Department of Biosciences, Division of Animal Physiology, University of Helsinki, 00014, Finland
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ABSTRACT |
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Lamsa, Karri,
J. Matias Palva,
Eva Ruusuvuori,
Kai Kaila, and
Tomi Taira.
Synaptic GABAA Activation Inhibits AMPA-Kainate
Receptor-Mediated Bursting in the Newborn
(P0-P2) Rat Hippocampus.
J. Neurophysiol. 83: 359-366, 2000.
The mechanisms of synaptic transmission in the rat
hippocampus at birth are assumed to be fundamentally different from
those found in the adult. It has been reported that in the CA3-CA1
pyramidal cells a conversion of "silent" glutamatergic synapses to
conductive -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) synapses starts gradually after P2. Further, GABA via its
depolarizing action seems to give rise to grossly synchronous yet slow
calcium oscillations. Therefore, GABA is generally thought to have a
purely excitatory rather than an inhibitory role during the first
postnatal week. In the present study field potential recordings and
gramicidin perforated and whole cell clamp techniques as well as
K+-selective microelectrodes were used to examine the
relative contributions of AMPA and GABAA receptors to
network activity of CA3-CA1 pyramidal cells in the newborn rat
hippocampus. As early as postnatal day (P0-P2), highly coherent spontaneous
firing of CA3 pyramidal cells was seen in vitro. Negative-going
extracellular spikes confined to periodic bursts (interval 16 ± 3 s) consisting of 2.9 ± 0.1 spikes were observed in stratum
pyramidale. The spikes were accompanied by AMPA-R-mediated
postsynaptic currents (PSCs) in simultaneously recorded pyramidal
neurons (7.6 ± 3.0 unitary currents per burst). In CA1 pyramidal
cells synchronous discharging of CA3 circuitry produced a barrage of
AMPA currents at >20 Hz frequencies, thus demonstrating a transfer of
the fast CA3 network activity to CA1 area. Despite its depolarizing
action, GABAA-R-mediated transmission appeared to exert
inhibition in the CA3 pyramidal cell population. The
GABAA-R antagonist bicuculline hypersynchronized the output of glutamatergic CA3 circuitry and increased the network-driven excitatory input to the pyramidal neurons, whereas the
GABAA-R agonist muscimol (100 nM) did the opposite.
However, the occurrence of unitary GABAA-R currents was
increased after muscimol application from 0.66 ± 0.16 s
1 to 1.43 ± 0.29 s
1. It was
concluded that AMPA synapses are critical in the generation of
spontaneous high-frequency bursts in CA3 as well as in CA3-CA1 transmission as early as P0-P2 in rat hippocampus.
Concurrently, although GABAA-R-mediated depolarization may
excite hippocampal interneurons, in CA3 pyramidal neurons it can
restrain excitatory inputs and limit the size of the activated neuronal population.
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INTRODUCTION |
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During development, neuronal activity shapes, and
is itself shaped, by emerging synaptic contacts. In the CA3-CA1
circuitry of the neonatal rat hippocampus AMPAfication (i.e.,
conversion of silent glutamatergic synapses to conductive
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA]
synapses) is proposed to be driven by spontaneous long-lasting
GABAA-R-mediated depolarizations (Durand
et al. 1996
; Hanse et al. 1997
). Repetitive
stimulation of afferents combined to postsynaptic depolarization has
indeed been found to induce functional connections from initially
silent AMPA-synapses in CA1 (Durand et al. 1996
), yet no
synaptic drives mimicked by the afferent stimulation in this scheme
have been reported in the newborn hippocampus. The build-up of
excitatory AMPA-R-mediated circuitry toward the end of the first
postnatal week is paralleled by a graded ontogenetic switch from
depolarizing GABAA responses to hyperpolarization
(Rivera et al. 1999
).
The idea of dormant fast glutamatergic transmission in the newborn rat
hippocampus was initially based on morphological studies demonstrating
a scarcity of identifiable dendritic synaptic contacts between
pyramidal cells during the first postnatal days. Electrophysiological recordings using single-pulse stimulation protocols also speak for
purely N-methyl-D-aspartate receptor
(NMDA-R)-based glutamatergic transmission in rat hippocampal slices
before P2-P3 (Durand et al. 1996;
Hsia et al. 1998
). However, it has been pointed out that
anatomic criteria may be misleading in judging the functional status of
developing synaptic contacts (Durand et al. 1996
;
Katz and Shatz 1996
). Further, because spike bursts
probably represent a basic element of the neural code (Lisman
1997
), adequate activation of hippocampal synapses may also
require natural, bursting-type firing pattern of neurons
(Dobrunz and Stevens 1999
; Selig et al.
1999
). In particular, this may be the case in the immature hippocampus, where the AMPA-R-mediated postsynaptic currents (PSCs) show a large variability in their quantal responses and thus in the
overall reliability of synaptic transmission (Hsia et al. 1998
).
Because of the proposed absence of effective fast glutamatergic
transmission, the absence of inhibitory GABAA
action has not been considered physiologically enigmatic, and GABA in
fact has frequently been suggested to be the major excitatory
transmitter in the newborn rat hippocampus (Ben-Ari et al.
1997; Holmes and Ben-Ari 1998
; but see
Dailey and Smith 1994
). It is, however, known from other
brain areas that postsynaptic inhibition is required for developmental
refinement of neuronal contacts (Hensch et al. 1998
).
Yet, the possible inhibitory aspects of
GABAA-R-mediated transmission in the newborn
hippocampus have received little attention. Functional inhibition may
still prevail despite depolarizing
GABAA-R-mediated responses (Jackson et
al. 1999
; Su and Chai 1998
; see also
Kaila et al. 1993
; Taira et al. 1997
).
GABAergic shunting conductances exert effective inhibition at
postsynaptic sites (e.g., Furshpan and Potter 1959
) and
desynchronize the discharge of pyramidal cells in the mature
hippocampus (Jackson et al. 1999
). Thus
GABAA-R-mediated conductances resulting from
endogenous activity in the neonatal hippocampus are likely to affect
membrane excitability and cable properties of individual neurons (see
Häusser and Clark 1997
; Staley and Mody
1992
).
The purpose of the present work was twofold. First, we wanted to
elucidate the role of synchronous discharge of pyramidal cell ensembles
in hippocampal transmission at P0-P2. Instead of using
traditional stimulation protocols, we chose to study spontaneous neuronal bursts, which are likely to represent the pattern of activity
occurring in the newborn rat hippocampus in vivo (Lahtinen et
al. 1999). Second, we examined the inhibitory aspects of
GABAA-R-mediated transmission in the
P0-P2 rat hippocampus.
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METHODS |
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Tissue preparation
P0-P2 Wistar rat pups were anesthetized by
hypothermia. Animals were decapitated, and brains were quickly removed
and dissected in iced (0-4°C), oxygenated (95%
O2-5% CO2) standard
solution containing (in mM) 124 NaCl, 3.5 KCl, 2.0 CaCl2, 25 NaHCO3, 1.1 NaH2PO4, 1.3 MgSO4, and 11 D-glucose. After
removing the cerebellum and the most frontal part of the neocortex, the
cerebral hemispheres were split. Hippocampi were then dissected from
the separated hemispheres, as described previously (Khalilov et
al. 1997). Isolated hippocampal structures were cut into
transverse slices (600-µm) using a McIlwain tissue chopper. For some
experiments intact structures were not sliced but were preserved as
whole hippocampus preparations (Khalilov et al. 1997
).
Preparations were kept in oxygenated standard solution at room
temperature (20-22°C) for
1 h before use.
Electrophysiological recordings
For experimental procedures, preparations were transferred to a
conventional submerged chamber (0.4 ml), laid on nylon mesh, and
anchored with platinum bars. Slices were superfused with oxygenated standard solution (32-33°C) at a rate of 3 ml/min and intact
P0-P1 hippocampal structures at a rate of 5-6 ml/min.
Extracellular field potential recordings were performed with
conventional NaCl-filled (150 mM) glass capillary microelectrodes (5-6
M). K+-selective microelectrodes were made
from double-barreled borosilicate glass pipettes, according to
techniques described in Voipio et al. (1994)
. The
reference barrel was filled with 150 mM NaCl, and the filling solution
in the silanized nonfilamented barrel was 150 mM NaCl and 3 mM KCl. A
short column of the ion sensor (60938, Fluka, Neu-Ulm, Germany) was
taken into the tip using slight suction. The resistance of the two
barrels was 5-10 M
and 5-10 G
, respectively. The electrode
responses were calibrated in terms of free concentration, and they had
a slope of 56-59 mV for a 10-fold change in
[K+]. Whole cell recordings were obtained by
using the method of Blanton et al. (1989)
. An Axoclamp
2B amplifier (Axon Instruments, Foster City, CA) was used in continuous
voltage-clamp mode. Cells were patched from CA1-CA3 area with 5-7 M
pipettes filled with a solution containing (in mM) 135 K-gluconate,
1-3 KCl, 10 HEPES, 2 Ca(OH)2, 5 EGTA, 2 Mg-ATP,
(pH 7.0 with NaOH). Gramicidin was used in experiments for perforated
patch recording (Ebihara et al. 1995
; Kyrozis and
Reichling 1995
). Gramicidin (Sigma, Deisenhofen, Germany) stock
solution was prepared in DMSO by dissolving 20 mg/ml. The tip of the
filamented borosilicate glass pipette (6-8 M
) was initially filled
with gramicidin-free pipette solution containing (in mM) 135 KCl, 10 HEPES, 2 CaCl2, 5 EGTA (pH 7.0 with NaOH).
Gramicidin was diluted in the solution of the same composition to a
final concentration of 20 µg/ml, and thereafter it was sonicated for
1 min in 10-s periods. No filtering of the solution was performed after
dissolving gramicidin. The remainder of the pipette was then backfilled
with the gramicidin-containing solution. New gramicidin solutions were
made every 2 h. After the cell membrane was patched, the series
resistance was routinely monitored throughout the experiment.
Measurements were started when a series resistance of 300 M
or lower
was reached (approximately 20-30 min after cell attachment). After
1 h, the series resistance stabilized to 60-80 M
. Cell input
resistance was measured by
50 pA currents steps (500 ms). Liquid
junction potential correction (
3 mV) was performed in all membrane
potential values reported in perforated-patch recordings. Electrical
stimuli (5-10 V, 0.1 ms) were delivered by a bipolar electrode placed
in the area CA1.
Drugs
Muscimol, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6-nitro-7-sulfamoylbenzoquinoxaline-2,3-dione (NBQX), and 2-amino-5-phosphonopentanoic acid (AP-5) were purchased from Tocris Cookson (Bristol, UK); bicuculline methiodide was obtained from Sigma. Drugs were dissolved directly to standard solution for bath application.
Data analysis
Spontaneous neuronal activity and stimulus evoked responses were recorded with a TEAC SR-31 tape recorder (data were low-pass filtered at 700 Hz), and digitized off-line at 2 kHz with a National Instruments AT-MIO-16-E-2 A/D board and LabView software (National Instruments). The spike latencies and amplitudes were determined from the field potential bursts with a peak detection algorithm (LabView) and verified visually. Spikes with an amplitude greater than three times the preburst standard deviation were accepted. Decay time constants of fast synaptic currents were estimated by a least-squares exponential fit. Currents appearing as monosynaptic were selected for the time constant estimation from late parts of the polysynaptic bursts. Data are given as mean ± SE. Care and use of animals conformed to the guidelines of the Helsinki University Animal Care Committee.
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RESULTS |
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AMPA-R-mediated CA3-CA1 transmission in P0-P2 hippocampus
Field potential recordings from stratum pyramidale in the area CA3
of P0-P2 rat hippocampal slices revealed negative-going shifts (interval 16 ± 3 s, n = 24 slices)
that were accompanied by bursts of 2.9 ± 0.1 spike-like
deflections (n = 106 bursts). In recordings from the
stratum radiatum the extracellular spikes were typically reversed to
positive-going changes, indicating that they were presumably generated
by a discharge of a subset of CA3 pyramidal cells (Fig.
1, A and B). To
determine whether the extracellular spikes reflect unit activity
(action potentials of single neurons) or synchronous discharges in a
neuronal ensemble, we performed paired field potential recordings using
100-µm interelectrode distance in CA3 s. pyramidale
(n = 3 slices). Cross-correlation analysis showed a
peak with ±1-ms phase lag indicating coincident spikes (c.f.
Draguhn et al. 1998) (n = 43 averages;
Fig. 1C). These results indicate periodic bursts of
synchronous firing in P0-P2 rat CA3 pyramidal cell
population.
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To examine the synaptic input to CA3 and CA1 neurons, we used the whole
cell voltage-clamp technique with a low concentration of chloride (1-3
mM) in the pipette filling solution. The glutamate AMPA-kainate-type
(hereafter referred to as AMPA) inward currents were studied in cells
clamped close to the reversal potential of
GABAA-R-mediated currents
(EGABAA, 80 mV) (c.f. Khazipov et al. 1997
). Inward unitary AMPA-R PSCs had a decay time constant (
decay) <3 ms and they were blocked by CNQX (20 µM) or NBQX (10 µM; n = 10 and 4, respectively), as
illustrated in Fig. 2, A and B. Recordings close to EGABAA showed
that AMPA-R PSCs in CA3 neurons were infrequent (0.20 ± 0.13 unitary PSCs/s
1, range 0.05-0.55
s
1) between the CA3 field potential bursts but
that they became strongly augmented when the bursts occurred. These
short periods (<500 ms) of coherent discharge in the pyramidal layer
were accompanied by a barrage of 7.6 ± 0.2 unitary AMPA-R PSCs in
CA3 neurons (n = 260 bursts, 10 cells). On the average,
73 ± 5% of the total AMPA-R-mediated input consisted of the
PSCs during the bursts (n = 10; Fig. 2B).
CNQX (20 µM) as well as NBQX (10 µM; 10 min; n = 10 and n = 4, respectively) abolished the CA3 field
potential bursts and strongly attenuated spontaneous
GABAA-R-mediated PSCs (see Bolea et al.
1999
; Gaiarsa et al. 1991
; Garaschuk et
al. 1998
; Hollrigel et al. 1998
; Strata
et al. 1997
), thus speaking for an active role of
AMPA-R-mediated recurrent excitation and recruitment of GABAergic
interneurons at P0-P2 (see Fig. 2A).
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To examine spontaneous activation of the excitatory synapses from CA3 pyramidal cells into CA1, we made simultaneous recordings of CA3 field potentials and of postsynaptic currents in whole cell clamped CA1 neurons (n = 4). As illustrated in Fig. 2C, CA1 neurons displayed a barrage of AMPA-R-mediated PSCs during the CA3 field potential burst. The bursting activity in CA1 is generated in CA3, as demonstrated by the disappearance of CA1 bursts after surgical isolation of CA3 from CA1 (Fig. 2D). The autocorrelation analysis from 72 barrages showed that AMPA-R PSCs were imposed to CA1 neurons with 10-50-ms intervals (Fig. 2E).
Interneurons inhibit CA3 pyramidal cells via GABAA-Rs in the P0-P2 hippocampus
On antidromic stimulation of the CA3-CA1 Schaffer collateral pathway in P0-P1 hippocampal slices, a single-pulse stimulus elicited in the CA3 field potential a response similar to the spontaneous bursts (2.2 ± 0.3 spikes; n = 46 bursts, 6 slices). As shown in Fig. 3A, after blockade of GABAA-R by bicuculline for >5 min (10 µM), the same stimulus generated a burst with 4.8 ± 0.5 spikes (P < 0.01, t-test). After a 15-min wash out of bicuculline and restoration of GABAA-R-mediated transmission, the number of extracellular spikes was reestablished to the control value of 1.9 ± 0.2 (n = 41 bursts, 5 slices).
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Gramicidin perforated-patch recordings demonstrated a network-driven
13.0 ± 1.0-mV (n = 30 cells) depolarization of
the CA3 neurons from the resting membrane potential
(Em = 67.5 ± 0.7 mV) during
the spontaneous field potential burst. A single-pulse antidromic
stimulation of Schaffer collaterals elicited a similar depolarization
(l3.5 ± 2.0 mV; Em =
69 ± 1.0 mV) with 1.6 ± 0.3 (range 0-4) action potentials
(n = 12 cells). In six neurons the response was
strongly intensified in bicuculline (10 µM) yielding a 28.0 ± 2.5-mV depolarization and generating 3.7 ± 0.7 (range 2-7)
action potentials. In three cells the network activity was abolished,
and only a monosynaptic response was elicited (16.0 ± 2.5 mV,
0-1 action potentials). In three cells no response was evoked in
bicuculline. These results demonstrate that a powerful inhibitory
effect of GABAA-R inputs on the glutamatergic CA3
recurrent excitation can frequently be observed in the hippocampus as
early as P0-P1. Figure 3B illustrates
augmentation of network-driven excitation of CA3 neurons by the
GABAA-R antagonist bicuculline in
P0-P1 hippocampal slices.
AMPA-R-mediated transmission is better preserved in the neonate rat
whole hippocampus preparation when compared with slices (Khalilov et al. 1997). Further, slices from septal and
temporal sites may differ in their properties of network activity
(Leinekugel et al. 1998
). Thus, to better appreciate the
contribution of AMPA-R-mediated transmission to the spontaneous
network activity, some of the experiments were performed using the
intact hippocampus in vitro preparation. Field potential recordings
from CA3 area showed that the spontaneous events were similar to those
occurring in slices. In P0 (n = 4) as well
as in P1 (n = 7) preparations, exposure to
bicuculline (10 µM) strongly augmented the spontaneous field potential responses during a 10-min exposure (see Fig.
4, A and B). In the
presence of bicuculline, the occurrence of the bursts (at 96 ± 40-s interval) was not seemingly influenced by the NMDA-R antagonist
AP-5 (40 µM) but became readily blocked by the AMPA-R antagonist CNQX
(20 µM; n = 3; data not shown). Activity-induced accumulation of extracellular potassium
([K+]o) serves as useful
indicator of epileptiform discharge (e.g., Swann et al.
1986
). Therefore, we performed some experiments with bicuculline using ion-selective double-barrel microelectrode recording of field potential and
[K+]o. These experiments
revealed that periods of synchronous activity in control conditions
were linked to a small increase in
[K+]o from the basal 3.5 mM to ~3.7 mM (n = 6 preparations), but epochs in
bicuculline were accompanied by prominent accumulation of
[K+]o up to 6.0 mM (n = 6 preparations), as depicted in Fig. 4A.
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After blockage of GABAA-Rs, whole cell recordings
demonstrated a strong increase in the amplitude of an average AMPA-R
PSC in the CA3 neurons from 27.0 ± 2.0 pA in control to 68.0 ± 3.5 pA in bicuculline (n = 112 and 182 PSCs,
respectively, P < 0.01, t-test). However,
the temporal properties of unitary PSCs were similar to control
(decay < 3 ms). This indicates that blockage of GABAA-R-mediated transmission strongly
enlarged the population of glutamatergic neurons firing synchronously
in the bursts (Fig. 4, B and C). Thus, the
results presented here suggest also that, as in the adult (Miles
and Wong 1987
), GABAA-R-mediated
activity plays a critical role in limiting network excitability in the CA3 area of newborn rat hippocampus (see Swann et al.
1989
).
Activation of GABAA-R by muscimol inhibits CA3 pyramidal cell firing while depolarizing postsynaptic cells
As shown by Fiszman et al. (1990), submicromolar
concentrations of the agonist, muscimol, induce a bicuculline-sensitive
depolarization in embryonic and early postnatal hippocampal neurons.
Field potential deflections and the accompanying spikes were abolished
by bath-applied muscimol (0.1-5 µM), as illustrated in Fig.
5A (n = 14).
However, during the early wash in of the agonist, the frequency of the bursts was transiently increased. With a low concentration (10 nM) of
muscimol, the rate of the field potential bursts was tonically increased to 155 ± 11% from control during a 10-min exposure
(mean occurrence in control 9.5 ± 0.6 bursts/min,
n = 5). After a >15-min wash out, the occurrence of
the bursts was restored to 90 ± 10% (n = 5). The
effect of muscimol was reversible at higher concentrations as well, and
the field potential deflections reappeared after a 10-min wash out.
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Whole cell recordings from CA3 neurons showed that bath-applied
100 nM muscimol (5 min) induced a steady outward current at clamping
potentials 30 mV, much positive to EGABAA
(
80 mV; n = 4). In parallel, the occurrence of
unitary GABAA-R-mediated PSCs increased from 0.66 ± 0.16 s
1 in the control to 1.43 ± 0.29 s
1 in 100 nM muscimol, thus indicating an increase in
spontaneous activity of GABAergic synapses (n = 4 slices; P < 0.05; t-test). These
results are illustrated in. Fig. 5, B and
C. Gramicidin perforated-patch recordings showed that
similar (5-min) exposure to 100-200 nM muscimol induced 8.0 ± 0.8 mV (range 2-13 mV) depolarization of CA3 neurons from their
resting membrane potential
67.0 ± 1.1 mV (n = 16; Fig. 5D). In the presence of bicuculline (10 µM)
the effect of muscimol was blocked (n = 3).
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DISCUSSION |
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It is generally accepted that endogenous activity plays a crucial
role in the formation of developing neuronal networks (see Goodman and Shatz 1993). In this context, the
spontaneous network activity in the newborn rat hippocampus has
received much attention (c.f. Hanse et al. 1997
;
Traub et al. 1998
). Yet, mechanisms underlying the early
network activity in the hippocampus have remained poorly understood
(c.f. O'Donovan 1999
). In the current study, we first addressed the question of whether the fast spontaneous spiking seen in
CA3 represents synchronous firing of pyramidal neurons. Having
established that this is the case, we then examined the synaptic
mechanisms driving these spontaneous events in CA3. In parallel, we
studied the synaptic inputs to CA1 to reveal the functional
connectivity between CA3-CA1 at birth. Finally, we investigated the
nature of GABAAergic transmission in the
endogenous activity of the newborn rat hippocampus.
The following are our central findings. 1) As early as P0, periodic activation of glutamate AMPA-Rs gives rise to synchronous recurrent bursting of CA3 pyramidal cells. The occurrence of AMPA PSCs is mainly restricted to these bursts. 2) During the coherent discharging of CA3, AMPA PSCs are also seen in CA1 pyramidal cells, where the PSCs display a barrage at high (>20-Hz) frequency. 3) GABAA-R-mediated transmission, despite its depolarizing postsynaptic action, is already a major inhibitory mechanism of the synchronous pyramidal cell activity at birth in the rat hippocampus.
AMPA-R-activation underlies the synchronous bursts of CA3 pyramidal cells and conveyes the excitation to CA1 at P0-P2
Notably, most of the studies investigating AMPA-R-mediated
transmission in the newborn brain have been performed at room
temperature using a single-pulse stimulation paradigm (c.f.
Bolshakov and Siegelbaum 1995; Durand et al.
1996
; Hsia et al. 1998
). However, lowering the
experimental temperature is known to effectively attenuate evoked
responses in CA3-CA1 synapses (Igelmund and Heinemann 1995
). Also, glutamate spillover and the consequent
extrasynaptic activation of NMDA-Rs is critically dependent on
temperature, the lowered temperature thus giving a biased view of the
relative contributions of AMPA and NMDA-Rs to synaptic transmission
(Asztely et al. 1997
). In this study, we decided to
investigate the synaptic inputs in immature CA3-CA1 circuitry during
spontaneous bursts. Interestingly, it was very recently pointed out
that a relevant way to study synaptic functioning in a neuronal network
is to use stimulation patterns mimicking the spike trains driving
synapses in vivo (Dobrunz and Stevens 1999
), thus making
the current approach even more appropriate.
In the light of the existing literature, the presence of strong
AMPA-R-mediated inputs to pyramidal cells at P0-P2 was
rather surprising. Intriguingly, the AMPA PSCs were temporally
restricted to the spontaneous bursts, and only rarely did AMPA PSCs
occur outside these events. Furthermore, the synaptic currents in CA1 showed a rhythmicity at gamma (20-100 Hz) frequencies reflecting a
synchronized output of the neonate CA3 circuitry (see Palva et
al. 1999). In the developing brain the spontaneous activity may
be critical for the formation of synaptic contacts (Katz and Shatz 1996
). Neuronal bursts capable of inducing synaptic
modifications would thus be an effective way to convey synaptic
transmission in the emerging CA3-CA1 circuitry. CA3 population activity
gives rise to long-lasting GABAergic conductance in CA1 neurons, yet with a tens- or hundreds-of-millisecond delay (Strata et al.
1997
). In terms of synapse induction, the sharply synchronous
AMPA-R-driven bursts could allow for finer tuning of neuronal connections.
GABAA-R-mediated inhibition in the newborn rat hippocampus
In the immature rat hippocampus GABAA-R
activation by afferent stimulation may evoke spiking in the
postsynaptic cell (Khazipov et al. 1997). Also, the
GABAAergic depolarization in the neonate hippocampal neurons is well established (Ben-Ari et al.
1989
; Rivera et al. 1999
). Our current
experimental approaches showed that although depolarizing in nature,
GABAA-R activation can be strongly inhibitory
also in the newborn rat pyramidal cells.
This conclusion is based on several findings. 1) The
amplitude of the AMPA-R PSC peak, which is determined by integration of
the synaptic inputs and therefore represents a good measure of
synchronously activated presynaptic glutamatergic neurons, was
increased in bicuculline. Also, the duration of the spontaneous CA3
bursts was much longer in the absence of GABAergic inhibition. 2) Experiments with antidromic stimulation of CA3 recurrent
excitatory loop showed that at P0-P1
GABAAergic transmission already restricts the
network-driven excitatory input to CA3 neurons. 3)
Simultaneous recordings of extracellular potential and
[K+]o demonstrated that
inhibition of GABAA-R-mediated transmission gave
rise to epileptiform synchronous activity. The effect of the
GABAA-R antagonist was even more accentuated in
the intact hippocampus preparation, probably because of better
preservation of the glutamatergic circuitry (Khalilov et al.
1997). This finding is in perfect agreement with the
observation that bicuculline induces seizures in the newborn rat in
vivo (Daval and Sarfati 1987
). 4)
AMPA-R-mediated transmission and the accompanying spike bursts were
abolished on activation of GABAA-Rs by muscimol.
However, whole cell recordings revealed that on muscimol application
the occurrence of GABAA-R PSCs was increased,
probably because of muscimol-induced depolarization and a consequent
increase of GABA release from interneurons.
Regarding the mechanisms of GABAA-R-mediated
inhibition in the neonate neurons, in all experiments in area CA3
gramicidin-perforated recordings showed only depolarizing responses to
the GABAA-R agonist muscimol. Therefore the
possibility that inhibition of the network would be mediated by a
subpopulation of the neurons in which GABA already induces
hyperpolarization seems unlikely. GABAergic inhibition by depolarizing
GABAA conductances is known to occur in neonate and adult rat dentate granular cells (Hollrigel et al.
1998; Staley and Mody 1992
). Thus, although
sometimes capable of triggering a spike, the
GABAA-R activation would not easily allow for
tight long-range synchronization, and thus coherent population firing, to ensue because of the effective shunting of the postsynaptic membrane. Further, the GABA-induced Ca2+ influx
could uncouple gap junctions and attenuate the fast network oscillations requiring electrical coupling in neonatal hippocampus (Draguhn et al. 1998
; Palva et al. 1999
;
Strata et al. 1997
). The high occurrence of
GABAA-R PSCs in P0-P2 neurons
indicates that spontaneous firing and thus "tonic" inhibition
(Häusser and Clark 1997
) is already a common
feature of GABAergic neurons at this age. Similar strategies for the
control of synaptic integration may thus be used in the newborn and in
the adult brain.
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ACKNOWLEDGMENTS |
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This study was supported by grants from the Academy of Finland and from the Sigrid Juselius Foundation.
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FOOTNOTES |
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Address for reprint requests: T. Taira, Dept. of Biosciences, Division of Animal Physiology, P. O. Box 17 (Arkadiankatu 7), FIN-00014 University of Helsinki, Finland.
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 1 June 1999; accepted in final form 16 September 1999.
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REFERENCES |
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