1Departments of Neurology and Neurosurgery, and Physiology, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada; and 2Centre Paul Broca and Institut National de la Santé et de la Recherche Médicale U109, 75014 Paris, France
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ABSTRACT |
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Barbarosie, Michaela, Jacques Louvel, Irène Kurcewicz, and Massimo Avoli. CA3-Released Entorhinal Seizures Disclose Dentate Gyrus Epileptogenicity and Unmask a Temporoammonic Pathway. J. Neurophysiol. 83: 1115-1124, 2000. We have investigated the propagation of epileptiform discharges induced by 4-aminopyridine (4-AP, 50 µM) in adult mouse hippocampus-entorhinal cortex slices, before and after Schaffer collateral cut. 4-AP application induced 1) ictal epileptiform activity that disappeared over time and 2) interictal epileptiform discharges, which continued throughout the experiment. Using simultaneous field potential and [K+]o recordings, we found that entorhinal and dentate ictal epileptiform discharges were accompanied by comparable elevations in [K+]o (up to 12 mM from a baseline value of 3.2 mM), whereas smaller rises in [K+]o (up to 6 mM) were associated with ictal activity in CA3. Cutting the Schaffer collaterals disclosed the occurrence of ictal discharges that were associated with larger rises in [K+]o as compared with the intact slice. Further lesion of the perforant path blocked ictal activity and the associated [K+]o increases in the dentate gyrus, indicating synaptic propagation to this area. Time delay measurements demonstrated that ictal epileptiform activity in the intact hippocampal-entorhinal cortex slice propagated via the trisynaptic path. However, after Schaffer collateral cut, ictal discharges continued to occur in CA1 and subiculum and spread to these areas directly from the entorhinal cortex. Thus our data indicate that the increased epileptogenicity of the dentate gyrus (a prominent feature of temporal lobe epilepsy as well), may depend on perforant path propagation of entorhinal ictal discharges, irrespective of mossy fiber reorganization. Moreover, hippocampal neuronal damage that is acutely mimicked in our model by Schaffer collateral cut, may contribute to "short-circuit" propagation of activity by pathways that are masked when the hippocampus is intact.
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INTRODUCTION |
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Entorhinal inputs to the dentate gyrus may be
instrumental in the reverberation and amplification of epileptiform
activity through the hippocampal-entorhinal loop (Paré et
al. 1992). The dentate gyrus may sustain ictal activity during
chronic epileptogenesis (Parent et al. 1997
;
Wuarin and Dudek 1996
). Indeed, dentate seizure discharges in "naive" animals are shorter as compared with those recorded from epileptic rats (Stringer and Lothman
1989
), lending support to the hypothesis that the dentate gyrus
may amplify seizures in chronic, animal models of epileptogenesis.
The dentate gyrus may also gate the propagation of epileptiform
discharge to the hippocampus (Collins et al. 1983;
Jones and Lambert 1990
; Walther et al.
1986
). However, adequate stimulation of the contralateral CA3
or the ipsilateral angular bundle can overcome the dentate resistance
to paroxysmal discharge generation, thus allowing robust ictal-like
discharges to occur (Stringer and Lothman 1989
;
Stringer et al. 1989
). This epileptiform activity most
likely results from perforant path propagation of ictal events originating in the entorhinal cortex. Accordingly, the entorhinal cortex is susceptible to generate seizure activity in patients with
temporal lobe epilepsy (Rutecki et al. 1989
;
Spencer and Spencer 1994
) and in models of epileptiform
discharge (e.g., Avoli et al. 1996a
; Bragdon et al. 1992
; Dreier and
Heinemann 1991
; Jones and Heinemann 1988
; Nagao et al. 1996
).
Propagation of entorhinal epileptiform discharges via the perforant
path has been the object of several studies. However, direct
propagation of epileptiform activity via the temporoammonic path
remains elusive. Monosynaptic, entorhinal layer III neuronal inputs
project directly to CA1 and subiculum (Steward and Scoville 1976; Witter et al. 1988
; cf. Soltesz and
Jones 1995
;). Maccaferri and McBain (1995)
have
shown that the entorhinal cortex-CA1 pathway is under the control of
oriens-alveus interneurons, which suppress the function of the
entorhinal inputs to the CA1 region. Thus under normal conditions,
entorhinal activity propagates to the hippocampus via the trisynaptic
circuit. At the same time, Schaffer collateral inputs activate CA1
pyramidal neurons, which thus excite oriens-alveus GABAergic
interneurons in this region and block the temporoammonic input.
In the present study, we have used a combined hippocampus-entorhinal
cortex slice obtained from mouse brain tissue to investigate the
propagation of ictal epileptiform discharges generated in the
entorhinal cortex. First, we have characterized the role of the dentate
gyrus in gating entorhinal ictal discharges toward the hippocampus
proper. Second, we have determined whether cutting the Schaffer
collaterals (and thus lifting control on the function of the
temporoammonic input) discloses propagation of entorhinal epileptiform
discharges via this pathway. We report here that in our model dentate
gyrus epileptogenicity depends on the occurrence of entorhinal
epileptiform discharges, not on synaptic reorganization of this
subfield. Additionally, we show that entorhinal epileptiform discharges
propagate directly to CA1 and subiculum only after Schaffer collateral
cut, suggesting that the temporoammonic path input is masked by the
trisynaptic propagation of epileptiform activity. Preliminary reports
of this work have appeared in abstract form (Barbarosie et al.
1997, 1998
).
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METHODS |
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Adult, male, CD-1 or Balb-C mice (25-35 g) were decapitated
under halothane anesthesia. Their brain was quickly removed and placed
in cold (1-4°C) oxygenated artificial cerebrospinal fluid (ACSF).
Horizontal slices were prepared following the the dissecting procedures
described in detail by Dreier and Heinemann (1991) for
rat combined hippocampus-entorhinal cortex slices, but were cut
(550-600 µm thick) with a vibratome (see also Avoli et al. 1996a
and Nagao et al. 1996
for previous rat
studies performed in our laboratory). Slices that included the
entorhinal cortex and the hippocampus proper (also comprising the
subiculum and the dentate gyrus) were then transferred to a tissue
chamber where they lay between oxygenated ACSF and humidified gas (95%
O2-5% CO2) at 32-33°C.
ACSF composition was (in mM) 124 NaCl, 2 KCl, 1.25 KH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose. 4-Aminopyridine (4-AP; 50 µM) was directly applied to the perfusing medium. All chemicals were
acquired from Sigma.
Field potential recordings were performed with ACSF-filled
microelectrodes (tip diameter, 8µm; resistance, 2-10 M).
Ion-selective microelectrodes were prepared according to the technique
described by Heinemann et al. (1977)
. Details concerning
the technique for measurements of ionic concentrations can be found in
other publications from our laboratory (e.g., Avoli et al.
1996a
,b
). Unless otherwise indicated, the recording electrodes
were positioned as follows: layers IV-V of the entorhinal cortex; the
granule cell layer of the dentate gyrus; CA3 and CA1 pyramidal layer;
and in continuation of the pyramidal layer in subiculum. A series of
cutting experiments were performed using a microknife to establish the
origin and the pathway used by epileptiform activity to propagate in
the slice (Avoli et al.1996a
; Barbarosie and
Avoli 1997
).
Field potential recordings as well as simultaneous field potential and [K+]o measurements were performed in >40 combined, mouse hippocampus-entorhinal cortex slices. The increases in [K+]o observed during the interictal and the ictal discharges induced by 4-AP were measured as maximal, absolute values. The time differences in ictal discharge onset were obtained from expanded traces of simultaneous field potential recordings, and measurements were made by taking into account the first sharp deflection of each trace at the beginning of the discharge (see Fig. 6, insets in b panels). Values in the text are expressed as means ± SD, and n represents the number of slices studied. Data were compared with the Student's t-test or the ANOVA test and were considered significantly different if P < 0.05.
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RESULTS |
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General features of 4-AP-induced epileptiform activity
Bath application of 4-AP to combined hippocampal-entorhinal slices
induced, within ~1 h, the appearance of spontaneous, CA3-driven interictal-like potentials (duration: <1 s; arrows in Fig.
1B, see also arrowheads in the
expanded recordings of Fig. 5B) and prolonged ictal-like
discharges of entorhinal cortex origin (duration: 20-50 s; continuous
line in Fig. 1B) (cf. Barbarosie and Avoli 1997). These potentials could be recorded simultaneously in all subfields (Fig. 1B). In the intact slice preparation, ictal
discharges disappeared over time, whereas interictal discharges
continued to be recorded during the experiment (cf. Barbarosie
and Avoli 1997
).
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Simultaneous field potential and [K+]o recordings revealed that rises in [K+]o, from a baseline value of 3.2 mM, were associated with both ictal and interictal discharges in the entorhinal cortex, dentate gyrus and in CA3 subfield (n = 22). In the intact slice preparation, the elevations in [K+]o seen during the ictal activity reached maximal values of 9.4 ± 2.1 mM (mean ± SD; n = 8) in the entorhinal cortex, 8.7 ± 1.9 mM (n = 7) in the dentate gyrus, and 5.1 ± 0.6 mM (n = 5) in CA3. In these same experiments, the interictal discharges were accompanied by increases in [K+]o that amounted to 4.0 ± 0.2 mM (n = 8) in the entorhinal cortex, 4.1 ± 0.2 mM (n = 8) in the dentate gyrus, and 3.5 ± 0.2 mM (n = 8) in CA3 (Fig. 1B).
Effect of Schaffer collateral cut on [K+]o elevations associated with 4-AP-induced epileptiform activity
After lesioning the Schaffer collaterals, hippocampal output
activity was suppressed in the entorhinal cortex and dentate gyrus, and
ictal events of entorhinal origin were either 1) disclosed when absent prior to Schaffer collateral cut, or 2)
prolonged in duration if they did occur prior to the lesion (cf.
Barbarosie and Avoli 1997). Figure
2 shows that the elevations in
[K+]o associated with
ictal discharges differed within the entorhinal cortex, the dentate
gyrus, and the CA3 subfield according to the position of the recording
electrodes. Therefore
[K+]o measurements were
performed in the deep layers of the entorhinal cortex, the granule cell
layer of the dentate sector, and in the pyramidal cell layer of CA3,
where the largest elevations in
[K+]o were observed in
coincidence with ictal activity. This type of laminar analysis also
revealed that, with the exception of dentate interictal activity, field
potential changed in polarity when recorded at different sites within a
given area. This evidence, along with the elevations in
[K+]o, indicates that
with the exception of interictal discharges in the dentate area, the
activity induced by 4-AP in different regions of the combined slice was
not volume conducted (Fig. 2B).
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We investigated how the increases in [K+]o associated with the ictal activity were affected by Schaffer collateral cut (Fig. 3, A and B). We found that after this lesioning procedure, [K+]o associated with ictal activity reached peak levels of 13.7 ± 2.5 mM (n = 7) in the entorhinal cortex, 12.0 ± 2.2 mM (n = 7) in the dentate gyrus, and 5.1 ± 0.6 mM (n = 5) in CA3. The ictal-associated [K+]o elevations were of comparable magnitudes in the dentate and entorhinal sectors (P > 0.05). In addition, in both regions, [K+]o rise associated with the ictal discharges was higher than prior to severing this pathway (Fig. 3C; P < 0.05). In contrast, [K+]o rise in CA3 was not significantly affected by the Schaffer collateral cut, and these rises remained significantly lower than in the entorhinal cortex and the dentate gyrus (Fig. 3C; P < 0.05).
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Our results show that 4-AP-induced epileptiform activity, in
particular ictal discharges in dentate and entorhinal sectors are
associated with prominent increases in
[K+]o (cf. Figs. 1-3).
Hence we investigated whether perforant path cut would affect the field
potential and the [K+]o
rises associated with ictal epileptiform discharges. Severing the
perforant path resulted in blockade of both the dentate ictal discharge
and the associated, local increase in
[K+]o, whereas it did not
influence the epileptiform discharge in the entorhinal cortex nor the
corresponding [K+]o rise
(Fig. 4; n = 3).
Therefore in our preparation, dentate paroxysmal discharges were
directly dependent on synaptic propagation of epileptiform activity
from the entorhinal cortex. As reported in the rat combined
hippocampus-entorhinal cortex slices (Avoli et al.
1996a; Nagao et al. 1996
), perforant path cut
also abolished the occurrence of ictal discharges in CA3 and CA1.
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Involvement of the temporoammonic path in ictal discharge propagation
We next examined the pathway used by ictal discharges to propagate to the hippocampus, before and after having lesioned the Schaffer collaterals. To this end, we performed simultaneous recordings in the dentate gyrus, CA3, CA1, and entorhinal cortex and determined the temporal characteristics of the onset of ictal discharge reverberation from expanded recording traces. In five experiments we found that in the intact slice preparation, when ictal discharges are still present, propagation occurred via the trisynaptic circuit (Fig. 5). As shown in Table 1, the onset delays measured in the different areas of these slices corresponded to entorhinal-dentate-CA3-CA1 propagation (Table 1).
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After Schaffer collateral cut, and thus after having disrupted the trisynaptic pathway, ictal discharges of entorhinal origin were still recorded in both CA1 area and subiculum (n = 8 and n = 7, respectively) where they were accompanied by rises in [K+]o (Fig. 6, panels in column a). By employing expanded recordings, and thus by measuring the discharge onset, we also discovered that seizure activity propagated to these regions directly, thus "short-circuiting" the trisynaptic path, at delays similar to those seen for the propagation to the dentate gyrus (Fig. 6, column b; Table 1).
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DISCUSSION |
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We have used field potential and
[K+]o recordings in the
combined, mouse hippocampal-entorhinal slice preparation to understand the propagation of epileptiform activity within the limbic system. Our
results can be summarized as follows. First, entorhinal seizures, which
are released from CA3 control, propagate to the dentate gyrus where
they induce robust ictal discharges comparable to those seen at the
site of origin (i.e., the entorhinal cortex). Second, epileptiform
discharges propagate via the trisynaptic circuit in the intact
hippocampal-entorhinal slice, whereas direct propagation to CA1 and
subiculum is uncovered by Schaffer collateral cut. We have previously
shown that Schaffer collateral cut, which mimics neuronal cell loss
observed in chronic animal models of epilepsy and temporal lobe
epilepsy (TLE) patients might be sufficient to disclose
recurrent seizures originating in the entorhinal cortex (Barbarosie and Avoli 1997).
Ictal discharge occurrence in the dentate gyrus
Considering hippocampal connections as strictly laminar in the
transverse plane represents an oversimplification of the limbic circuit. For instance, the perforant path recruits the dentate gyrus
via longitudinal connections that are topographically organized (Amaral and Witter 1989). In addition,
electrophysiologists studying limbic epileptiform discharges in vivo
have emphasized the importance of reciprocal connectivity among the
hippocampus and entorhinal cortex that, in most in vitro slice
preparations is lost (e.g., Paré et al. 1992
).
This may explain findings obtained from normal hippocampal-entorhinal slices, in which ictal discharges, generated in the entorhinal cortex, do not appear to propagate readily to the
dentate gyrus via the perforant path (Behr et al. 1998
;
Jones and Lambert 1990
; Mody et al.
1988
). Our results show that entorhinal ictal discharges that
are associated with rises in
[K+]o can occur in
otherwise "normal" slices, and do propagate to both dentate gyrus
and CA3 area. Furthermore, the dentate ictal activity (as measured in
terms of associated rise in
[K+]o), correlates
directly with the occurrence and strength of these events at the site
of onset, i.e., the entorhinal cortex. By measuring [K+]o we also ruled out
the possibility that field potential epileptiform discharges recorded
in the hippocampal areas represented volume conduction phenomena. This
is also supported by the inversion of polarity of the field potential
discharges observed with laminar analysis and by the blockade of ictal
discharges following lesion of the perforant path.
The spontaneous potentials induced by 4-AP were seen in CA3, entorhinal
cortex, and dentate gyrus, thus indicating that these limbic areas were
all recruited to give rise to epileptiform activity. Indeed, the robust
ictal discharges and the associated elevations in
[K+]o recorded in the
dentate gyrus were reminiscent of the maximal dentate
activation described by Stringer et al. (1989).
Maximal dentate activation has been reproduced in vitro in
low [Ca+2]o and high
[K+]o medium (Pan
and Stringer 1996
, 1997
; Patrylo et al.
1994
; Schweitzer et al. 1992
). In that model,
dentate ictal activity depends primarily on intrinsic granule cell
properties and ephaptic, nonsynaptic mechanisms because epileptiform
dicharges are unaffected by excitatory amino acid receptor antagonists.
Herein, we have shown that under physiological concentrations of
Ca2+ and K+, 4-AP can
induce ictal discharges in the entorhinal cortex, and that these events
propagate synaptically to the hippocampal areas. Accordingly, cutting
the perforant path abolished both the occurrence of ictal discharges
and the associated increases in
[K+]o in the dentate
gyrus, demonstrating synaptic propagation of the epileptiform activity.
4-AP-induced ictal events in the adult entorhinal cortex and
hippocampus are dependent on
N-methyl-D-aspartate (NMDA) receptor activation
(Avoli et al. 1996a).
[K+]o during 4-AP-induced ictal epileptiform activity
Ictal discharges in the entorhinal cortex and dentate gyrus
are associated with elevations in
[K+]o that are
significantly larger than in CA3. The
[K+]o increases
associated with the dentate ictal discharge (that correlate well with
the [K+]o rises observed
in the entorhinal cortex) can be explained through the high density of
granule cells and the low extracellular space in this region. On the
other hand, the low [K+]o
associated with ictal discharge in CA3 versus dentate gyrus may be due
to the reported preferential activation of interneurons by mossy
fibers. Accordingly, the increased granule cell activity may suppress
the excitability of the CA3 recurrent system (Acsady et al.
1998). Alternatively, presynaptic activation of metabotropic receptors may limit the bursting capacity of CA3 neurons and thus give
rise to a smaller ictal discharge in CA3 (Scanziani et al. 1997
). Moreover, bursting behavior of CA3 pyramidal
neurons is tightly dependent on the pool size of available synaptic
vesicles for release (Staley et al. 1998
). These
mechanisms may act in concert to protect excessive neuronal activity
and damage to this region, which is critical in controlling the
generation of ictal discharges within the entorhinal cortex
(Barbarosie and Avoli 1997
).
Laminar analysis of the ictal discharges recorded in CA3 indicates that
in the combined hippocampus-entorhinal cortex slice, the largest
[K+]o rises occur in the CA3 pyramidal cell
layer. This is in agreement with a study made in juvenile,
"isolated" rat hippocampal slices treated with 4-AP (Avoli
et al. 1996b). In this preparation the [K+]o elevations associated with ictal
discharges in CA3 attain maximal values in the pyramidal layer.
However, they are preceded by an initial increase in
[K+]o (which trigger them and coincides with
a GABA-mediated synchronous potential) that was even greater than what
was seen during the discharge and attained largest value in stratum
radiatum. Our present findings indicate that, although ictal discharges
in CA3 were associated with the largest [K+]o
in the pyramidal layer, they were probably not initiated by an initial,
GABA-mediated rise in [K+]o, as was the case
in juvenile CA3. Rather, they simply resulted from propagation through
the dentate gyrus of an ictal event originating in the entorhinal cortex.
Mossy fiber sprouting and chronic models of TLE
Mossy fiber reorganization and increased epileptogenicity of
dentate granule cells is a hallmark of human TLE (Houser et al. 1990; Sutula et al. 1989
) and chronic animal
models of TLE (Behr et al. 1998
; Cavazos et al.
1991
; Represa et al. 1989
; Sutula et al.
1988
; Wuarin and Dudek 1996
). Parent et
al. (1997)
have also reported that ectopic granule cell
neurogenesis is associated with sprouting in this region and correlates
with increased seizure activity. However, blocking mossy fiber
sprouting does not interfere with the appearance of recurrent epileptic
seizures (Longo and Mello 1997
, 1998
).
Our results demonstrate that robust ictal discharges do occur in the
dentate gyrus of "normal" slices (and hence in the absence of
synaptic reorganization). Hence, although the dentate gyrus lacks the
recurrent excitatory connections to support generation of seizure
activity, our data show that it can be fully activated when entorhinal
inputs impinge on it.
We have reported that CA3 neuronal damage, another hallmark of TLE
(Ben-Ari 1985), mimicked by Schaffer collateral cut of an intact hippocampal-entorhinal slice, may be sufficient to disclose the generation of recurrent entorhinal seizures (Barbarosie and Avoli 1997
). Therefore increased dentate gyrus
epileptogenicity, evident in the chronic condition, may also result
from the propagation of uncovered entorhinal cortex seizure activity.
Role of a temporoammonic pathway in epilepsy
It has been demonstrated that under normal conditions, depression
of synaptic transmission between areas CA3 and CA1 discloses the
operativity of a perforant path projection from the entorhinal cortex
to the CA1 region (Maccaferri and McBain 1995). In our experiments, Schaffer collateral cut (which blocks CA3-driven excitatory inputs to both CA1 pyramids and interneurons) is sufficient to "short-circuit" the propagation of 4-AP-induced epileptiform activity via the entorhinal-CA1 path.
In chronic animal models of TLE, entorhinal layer III neurons are
preferentially lost (Du et al. 1995), and inhibitory
postsynaptic potentials appear impaired in both superficial and deep
entorhinal layers in slices obtained from such animals (Bear et
al. 1996
; Fountain et al. 1998
). It is
attractive to postulate that CA1 pyramidal cell hyperexcitability
observed in epileptic tissue (Williams et al. 1993
) may,
at least in part, be conferred via the direct excitation of CA1
pyramidal cells by disinhibited entorhinal neurons. Hence CA3 neuronal
cell loss, and Schaffer collateral damage, also observed in models of
TLE (Ben-Ari 1985
), in addition to potential
disinhibition of the entorhinal cortex, may contribute to disclose a
direct entorhinal-CA1 pathway.
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ACKNOWLEDGMENTS |
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We thank T. Papadopoulos for secretarial assistance.
This study was supported by the Medical Research Council of Canada (Grant MT-8109), the Savoy Foundation, and the Hospital for Sick Children (Grant XG-93056). M. Barbarosie is the recipient of a Fonds de la Recherche en Santé du Québec studentship.
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FOOTNOTES |
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Address for reprint requests: M. Avoli, 3801 University, Montreal, Quebec H3A 2B4, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 May 1999; accepted in final form 25 October 1999.
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REFERENCES |
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