1Departamento de Fisiologia, Instituto de Bioingenieria, Universidad Miguel Hernandez, Campus de San Juan, 03550 San Juan, Alicante, Spain; and 2Neuroscience Program and Istituto Nazionale Fisica della Materia Unit, International School for Advanced Studies, 34014 Trieste, Italy
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ABSTRACT |
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Bolea, Sonia,
Elena Avignone,
Nicola Berretta,
Juan V. Sanchez-Andres, and
Enrico Cherubini.
Glutamate controls the induction of GABA-mediated giant depolarizing
potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are
generated by the interplay of the depolarizing action of GABA and
glutamate. In this study, single and dual whole cell recordings (in
current-clamp configuration) were performed from CA3 pyramidal cells in
hippocampal slices obtained from postnatal (P) days P1- to P6-old rats
to evaluate the role of ionotropic glutamate receptors in GDP
generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)
(10-40 µM) completely blocked GDPs. However, in the presence of
CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 µM) or (RS)--amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 µM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 µM). In the
presence of GYKI 53655, both kainic or domoic acid (0.1-1 µM) were
unable to induce GDPs. In contrast, bath application of
D-(
)-2-amino-5-phosphonopentanoic acid (50 µM) or
(+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 µM) produced only a 37 ± 9% (SE) and 36 ± 11% reduction in GDPs frequency, respectively. Cyclothiazide,
a selective blocker of AMPA receptor desensitization, increased GDP
frequency by 76 ± 14%. Experiments were also performed with an
intracellular solution containing KF to block GABAA
receptor-mediated responses. In these conditions, a glutamatergic
component of GDP was revealed. GDPs could still be recorded synchronous
with those detected simultaneously with KCl-filled electrodes, although
their amplitude was smaller. Similar results were found in pair
recordings obtained from minislices containing only a small portion of
the CA3 area. These data suggest that GDP generation requires
activation of AMPA receptors by local release of glutamate from
recurrent collaterals.
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INTRODUCTION |
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A peculiar characteristic of the hippocampus
of neonatal rats is the presence of spontaneous network-driven
GABA-mediated oscillatory events, the so-called giant depolarizing
potentials (GDPs) (Ben Ari et al. 1989; Xie et
al. 1994
). These appear to be synchronous within the entire
hippocampus (Menendez de la Prida et al. 1998
;
Strata et al. 1997
) and are generated by GABA (acting on
GABAA receptors) that at this early developmental stage is depolarizing and excitatory (Cherubini et al. 1991
).
GABA-induced depolarization represents a general feature of postnatal
development (Ben Ari et al. 1989
; Chen et al.
1996
; Cherubini et al. 1991
; Kaneda et
al. 1996
; Owens et al. 1996
; Rohrbough
and Spitzer 1996
; Serafini et al. 1995
;
Wu et al. 1992
). GABA, through its depolarizing action, activates voltage-dependent calcium channels, thereby increasing the levels of [Ca2+]i
(Leinekugel et al. 1995
). Therefore GDPs are strictly
associated with spontaneous calcium oscillations occurring in groups of
neighboring cells (Garaschuk et al. 1998
). These highly
correlated calcium signals are thought to be essential for
consolidation of synaptic connections and development of the adult
neuronal network (Shatz 1990
). However, GDPs need a
glutamatergic drive for their expression. In previous studies from this
(Strata et al. 1995
) and other laboratories (Ben
Ari et al. 1989
; Gaiarsa et al. 1991
) it has
been shown that GDPs can be modulated by activation of both ionotropic
and metabotropic glutamate receptors. More recently, it was suggested
that synchronization is determined by cooperation of excitatory
GABAergic connections between interneurons and glutamatergic
connections presumably from pyramidal cells to interneurons
(Khazipov et al. 1997
). Hence in interneurons a
glutamatergic component of the GDP could be revealed after
intracellular blockade of GABAA receptors with an internal
solution containing fluoride (Khazipov et al. 1997
). In
particular, it was suggested that the main contribution to GDP
generation is provided by N-methyl-D-aspartate
(NMDA) receptors that would be activated after GABA-induced
depolarization, whereas AMPA receptors would play only a minor role
(Ben Ari et al. 1997
; Leinekugel et al.
1997
; McLean et al. 1995
).
The current experiments were undertaken to further elucidate the role
of glutamatergic receptors in GDP generation. In contrast to previously
published data (Khazipov et al. 1997; Leinekugel et al. 1997
) it was found that AMPA receptors play a major role in neuronal synchronization.
Part of this work was reported in preliminary form (Bolea et al.
1998).
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METHODS |
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Slice preparation
Experiments were performed on hippocampal slices obtained from
postnatal P1-P6 Wistar rats (P0 is the day of birth) according to the
methods already described (Strata et al. 1995). Briefly, animals were decapitated after being anesthetized with intraperitoneal injection of urethan (2 g/kg). The brain was quickly removed from the
skull, and the hippocampi were dissected free. Transverse 600-µm
thick slices were cut with a tissue chopper and maintained at room
temperature (22-24 °C) in oxygenated artificial cerebrospinal fluid
(ACSF) containing (in mM) 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 1.3 MgCl2, 2 CaCl2, 25 NaHCO3, and 11 glucose, pH 7.3, saturated with 95% O2-5% CO2. After
incubation in ACSF for
1 h, an individual slice was transferred to a
submerged recording chamber, continuously superfused at 33-34°C with
oxygenated ACSF at a rate of 3 ml/min.
Patch-clamp whole cell recordings
Spontaneous or evoked GDPs were recorded in the whole cell
configuration of the patch-clamp technique (current-clamp mode) with a
standard amplifier (Axoclamp 2B, Axon Instruments; Foster City, CA).
Patch electrodes had a resistance of 3-6 M when filled with an
intracellular solution containing (in mM) 140 KCl or KF, 1 MgCl2, 1 NaCl, 1 EGTA, 5 HEPES, and 2 K2ATP; pH
was adjusted to 7.3 with KOH. GDPs were evoked with bipolar twisted
NiCr-insulated electrodes (50 µm OD) placed in the hilus. Pulses of
variable amplitude (5-15 V, 100-µs duration) were applied. Membrane
input resistance was measured from the amplitude of small
hyperpolarizations (300-ms duration) evoked by passing current pulses
across the cell membrane.
Drugs
Drugs were dissolved in ACSF and applied in the bath by changing
the superfusion solution to one that differed only in its content of
drug(s). The ratio of flow rate to bath volume ensured complete
exchange within 1 min. Drugs used were
(RS)--amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA),
(+)-3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP), or
D-(
)-2-amino-5-phosphonopentanoic acid
(D-AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), domoic acid, kainic
acid, GABA, and bicuculline (all purchased from Tocris Cookson;
Bristol, UK); kynurenic acid, TTX, and
6-chloro-3,4dihydro-3-[2-norbornen-5-yl]
2H-1,2-4benzothiadiazine-7-sulfonamide 1,1-dioxide (cyclothiazide; from Sigma; Milano, Italy); GYKI 52466 (from RBI); and GYKI 53655 (from Lilly; Indianapolis, IN). If not
otherwise stated, data are expressed as means ± SE.
Data acquisition and analysis
Data of spontaneous and evoked GDPs were stored on a magnetic tape and transferred to a computer after digitization with an A/D converter (Digidata 1200). Data acquisition was done with pClamp (Axon Instruments; Foster City, CA), and the amplitude and frequency of GDPs were analyzed with Axoscope (Axon Instruments; Foster City, CA).
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RESULTS |
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Stable whole cell recordings (in current-clamp configuration)
lasting >30 min were obtained from 120 CA3 hippocampal pyramidal cells
in slices from P1- to P6-old rats that exhibited spontaneous GDPs. They
occurred at the frequency of 5.2 ± 0.43 GDPs/min and consisted of
large (30-50 mV) depolarizing potentials lasting 400-700 ms, which
triggered action potentials, followed by an afterhyperpolarization. In
previous experiments (Ben Ari et al. 1989), it was
reported that GDPs are network-driven events generated by a large
population of neurons firing synchronously because 1) their
frequency is independent of the membrane potential, 2) they
are synchronous in pair of CA3 recordings, 3) they are
reversibly blocked by a high-magnesium, low-calcium solution, and
4) they are abolished by TTX. These events are
GABAA mediated because they are reversibly blocked by
bicuculline. In a first set of experiments, the role of ionotropic
glutamate receptors in GDP generation was reexamined, taking also
advantage of new pharmacological tools. Then, in a second series of
experiments, the conductances activated during GDPs in pyramidal cells
were studied after intracellular blockade of
GABAA-receptor-evoked responses with fluoride.
AMPA receptor activation is necessary for GDP induction
To test the role of AMPA receptors in GDPs induction, the
selective AMPA-kainate receptor antagonist CNQX (10-40 µM) was
applied (n = 11). As reported earlier (Gaiarsa
et al. 1991), this compound completely blocked spontaneous GDPs
(data not shown), even in rats as young as P2. A full recovery was
obtained 10-15 min after wash. However, in the presence of CNQX, it
was still possible to re-induce the appearance of spontaneous GDPs with
GABA (20 µM) or AMPA (5 µM) and to evoke GDPs by focal stimulation.
The effect of AMPA was usually associated to a 5- to 12-mV membrane depolarization. Both GDPs as well as membrane depolarization
disappeared when AMPA was applied in the presence of bicuculline (10 µM, data not shown), suggesting that both phenomena were due to the
release of GABA after an incomplete block of AMPA receptors with CNQX. Similar results were obtained with kynurenic acid (2 mM,
n = 8), DNQX (20 µM, n = 2), and GYKI
52466 (20-100 µM, n = 6). The possibility to
synchronize again the network with AMPA suggests that AMPA receptors
were not completely blocked by the selective antagonists used. To check
whether this hypothesis was correct, a more potent and selective AMPA
antagonist, GYKI 53655, was tested (Lerma et al. 1997
).
GYKI 53655 (50-100 µM) blocked spontaneous GDPs as well as those
evoked by electrical stimulation of the hilus. Moreover, AMPA (5 µM)
or GABA (20 µM) application were unable to re-induce GDPs (Fig.
1). These data suggest that the
glutamatergic drive via AMPA receptors is essential for GDP generation.
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To further examine the role of AMPA receptors in GDPs induction,
experiments were performed in the presence of cyclothiazide, a
selective blocker of AMPA receptor desensitization (Partin et al. 1993). In the presence of cyclothiazide (20 µM) GDPs
frequency increased by 76 ± 14% (n = 6, Fig.
2). Kainate receptors were not apparently
involved in GDPs induction because in the presence of GYKI 53655 both
kainate and domoic acid (0.1-1 µM) failed to induce GDPs
(n = 3, data not shown).
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In contrast to AMPA receptor antagonists, the selective NMDA receptor antagonist D-AP5 (50 µM) applied in the bath for 10-15 min produced only a reduction of GDP frequency (Fig. 3). Occasionally a complete blockade was observed during the first minutes of drug application. Later on, in the continuous presence of D-AP5, GDP frequency slowly returned toward control values. On average a 35% reduction in GDP frequency was measured along the time of drug application. Similar results were obtained with CPP (20 µM). No significant changes in the shape of GDPs were observed during application of CPP or D-AP5. These data suggest that, although NMDA receptors may contribute to GDP generation, they are not essential for their induction. However, in the presence of CNQX, D-AP5 was able to prevent evoked GDPs or re-induction of spontaneous oscillations by GABA and AMPA (data not shown), indicating that in particular conditions also NMDA receptors may contribute to network synchronization.
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Intracellular blockade of GABAA receptor with fluoride reveals an AMPA-mediated component of GDPs
In a previous work aimed at understanding synchronization of
interneuronal network (Khazipov et al. 1997), whole cell
recordings were made from interneurons dialyzed with the poorly
permeable anion F
, which produces a block of
GABAA receptors (Bormann et al. 1987
). We
used a similar approach to see whether in CA3 pyramidal cells glutamate
receptors are activated during GDPs. To this purpose we tested
1) GDP reversal potential immediately and 30-40 min after a full whole cell access was achieved with a F
containing solution and 2) the pharmacological
sensitivity of GDPs to GABAA and ionotropic NMDA and
non-NMDA glutamate receptor antagonists. Immediately (3-5 min) after
breaking into whole cell configuration, the reversal of evoked and
spontaneous GDPs was
45.7 ± 6.1 mV (n = 28)
and
40.8 ± 6.2 mV (n = 11), respectively (Fig. 4, A and
B). Plots of amplitude-voltage relationships were always linear. The slope of the regression lines through different data
points was
0.58 ± 0.19 (n = 28) for evoked
and
0.67 ± 0.14 (n = 11) for spontaneous
GDPs. After 40-min dialysis with an intracellular F
containing solution a complete block of GABA-evoked responses in the
presence of TTX (1 µM) was observed (Fig. 4C). The
block of GABAA receptors was associated with a significant
shift of the reversal toward more positive values. The reversal of the evoked and spontaneous GDPs was
14.1 ± 11.4 mV
(n = 17) and
15.8 ± 13.1 mV
(n = 6), respectively (Fig. 4, A and
B). Changes in the reversal were associated with a
reduction in the slope of the regression lines, indicating a reduction
of GDP amplitude (this was
0.29 ± 0.12, n = 17, for evoked and
0.31 ± 0.13, n = 6, for
spontaneous GDPs). In the majority of cells the amplitude-voltage relationship was linear, whereas in four cases a rectification at more
negative potentials was observed. The shift of the reversal potential
toward more positive values as well as the reduction of the slope in
the amplitude-voltage relationship suggest the presence of a different
component unmasked after intracellular blockade of GABAA
receptors with F
. To understand the nature of this
component, spontaneous and evoked GDPs were recorded at different
membrane potentials in the presence of AMPA and NMDA receptor
antagonists. Bath application of CNQX (10-20 µM) completely
abolished the residual component of spontaneous GDPs and in 8 of 11 completely blocked the evoked GDPs. D-AP5 (50 µM) reduced
the residual component only in 3 of 11 (Fig.
5, A-C).
In the presence of D-AP5 and CNQX GDPs could not be evoked.
It could be that the glutamatergic component of the GDP, which remains
after blockade of GABAA receptor, is amplified as the
result of the interference of intracellular F
with
phosphatases (Andrews and Babior 1984
), enzymes that
regulate receptor desensitization (Yakel 1997
). To test
this hypothesis, the amplitude of AMPA responses recorded in TTX (1 µM) with a F
containing intrapipette solution was
compared with that of responses recorded with a KCl solution. As shown
in Fig. 5D, in F
, AMPA responses were not
significantly (P > 0.08) different from those
recorded in KCl.
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Pair recordings performed from adjacent CA3 pyramidal cells
(n = 21) with two different intrapipette solutions
showed that GDPs recorded with KF and KCl intrapipette solutions were
still synchronous in the two cells, confirming their combined
glutamatergic and GABAergic nature (Fig.
6). However, a clear difference in the
shape of GDPs recorded with KF or KCl was observed. As depicted in Fig.
6, in KF GDPs were smaller in amplitude and exhibited a slower rising
phase that often did not reach the threshold for action potential
generation. Moreover, when GABA (20 µM) was applied in the presence
of kynurenic acid (2 mM), known to abolish spontaneous GDPs
(Strata et al. 1995), this amino acid was able to
re-induce GDPs only in the cell recorded with KCl electrode (Fig. 6).
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Source of glutamatergic drive needed to synchronize GABAergic interneurons
Glutamate may be released from mossy fibers, known to make
synaptic contacts with the proximal dendrites of CA3 pyramidal neurons
and with GABAergic interneurons (Acsády et al.
1998). In early postnatal days, when the present experiments
were performed, mossy fibers were not completely developed
(Gaarskjaer 1986
) and therefore their contribution to
GDPs induction appears to be modest. Other sources of glutamate are the
associative-commisural fibers coming from the controlateral hippocampus
as well as from collaterals of principal cells. To examine whether GDPs
can be generated locally by these fibers, in three slices, a small part
of the CA3 region was isolated with a knife cut from the rest of the
hippocampus, and pair recordings were performed from two CA3 adjacent
pyramidal cells with two intrapipette solutions containing KF and KCl,
respectively. In these conditions GDPs with characteristics similar to
those already reported could be still recorded synchronously in the two
cells (Fig. 7). This suggests that a
local population of pyramidal cells and interneurons is sufficient to
generate GDPs and that collaterals of principal cells are probably the
main source of glutamatergic drive.
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DISCUSSION |
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In line with a previous report (Ben Ari et al.
1997), the current experiments clearly show that GDPs are
generated by the synergistic action of glutamate and GABA. However, in
contrast with earlier findings on interneurons (Khazipov et al.
1997
; Leinekugel et al. 1997
), they demonstrate
that the release of GABA is triggered by glutamate acting mainly on
AMPA type of receptors and that the ionotropic glutamatergic component
of GDPs, revealed after blockade of GABAA-evoked responses
with intracellular F
, is predominantly of the non-NMDA type.
AMPA receptor activation is necessary for GDP induction
AMPA receptor activation was necessary for GDP induction as proved
by the observation that AMPA receptor antagonists were fully effective
in blocking GDPs, whereas NMDA receptor antagonists only produced a
transient reduction in their frequency (see also Gaiarsa et al.
1991). In agreement with the current data, it has been recently
shown, with fluorometric calcium imaging techniques, that CNQX is able
to completely block spontaneous oscillatory calcium transients,
associated to GDPs, recorded in the CA1 region of the hippocampus
during early postnatal development (Garaschuk et al.
1998
). In previous studies a complete block of GDPs with NMDA
receptors antagonists was reported. The same block was occasionally observed only during the first 2-3 min of drug application. At an
early stage of development, at least in the CA1 area, glutamatergic synapses containing only NMDA receptors were described (Durand et al. 1996
). However, the observations that at P2 it is
already possible to block the entire network with CNQX and to increase GDPs frequency with cyclothiazide indicate that also AMPA receptors are
present and functional at this early developmental period. The
contribution of NMDA receptors to network synchronization is suggested
by the experiments in which GDPs evoked by GABA or AMPA in the presence
of CNQX were abolished by addition of D-AP5 or CPP.
However, it should be stressed that in these cases AMPA responses were
not completely abolished, probably because of AMPA receptors localized
on basket cells, not entirely blocked by CNQX. Therefore we suggest
that functional AMPA receptors would provide the depolarization
sufficient to remove the magnesium block and to activate NMDA receptor
channels present on interneurons. Further evidence in favor of the
involvement of AMPA receptor in GDP induction is given by the
experiment with GYKI 53655, a more-selective AMPA receptor antagonist
(Lerma et al. 1997
). In these cases, the impossibility to re-activate the network with GABA or AMPA suggest that network synchronization may involve AMPA receptors localized on GABAergic interneurons, having, in comparison with principal cells, distinct biophysical and pharmacological properties. Striking differences in the
functional characteristics of AMPA receptors in basket and principal
cells were reported (Isa et al. 1996
; Koh et al. 1995
; for review see Freund and Buzsaki
1996
). These consist of larger single-channel conductance,
rapid desensitization kinetics (see also Livsey et al.
1993
), and in the majority of the cases a higher permeability
to calcium (Burnashev et al. 1995
; Koh et al.
1995
). This latter issue appears to be particularly interesting in view of the role that this divalent cation has in synaptic plasticity processes during development. Thus the rapid transient increase in calcium flux through AMPA receptor channel at rest would
temporally summate to that entering the cell through GABA-mediated activation of voltage-dependent calcium channels. As suggested by
recombinant studies (Burnashev et al. 1992
), different
subunit composition may underline differences in function.
Interestingly, in a recent study on hippocampal slices obtained from
16- to 20-day-old rats, it has been shown that only interneurons
activated by the mossy fibers bear polyamine toxin
(philanthotoxin)-sensitive AMPA receptors, presumably calcium permeable
(Tóth and McBain 1998
). Further support to the
idea that glutamate triggers the release of GABA from GABAergic
interneurons through AMPA receptor subtypes is given by the experiments
in which during superfusion of CNQX, AMPA-evoked membrane
depolarization and associated increase in frequency of GDPs are blocked
by bicuculline. Moreover the double-recording experiments (with KCl or
KF), in which in the presence of kynurenic acid it was not possible to
induce GDPs in the cell recorded with KF, strongly indicate that AMPA
receptors on pyramidal cells are blocked. The involvement of kainate
receptors in GDP generation can be excluded on the basis of the
impossibility to synchronize the network when kainic or domoic acid are
applied in the presence of GYKI 53655.
AMPA receptor-mediated component of GDPs could be revealed after intracellular blockage of GABAA receptor with fluoride
After dialyzing principal cells with F (see also
Khazipov et al. 1997
) in concomitance with a complete
block of GABAA-induced responses, a residual component with
slower kinetics can be revealed. This component appears to be
glutamatergic in origin, as suggested by the shift in the reversal
potential toward more positive values and by the change in the slope of
the amplitude-voltage plot, obtained after dialyzing the cell with
F
for
30-40 min. The observed shift in reversal was
similar to that already reported for GDPs in interneurons
(Khazipov et al. 1997
) and was associated, in the
majority of the cases, with a linear amplitude-voltage relationship.
This indicates the activation of a non-NMDA receptor type, unmasked
after intracellular blockade of GABA. Further support in favor of
activation of AMPA receptor type in evoked GDPs is given by
pharmacological experiments in which, in 8 out of 11 cases, CNQX was
able to completely block the residual component. In this respect our
data differ from those reported earlier by Khazipov et al. (1997)
and
Leinekugel et al. (1997)
. However, in those reports, the assumption
that the residual part of GDPs (which remains after intracellular
dialysis with F
) is NMDA dependent was based only on the
characteristic voltage dependency of the responses. No pharmacological
tools were used to dissect out the different glutamatergic components.
On the other hand, the lack of effect of CNQX in blocking spikes
recorded in cell-attached configuration and supposed to represent
interneuronal GDPs reported in those experiments (Khazipov et
al. 1997
; Leinekugel et al. 1997
) has, as
pointed out by the same authors, strong limitations. In the current
experiments, the similarity in the dose-response curves for AMPA,
obtained with intracellular solutions containing KCl and
F
, respectively, allows to exclude the possibility that
the residual AMPA-mediated component of GDPs is modified as the result
of the interference of F
with some phosphorylation
processes (Andrews and Babior 1984
).
Source of glutamatergic drive needed to synchronize GABAergic interneurons
As already shown by Khazipov et al. (1997), Garaschuk et al.
(1998)
, and Menendez de la Prida et al. (1998)
, GDPs were still present
in small CA3 islands isolated from the rest of the hippocampus, implying that a local circuit comprehensive of a relatively small population of principal cells and interneurons was sufficient to
generate them. In these cases, the glutamatergic input presumably originates from collaterals of principal cells because the mossy fibers
are disconnected from the granule cells of the dentate gyrus. Also the
glutamatergic component revealed in pyramidal cells recorded with KF is
presumably due to glutamate release from recurrent collaterals. In
summary, these data show that GDP generation requires activation of
AMPA receptors by local release of glutamate from recurrent
collaterals. Other glutamatergic components can also be involved but do
not seem to be essential.
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ACKNOWLEDGMENTS |
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This work was partially supported by a grant from Consiglio Nazionale delle Ricerche, Ministero dell'Universita' e Ricerca Scientifica e Tecnologica to E. Cherubini and by a grant from the Fondo de Investigacion Sanitaria to J. V. Sanchez-Andres. S. Bolea was supported by a fellowship from the Ministerio de Educacion y Cultura. N. Berretta was supported by a fellowship from Novartis Pharma. S. Bolea and E. Avignone contributed equally to this work.
Present address of N. Berretta: IRCCS, S. Lucia, Via Ardeatina 306, 00178 Roma, Italy.
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FOOTNOTES |
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Address for reprint requests: E. Cherubini, International School for Advanced Studies, Via Beirut 2-4, 34014 Trieste, Italy.
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 9 October 1998; accepted in final form 19 January 1999.
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REFERENCES |
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