonists, epileptiform discharge peak amplitude and area were
calculated. Peak amplitude was defined as the greatest absolute value
after the stimulus artifact. Area was calculated by zeroing the
baseline to the average of 50 ms just before the stimulus artifact,
then integrating the trace after the stimulus artifact.
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All drugs were bath applied and each cell served as its own control.
Concentrated stocks of APV (Tocris) were made in deionized water.
Ifenprodil (Sigma) was dissolved in DMSO to make concentrated stocks.
These stocks were stored at 20 C.
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RESULTS |
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Voltage dependence of the epileptiform discharge
Previous investigations have shown that there is a
hyperexcitable zone adjacent to the microsulcus in freeze-lesioned
cortex (Hablitz and DeFazio 1998; Jacobs et al.
1999
). Synaptic activation of deep layers adjacent to the
lesion evoked a long-lasting epileptiform discharge that was monitored
in layer II/III pyramidal cells 1-2 mm lateral to the lesion using
whole cell voltage clamp techniques (Fig. 1). The voltage dependence of
the epileptiform discharge was examined by stepping the membrane
potential between
80 and 20 mV from a holding potential of
60 mV.
The voltage step was 1 s in duration, starting 400 ms before the
stimulus. In 22 of 26 cells, a biphasic current waveform was apparent
at membrane potentials between
40 and
20 mV, whereas the remaining
recordings were monophasic with little or no current at 0 mV. Outward
currents at these potentials are most likely caused by synaptic
activation of chloride-permeable GABAA receptors
that reverse near
70 mV (compared with 0 mV for the expected reversal
of excitatory synaptic currents). Figure 1C shows
epileptiform discharges evoked at potentials from
80 mV to 20 mV.
Both inward and outward currents were present at step potentials
between -60 mV and
40 mV, whereas an outward component predominated
at 0 mV. Plots of current amplitude as a function of voltage for the
time points indicated by the dashed lines are shown in Fig.
1D. The early component did not change polarity until the
voltage step exceeded 0 mV, as would be expected for an excitatory
postsynaptic current under the present recording conditions. The late
component reversed near
50 mV, closer to the reversal potential for
chloride. Exact reversal potentials for the two components could not be
measured because of overlapping excitatory and inhibitory components.
Pharmacological blockade of individual components was not attempted
because this would alter the excitability of the local circuits
responsible for generating the epileptiform discharge.
NMDAR antagonists raise epileptiform discharge threshold
NMDAR antagonists have been shown to either block a late recurrent
component of the evoked epileptiform discharge (Luhmann et
al. 1998) or abolish the discharge entirely (Jacobs et
al. 1999
). In the present experiments, bath application of APV
at concentrations of 2-10 µM reversibly raised the threshold for evoking epileptiform discharges, in addition to reducing peak amplitude
and response area in lesioned animals. Figure
2A illustrates the multiple
effects of antagonist on evoked epileptiform discharges. In the
presence of 10 µM APV, the epileptiform discharge was reduced in
amplitude and decayed more rapidly. These effects were reversible. Epileptiform discharges can show trial-to-trial variation in peak amplitude and area. A >25% change in the measured properties was considered significant because this represented a change in threshold greater than that produced by the minimum stimulus duration increment employed in determining input-output curves. APV caused a reduction of
>25% in the peak amplitude in 6 of 14 recordings and in the area in 9 of 14 recordings. A reduction in area without a >25% change in
amplitude was observed in 3 of 9 cells.
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APV also raised the threshold for evoking an epileptiform discharge. A plot of response area as a function of stimulus duration is shown in Fig. 2B. In both control and wash, the epileptiform discharge could be reliably evoked at a 100 µsec stimulus duration. In APV, a stimulus of 180 µsec was required to evoke a discharge. APV increased the threshold for evoking an epileptiform discharge by >25% in 8 of 9 slices tested. As shown in Fig. 2B, elevated stimulus intensity decreased the area of the discharge, but not to the same extent as APV.
To examine the possibility of enhanced expression of
NR2B-containing NMDARs, the polyamine-site antagonist ifenprodil was tested. The effects of ifenprodil (10 µM), which has a 400-fold greater affinity at NR2B-containing receptors (Williams
1993), on epileptiform discharges are shown in Fig.
3. Epileptiform discharges in this cell
exhibited an early fast component followed by a multiphasic late
component. The fast component was unaffected by ifenprodil whereas peak
amplitude and area of the late component of the epileptiform discharge
were decreased. The time course of this experiment is shown in Fig.
3B. After two stimuli at 100 µsec, a series of stimuli at
20, 40, 60, 80, 100, 150, and 200 µsec was delivered to determine threshold (a). Threshold for this cell was 60 µsec. A stimulus duration of 100 µsec was employed before and during the application of 10 µM ifenprodil. At (b) and (c), a second threshold determination was made. Epileptiform discharges could not be evoked at stimulus durations <100 µsec. One stimulation at 100 µsec resulted in a failure (arrow). Peak amplitude (n = 4 of 8 slices) and
area (n = 8 of 8 slices) of epileptiform discharges
were reduced by ifenprodil. Ifenprodil raised the threshold >25% in
all recordings examined for threshold effects (n = 6 slices). Thus the NR2B-selective antagonist had effects similar to
those observed in APV.
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Partial disinhibition reveals differences in sensitivity to ifenprodil in lesioned and nonlesioned cortex
Partial disinhibition resulting from bath application of low doses
of bicuculline produces a hyperexcitable state characterized by
propagating epileptiform discharges (Chagnac-Amitai and Connors 1989). In brain slices bathed in 2 µM bicuculline, we
examined the ifenprodil sensitivity of the evoked epileptiform
discharge threshold in slices from lesioned and nonlesioned animals.
Bicuculline significantly enhanced the peak amplitude and area of the
epileptiform discharge in lesioned animals (Fig.
4). No significant differences in
bicuculline-induced epileptiform discharges in slices from nonlesioned
and lesioned animals were observed. In slices from nonlesioned animals,
bath application of 10 µM ifenprodil reduced the area of the
epileptiform discharge by >25% in 2 of 7 cells. Figure
5A illustrates the effect of
ifenprodil on bicuculline-induced epileptiform discharges in a slice
from a nonlesioned animal. In this cell, both the area and the peak
amplitude were reduced >25%. The time course of the experiment is
shown in Fig. 5B. Symbols near zero represent responses to
subthreshold stimulation. The threshold was unaffected by ifenprodil in
this cell. Overall, ifenprodil had little effect on the threshold
(<25% increase in threshold) for evoking the epileptiform discharge
(n = 5 of 6 slices tested).
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In contrast, ifenprodil raised the threshold of the bicuculline-induced epileptiform discharge in slices from lesioned animals, as shown in Fig. 6. For this recording, stimuli of 60 µsec duration reliably elicited epileptiform discharges (Fig. 6A), but in the presence of ifenprodil, stimuli of 100 µsec or greater were required to evoke the epileptiform discharge (Fig. 6B). After washout, the 60 µsec stimulus again reliably evoked the epileptiform discharge. In lesioned animals, ifenprodil raised the threshold by >25% in 5 of 7 slices examined, but had little effect (<25%) on peak or area of the epileptiform discharge (n = 7 of 7). These results are summarized in Table 1 and Fig. 7. The mean of the percent changes observed for each experimental group is shown in Fig. 7. On average, ifenprodil had significantly less effect on threshold in control animals (P < 0.05) in bicuculline. The amplitude and area of the discharges evoked in slices from control and lesioned animals in bicuculline were also less sensitive to ifenprodil. The number of slices is the same as that given in Table 1.
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DISCUSSION |
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Three main findings were obtained in the present experiments. The voltage dependence of epileptiform discharges indicated that these paroxysmal events were produced by a complex pattern of excitatory and inhibitory inputs. NMDAR antagonists were found to be effective in raising the threshold for evoking epileptiform discharges. Finally, the differential ifenprodil sensitivity of threshold for evoked bicuculline-induced discharges in control and lesioned animals indicated functional differences in NMDARs expressing NR2B subunits.
The presence of a GABAA receptor-mediated
component in epileptiform discharges in the freeze lesion model is
consistent with previous observations. These earlier studies, using
local stimulation <0.5 mm distal to the recording site, demonstrated
the presence of inhibitory potentials in layer II/III pyramidal cells
(Luhmann et al. 1998) and early and late inhibitory
currents in layer 5 pyramidal cells (Prince et al.
1997
). Our data demonstrate that even during the propagation of
epileptiform discharges (1-2 mm from the stimulation site), a
prominent inhibitory component is detectable in layer II/III pyramidal
cells. In addition, studies of inhibitory postsynaptic currents (IPSCs)
in brain slices from lesioned animals have indicated an enhancement of
unitary inhibitory events (DeFazio and Hablitz 1999
) and
enhanced excitatory drive on inhibitory cells (Prince et al.
1997
). Although disinhibition has often been suggested as a
basis for epileptogenesis (Ribak and Reiffenstein 1982
;
Schwartzkroin and Prince 1980
), epileptiform activity
can be observed when inhibitory synaptic activity is normal
(Malouf et al. 1990
) or enhanced (Rutecki et al.
1987
). The occurrence of complex long-lasting epileptiform
discharges in dysplastic neocortex suggests alterations in local
excitatory circuits. The presence of some degree of inhibitory control
may explain the failure to observe spontaneous epileptiform activity in
this model.
The effects of NMDAR antagonists on the threshold of the epileptiform
discharge indicate that NMDARs play an important role in the initiation
and/or propagation of epileptiform discharges. In lesioned cortex,
NMDAR antagonists potently raised the threshold for evoking discharges
in both the intrinsic hyperexcitabe state and after partial
disinhibition. These results may help to resolve the apparent
differences reported in previous studies of APV effects in this animal
model. A previous study (using relatively local stimulation, ~0.5 mm
lateral to the lesion) demonstrated that APV at concentrations >20
µM blocked only a late component of the discharge (Luhmann et
al. 1998). Our study suggests that APV-induced changes in the
threshold for evoking the propagating discharge could lead to an
apparent abolition of the late component with little effect on the
local (early) response if only a single stimulation intensity was
tested. Another group reported that 100 µM APV completely blocked
late field potential discharges recorded at 2, 4, and 8 times threshold
stimulus intensities (Jacobs et al. 1999
). An early
component representing the locally evoked postsynaptic potential and
population action potential was not obviously affected by 100 µM APV
(see Fig. 10 in Jacobs et al. 1999
). The combination of
these results with the present study suggests that NMDARs play an
important role in the initiation and propagation of the
epileptiform discharge.
The inhibitory effect of ifenprodil, an antagonist with higher affinity
for receptors containing the NR2B subunit, was unexpected because NR2A
receptors are thought to predominate at the stage of development
examined in this study (Flint et al. 1997; Sheng et al. 1994
). Although ifenprodil sensitivity has been
demonstrated in a subpopulation of isolated neocortical cells from
animals of this age group (Kew et al. 1998
), differences
between control and lesioned animals under conditions of partial
disinhibition support the hypothesis of alterations in NMDAR subunit
composition. These results suggest that NR2B subunit-containing NMDARs
predominate in at least a subset of the cells responsible for the
initiation of the discharge, possibly conferring hyperexcitability
through the prolongation of excitatory postsynaptic currents (EPSCs). The lack of effect of ifenprodil on the discharge observed in the
presence of bicuculline in slices from control and lesioned animals
suggests that such changes in NMDAR subunit composition are not widespread.
Studies of resected human dysplastic cortex have demonstrated the
presence of NR2 subunit immunoreactivity in cortical resections characterized as "spiking" with subdural electrodes. The
immunoreactivity was localized to "dysplastic neurons," or cells
that appear disordered compared with the normal laminar structure of
cortex (Ying et al.1998). It should be noted that the
morphological abnormalities observed in human polymicrogyria and this
freeze-lesion model represent only one type of cortical dysplasia.
Consequently, our results cannot be directly compared with the
dysplasia observed in human epilepsy. However, our data support the
hypothesis that a subset of cells near the lesion and
responsible for the initiation of the discharge are sensitive to
ifenprodil. These cells could represent the "dysplastic" neurons in
human tissue that are observed to express NR2 immunoreactivity.
NMDARs are known to participate in local recurrent excitation in
neocortex (Hess et al. 1994; Sutor and Hablitz
1989
). Abnormalities in these horizontal connections could also
contribute to hyperexcitability. Alterations in gene expression and
changes in receptor subunits have been reported in several models of
epilepsy. Metabotropic glutamate receptors are differentially modulated
in the kindling model of temporal lobe epilepsy (Akbar et al.
1996
; Neugebauer et al. 1997
). Our previous
studies on miniature IPSCs in dysplastic cortex demonstrated a
predominance of
2 over
1 subunits (DeFazio and
Hablitz 1999
), a pattern found in immature neocortex. Those observations, coupled with the present findings with NMDARs, suggest that a delay or arrest of the developmental pattern of neurotransmitter receptor expression on neocortical cells may be a hallmark of freeze-induced cortical dysplasia.
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ACKNOWLEDGMENTS |
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The authors thank A. Margolies for excellent technical assistance and S. Keros for helpful comments on the manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-22373 and by the American Epilepsy Society with support from the Milken Family Foundation.
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FOOTNOTES |
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Address reprint requests to J. J. Hablitz.
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 13 July 1999; accepted in final form 23 September 1999.
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REFERENCES |
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