Neurobiologie et Diversité Cellulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7637, Ecole Supérieure de Physique et de Chimie Industrielles de la ville de Paris, 75231 Paris Cedex 5, France
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ABSTRACT |
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Angulo, Maria Cecilia,
Jean Rossier, and
Etienne Audinat.
Postsynaptic Glutamate Receptors and Integrative Properties
of Fast-Spiking Interneurons in the Rat Neocortex.
J. Neurophysiol. 82: 1295-1302, 1999.
The
glutamate-mediated synaptic responses of neocortical pyramidal cell to
fast-spiking interneuron (pyramidal-FS) connections were studied by
performing paired recordings at 30-33°C in acute slices of 14- to
35-day-old rats (n = 39). Postsynaptic fast-spiking (FS) cells were recorded in whole cell configuration with a patch pipette, and presynaptic pyramidal cells were impaled with sharp intracellular electrodes. At a holding potential of 72 mV (near the
resting membrane potential), unitary excitatory postsynaptic potentials
(EPSPs) had a mean amplitude of 2.1 ± 1.3 mV and a mean width at
half-amplitude of 10.5 ± 3.7 ms (n = 18).
Bath application of the N-methyl-D-aspartate
(NMDA) receptor antagonist D(
)2-amino-5-phosphonovaleric acid (D-AP5) had minor effects on both the amplitude and
the duration of unitary EPSPs, whereas the
-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainate
receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) almost
completely blocked the synaptic responses. In voltage-clamp mode, the
selective antagonist of AMPA receptors
1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI 53655; 40-66 µM) blocked 96 ± 1.9% of
D-AP5-insensitive unitary excitatory postsynaptic currents
(EPSCs), confirming the predominance of AMPA receptors, as opposed to
kainate receptors, at pyramidal-FS connections (n = 3). Unitary EPSCs mediated by AMPA receptors had fast rise times
(0.29 ± 0.04 ms) and amplitude-weighted decay time constants
(2 ± 0.8 ms; n = 16). In the presence of intracellular spermine, these currents showed the characteristic rectifying current-voltage (I-V) curve of
calcium-permeable AMPA receptors. A slower component mediated by NMDA
receptors was observed when unitary synaptic currents were recorded at
a membrane potential more positive than
50 mV. In response to short
trains of moderately high-frequency (67 Hz) presynaptic action
potentials, we observed only a limited temporal summation of unitary
EPSPs, probably because of the rapid kinetics of AMPA receptors and the
absence of NMDA component in these subthreshold synaptic responses. By
combining paired recordings with extracellular stimulations
(n = 11), we demonstrated that EPSPs elicited by
two different inputs were summed linearly by FS interneurons at
membrane potentials below the action potential threshold. We estimated
that, in our in vitro recording conditions, 8 ± 5 pyramidal cells
(n = 18) should be activated simultaneously to make
FS interneurons fire an action potential from
72 mV. The low level of
temporal summation and the linear summation of excitatory inputs in FS
cells favor the role of coincidence detectors of these interneurons in
neocortical circuits.
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INTRODUCTION |
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In the CNS, the dendrites of different neuronal
types can receive thousands of synaptic inputs. The passive cable
theory predicts that the sum of these inputs would be algebraic if they
are electrically isolated or attenuated if they are electrically close
(see Spruston et al. 1994, for review). Indeed, the
proximity between synaptic inputs can lead to a change in the ionic
driving force or to a shunt of the synaptic currents by decreasing the
input resistance of the membrane. In addition to these passive
properties, active membrane mechanisms can also influence the
processing of information, and the final integration of synaptic inputs
depends on nonlinear interactions dictated by the distribution and
activation of voltage-gated channels in dendrites (see Yuste and
Tank 1996
, for review).
Although theoretical models aiming to understand passive and active
integrations of different synaptic inputs exist (Jaslove 1992; Mel 1993
; Softky 1994
),
reliable experimental tests of their predictions are sparse. In
cortical structures, the summation of subthreshold excitatory synaptic
inputs by pyramidal cells has recently been studied (Cash and
Yuste 1998
; Urban and Barrionuevo 1998
), but
there is still a lack of information about the integrative properties
of other types of cortical neurons. In addition, most experiments have
been done with extracellular stimulation, i.e., by activating
simultaneously several different presynaptic fibers, or by mimicking
synaptic inputs with local applications of exogenous glutamate
(Cash and Yuste 1998
; Langmoen and Andersen
1983
; Skydsgaard and Hounsgaard 1994
;
Urban and Barrionuevo 1998
). Under these conditions, the
contribution of active mechanisms to the integration of synaptic
potentials generated by the activation of unitary inputs is not easily resolved.
At excitatory glutamatergic synapses, the postsynaptic
depolarization is primarily due to the activation of ionotropic
-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and
N-methyl-D-aspartate (NMDA) receptors. The
block of NMDA channels by magnesium ions at hyperpolarized potentials
results in the marked nonlinear properties of these receptors. The
integration of a given synaptic excitatory input will therefore be
affected by the presence of nearby voltage-gated channels (see above)
and also by the relative proportions of NMDA and AMPA receptors at these synapses (Thomson 1997
; see Thomson and
Deuchars 1995
for review).
The aim of the present work was to examine by means of paired
recordings the characteristics of unitary excitatory connections between pyramidal cells and fast-spiking (FS) interneurons, a subpopulation of nonpyramidal cells (Cauli et al. 1997;
Kawaguchi and Kubota 1993
; McCormick et al.
1985
). We first characterized the postsynaptic properties of
excitatory postsynaptic potentials (EPSPs) and excitatory postsynaptic
currents (EPSCs) mediated by glutamate receptors at pyramidal cell to
FS interneuron connections in layer V of rat motor cortical slices. We
then analyzed how different excitatory inputs were integrated by FS
cells. We found that the summation of excitatory synaptic responses was
linear, and we could therefore estimate the number of pyramidal cells needed to be activated to make a FS cell fire.
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METHODS |
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Slice preparation
Wistar rats (20 ± 6 postnatal day-old; range 14-35) were
anesthetized by an intraperitoneal injection of Ketamine (65 mg/kg) and
Xylazin (14 mg/kg) and decapitated. Brains were quickly removed and
300-µm thick parasagittal sections of cerebral motor cortex were
prepared as previously described (Cauli et al. 1997).
The slices were incubated for 1 h in a physiological extracellular saline solution containing (in mM) 121 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 20 glucose, and 5 pyruvate and bubbled with a mixture of 95% O2-5%
CO2. For recordings, they were transferred to a
chamber and perfused at 1-2 ml/min with the same physiological extracellular saline solution at 30-33°C.
Paired recordings
Pyramidal-FS connections were examined by paired recordings as
previously described (Angulo et al. 1999). Briefly,
postsynaptic FS interneurons in layer V of the motor cortex were
recorded with patch pipettes (resistance 3-5 M
) pulled from
borosilicate glass tubing and filled with an internal solution
containing (in mM) 144 K-gluconate, 3 MgCl2, 0.2 EGTA, and 10 HEPES (pH 7.2-7.4, 300 mosM). In five experiments, we
also included 4 mM ATP and 0.4 mM GTP. Postsynaptic FS cells were
initially visualized using videomicroscopy with Nomarski optics under
infrared illumination (Stuart et al. 1993
) and later
identified by the characteristics of their action potential discharges
according to the procedure reported by Cauli et al.
(1997)
. Only FS neurons with multipolar shapes and membrane
potentials more negative than
60 mV were included in the sample. All
membrane potentials were corrected for a junction potential of
12 mV.
After whole cell recordings were established, presynaptic pyramidal
cells were impaled with sharp intracellular microelectrodes filled with
3 M KCl (resistance 40-80 M) and identified by their characteristic
action potential firing (Connors and Gutnick 1990
; McCormick et al. 1985
). Unitary EPSCs and EPSPs were
induced in FS cells by triggering action potentials in presynaptic
pyramidal cells with depolarizing pulses (3 ms, 1.5 nA). Means of
elicited synaptic responses were obtained by averaging the traces after the presynaptic action potential was aligned using automatic peak detection. The stability of the recordings during the time course of
the experiment was tested by plotting the amplitudes of individual synaptic responses against time. Only connections showing stable EPSC
or EPSP amplitudes and probablities of response were further analyzed.
From 50 to 100 traces were averaged to measure parameters of mean
synaptic responses.
A Cs-gluconate internal solution was used to minimize the voltage-clamp
error in experiments aimed to study the current-voltage (I-V) curves of AMPA and NMDA receptor synaptic components
of unitary EPSCs. However, to obtain the firing pattern of postsynaptic FS cells, the tip of the patch pipette was filled with the K-gluconate internal solution (see above) and backfilled with a solution of the
following composition (in mM): 125 Cs-gluconate, 3 MgCl2, 10 EGTA, 10 HEPES, 4 ATP, and 0.4 GTP (pH
7.2-7.4, 300 mosM). Spermine (100 µM) was also included to maintain
the voltage dependence of AMPA receptors (Bowie and Mayer
1995; Kamboj et al. 1995
; Koh et al.
1995
). The dialysis of the recorded cell with the Cs-gluconate solution could be observed from the broadening of the action potentials after ~15 min of recordings.
Paired recordings were combined with extracellular stimulations (100 µs; 0.02-0.04 mA) using a monopolar electrode placed in layer III or VI of the motor cortex to determine how the responses elicited by two independent excitatory inputs were integrated by FS interneurons. The position of the extracellular electrode and the stimulation intensities were adjusted to elicit pure excitatory responses; in our recording conditions, inhibitory synaptic currents were outwardly directed and thus could be distinguished from excitatory responses. The extracellular electrode was also used to establish the action potential threshold of FS interneurons. Stimulation intensities were increased until synaptic potentials induced in FS cells triggered an action potential. The threshold was measured when 50% of the responses elicited an action potential.
Data collection and analysis
Whole cell current-clamp (mode I-Clamp fast) and voltage-clamp recordings of postsynaptic FS cells were obtained using a patch-clamp amplifier (Axopatch 200A, Axon Instruments) and filtered at 5 or 2 kHz. Series resistances were monitored throughout the experiments and compensated only in experiments aimed to assess the independence of two inputs. Intracellular current-clamp recordings of presynaptic pyramidal cells were obtained using an intracellular amplifier (Neuro Data, Instruments Corp.). Digitized data were acquired and analyzed using Acquis1 software (Gérard Sadoc, CNRS, Gif-sur-Yvette, France).
Means of evoked unitary synaptic responses were obtained by averaging the traces after the presynaptic action potentials were aligned using automatic peak detection. Traces not showing postsynaptic responses larger than 150% of the noise level were considered as failures. The mean rise times (20-80%) of EPSCs and EPSPs were calculated by averaging the synaptic responses excluding failures. All statistical data are given as means ± SD.
The AMPA receptor-mediated synaptic current was obtained by
subtracting the insensitive 6,7-dinitroquinoxaline-2,3-dione (DNQX) or
2,3-dioxi-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX) NMDA receptor-mediated synaptic current from the
control current. The rectification index (RI) of the AMPA receptors was calculated from I-V curves with the following expression
(Angulo et al. 1997; Ozawa et al. 1991
)
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The kinetics of AMPA and NMDA receptor components were determined
by fitting the EPSCs with one or two exponential functions at different
membrane potentials. When the decay was best fitted by two exponential
functions, an amplitude-weighted decay time constant () of AMPA
receptor-mediated synaptic currents was calculated by summing the two
time constants, weighted by their fractional amplitude contribution.
For comparison between cells,
NMDA were determined by fitting the decay of the slow component of the EPSC recorded at holding potentials between +38 and +48 mV in control experiments (without DNQX or NBQX). Because the kinetics of the AMPA
receptor current is faster than that of the NMDA receptor current, the
contamination by the AMPA receptor component during the decay phase of
the NMDA receptor component is negligible.
CNQX, DNQX, and NBQX were purchased from Tocris Cookson (Bristol) and the GYKI 53655 was a gift from Egis (Budapest).
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RESULTS |
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The glutamate-mediated synaptic responses of neocortical pyramidal cell to FS interneuron connections were examined by using paired recordings (n = 39). Presynaptic pyramidal cells were impaled with sharp intracellular microelectrodes, and unitary EPSCs and EPSPs were recorded in FS cells in the whole cell configuration with patch pipettes in layer V of the motor cortex (see METHODS).
Presynaptic and postsynaptic cells were first identified by the
kinetics of their action potential firing. Figure
1A1 illustrates the firing
behaviors of a pyramidal cell (top panel) and a FS interneuron (bottom panel), which were synaptically coupled.
Presynaptic pyramidal cells showed slow action potentials and a strong
and rapid accommodation of their action potential firing induced by depolarizing current pulses (see Connors and Gutnick
1990, for review). Postsynaptic FS interneurons had fast action
potentials and limited accommodation of their action potential firing
at all tested stimulation intensities (Cauli et al.
1997
; Kawaguchi 1995
; Kawaguchi and
Kubota 1993
). A detailed description of the firing kinetics and
morphology of pyramidal-FS connections considered in the present work
has been previously reported (Angulo et al. 1999
).
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Unitary AMPA and NMDA receptor-mediated postsynaptic responses at pyramidal-FS connections
Unitary EPSPs were induced in FS interneurons by eliciting action
potentials in the presynaptic pyramidal cells at a stimulation rate of
1 Hz (Fig. 1A2). Postsynaptic potentials were characterized by brief latencies (0.6 ± 0.2 ms, mean ± SD,
n = 13) and fast rise times (0.76 ± 0.3 ms,
n = 18; see METHODS). At a
membrane potential of 72 mV, the average amplitude and width at
half-amplitude of the EPSPs had mean values of 2.1 ± 1.3 mV
(0.35-5 mV) and 10.5 ± 3.7 ms (4.6-18.5 mV; n = 18), respectively. We did not find any correlation between the rise
time and the EPSP amplitude or width at half-amplitude (data not
shown), suggesting that the variability of the EPSPs was not markedly
caused by cable attenuation of the responses over the dendrites of FS cells.
We then tested the effect of different glutamate receptor antagonists
to identify the postsynaptic receptors underlying the unitary EPSPs at
pyramidal-FS connections. Near the resting membrane potential of FS
interneurons (76 ± 5 mV; n = 38), the
AMPA/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) almost completely blocked the EPSPs (Fig. 1B1). In
contrast, bath application of the NMDA antagonist
D(
)2-amino-5-phosphonovaleric acid (D-AP5; 50 µM) had only minor effects on the EPSPs (data not shown). This
antagonist did not affect significantly either the amplitude or the
width at half-amplitude of the EPSPs when the postsynaptic cells were
depolarized to
60 mV (P > 0.05, Wilcoxon t-test; Fig.1B2). More depolarized membrane
potentials were not tested in current-clamp because FS interneurons had
a low action potential threshold (
55 ± 3 mV, n = 6; see METHODS). These results indicate that, at
subthreshold postsynaptic membrane potentials, NMDA receptors do not
contribute significantly to the synaptic transmission at pyramidal-FS connections.
To further characterize the postsynaptic receptors involved at these
connections, we studied the kinetics and voltage dependence of unitary
EPSCs. Figure 2A illustrates
the average unitary EPSCs of a FS interneuron at different membrane
potentials. At a holding potential of 72 mV, the average EPSC
excluding failures rose and decayed rapidly. In most of the
connections, the decay was best fitted with a double exponential
function showing a predominant fast component (Fig. 2D). For
16 connections, the rise time and amplitude-weighted decay time
constant of the fast synaptic component had mean values of 0.29 ± 0.04 ms and 2 ± 0.8 ms, respectively (see METHODS).
At this potential, 10 µM of the AMPA/kainate receptor antagonists
NBQX or DNQX blocked 98 ± 2% of the synaptic currents (n = 5; Fig. 2B). The selective antagonist
of AMPA receptors GYKI 53655 (40-66 µM) also blocked 96 ± 1.9% of D-AP5-insensitive responses, confirming the
predominance of AMPA receptors, as opposed to kainate receptors, at
pyramidal-FS connections (n = 3; data not shown).
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As shown in Fig. 2C, the I-V plot of the AMPA
receptor EPSCs showed a pronounced inward rectification with a RI of
0.38 ± 0.1 (n = 9, see METHODS). The
fast kinetics and the inward rectification of this EPSC component was
consistent with the activation of calcium-permeable AMPA receptors
lacking GluR2 subunits (see Jonas and Burnashev 1995,
for review; Angulo et al. 1997
; Lambolez et al.
1996
).
At positive holding potentials, unitary EPSCs showed an additional
slower component (Fig. 2A, inset). This component was
insensitive to NBQX (Fig. 2B) and had a mean rise time of
7 ± 0.5 ms and a decay time constant of 52 ± 9 ms
(n = 4; Fig. 2E). The I-V plot of
the average slow EPSC had a region of negative slope between 60 mV
and
20 mV (Fig. 2C), characteristic of NMDA
receptor-mediated currents (Mayer et al. 1984
;
Nowak et al. 1984
).
We estimated the relative proportion of NMDA and AMPA
receptor-mediated synaptic components by calculating the ratio of
gNMDA(+40
mV)/gAMPA(60 mV), which
was 19 ± 1% (n = 3; see METHODS).
These results indicate that calcium-permeable AMPA receptors and NMDA receptors are present at pyramidal-FS connections. However, when the membrane potential of the interneuron is below the action potential threshold, synaptic transmission is mainly mediated by AMPA receptors.
Integration of excitatory inputs in FS interneurons
We previously reported that either depression or facilitation of
synaptic currents can occur at pyramidal cell to FS interneuron connections when pairs of presynaptic action potentials are applied (Angulo et al. 1999). We therefore tested how unitary
EPSPs triggered by train of moderately high-frequency presynaptic
action potentials were temporally integrated at connections showing
paired-pulse depression (Fig.
3A) and paired-pulse
facilitation (Fig. 3B). At both types of connections, we
observed a limited temporal summation of unitary EPSPs when presynaptic
action potentials were delivered every 15 ms at a stimulation rate of
0.2 Hz (Fig. 3). The rapid time course of unitary EPSPs prevented
significant temporal summation at these connections. Together with the
small mean amplitude of unitary EPSPs, the lack of substantial temporal
summation indicates that several presynaptic pyramidal cells must fire
simultaneously to induce the discharge of a FS interneuron.
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We therefore determined how FS cells integrate the responses elicited by two independent inputs by combining paired recordings with extracellular stimulations using a monopolar electrode placed in layer III or VI of the motor cortex.
We first tested the independence of these two inputs in voltage-clamp mode using two different procedures. First, as shown in Fig. 4A1, the EPSC induced by the extracellular stimulation (extracellular EPSC) and the EPSC elicited by the activation of the presynaptic pyramidal cell (unitary EPSC) were first elicited alternately and then simultaneously. The predicted sum of the two EPSCs elicited alternately was compared with the experimental sum obtained by simultaneous stimulation of the two inputs (Fig. 4A2). The independence of both inputs was assumed when there was <10% difference between the amplitude of the predicted and the experimental sum of the EPSCs (n = 5; Fig. 4A2). However, this protocol could lead to exclusion of cases where nonlinear summation of independent inputs arises from a large shunt of the membrane caused by one of the inputs. Therefore the independence of the inputs was tested in six other experiments with a second protocol. The paired-pulse response characteristics of the unitary EPSC were first examined using pairs of single action potentials elicited in the presynaptic pyramidal cell by applying two short depolarizing current pulses separated by 50 ms (Fig. 4B1; top light traces). In all tested pyramidal-FS connections, the amplitude of the first EPSC (EPSC1) was larger than that of the second EPSC (EPSC2), indicating that these connections displayed a paired-pulse depression in our recording conditions (Fig. 4B1; top light traces). The mean paired-pulse ratio (EPSC2 amplitude/EPSC1 amplitude) was 0.7 ± 0.18. After the paired-pulse characteristics of the unitary connections were established, we determined whether the unitary input was depressed by a preceding extracellular stimulation (Fig. 4B1; bottom dark traces). The extracellular input was therefore stimulated 50 ms before testing the unitary input (Fig. 4B1; bottom dark traces). The independence of both inputs was assumed when there was <10% difference between the unitary EPSC1 obtained with or without a preceding extracellular stimulation (Fig. 4B1, inset).
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Once the independence of the two inputs was established, we switched to the current-clamp mode to compare the predicted sum corresponding to the algebraic summation of the two EPSPs elicited alternately to the experimental sum obtained by stimulating the two inputs simultaneously (Fig. 4, A and B, right). As shown in the examples of Fig. 4, the EPSPs of the predicted and experimental sums were well superimposed, suggesting a linear summation of the two synaptic responses (Fig. 4, A4 and B3).
For 11 recorded FS interneurons, the amplitude and width at half-amplitude of the predicted and experimental sums obtained at different membrane potentials were not significantly different and were correlated with linear coefficients of regression of 0.99 and 0.93, respectively (Fig. 5, A1 and A2; P > 0.05, Wilcoxon t-test). These data indicate that, in FS cells, the integration of the responses elicited by independent inputs was linear and that the kinetics of EPSPs did not change during the summation of different responses.
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To determine whether the linearity of the experimental sum of the EPSPs
was dependent on the membrane potential, we plotted the ratio
experimental/predicted sum amplitudes as a function of the potential
(from 85 to
57 mV; Fig. 5B1). We did not observed any
significant correlation between this ratio and either the holding
potential (
) or the potential at the peak of the response (
;
coefficients of regression of
0.01 and
0.02, respectively). The
ratio experimental/predicted EPSP amplitude was also independent of the
amplitude of the unitary EPSP (Fig. 5B2; coefficient of regression of
0.22) and of the extracellular EPSP (data not shown; coefficient of regression of 0.2) and had a mean value of 103 ± 6% (n = 11). These results show that the responses
elicited by two independent inputs were integrated linearly by FS cells even near the action potential threshold.
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DISCUSSION |
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Paired recordings in neocortical slices showed that both calcium-permeable AMPA and NMDA receptors mediate the synaptic responses at pyramidal-FS connections. The contribution of NMDA receptors was, however, almost negligible at subthreshold potentials. By combining paired recordings with extracellular stimulations, we also demonstrated that the sum of synaptic responses elicited simultaneously by two independent excitatory inputs in FS cells is algebraic at subthreshold potentials.
Glutamate synaptic receptors at pyramidal-FS connections
It has been shown previously that neocortical FS interneurons
express a relatively low proportion of the AMPA receptor subunit GluR2
(Lambolez et al. 1996). It is now clearly established
from studies on recombinant and native receptors, that AMPA receptors lacking this subunit have a large single-channel conductance, an
inwardly rectifying I-V curve, and a high calcium
permeability (see Jonas and Burnashev 1995
, for review).
Our present data demonstrate that, as established for extrasynaptic
receptors of FS cells (Angulo et al. 1997
), AMPA
receptors contributing to synaptic transmission at pyramidal-FS
connections displays an inward rectification. The good correlation
between the rectification index and the calcium permeability of native
AMPA receptors (Iino et al. 1994
; Itazawa et al.
1997
) allows us to estimate the permeability ratio of
Ca2+ over monovalent cation of AMPA receptors at
pyramidal-FS synapses to be close to 1, i.e., almost 10 times higher
than that of nonrectifying receptors. The activation of pyramidal-FS
synapses is thus always associated with a Ca2+
influx in the postsynaptic FS cell, through AMPA receptors at hyperpolarized membrane potentials and through NMDA receptors at
depolarized potentials (see Fig. 2).
Integration of pyramidal cell inputs in FS interneurons
Unitary EPSPs at pyramidal-FS connections described in the present
work like those reported previously for most of adult neocortical pyramidal-interneuron connections (Buhl et al. 1997;
Deuchars and Thomson 1995
; Thomson 1997
;
Thomson et al. 1993
, 1995
) had a shorter
duration than EPSPs at adult pyramidal-pyramidal pairs (Deuchars
et al. 1994
; Thomson 1997
). Several parameters
such as the substantial contribution of NMDA receptors (Jones
and Baughman 1988
; Thomson et al. 1988
), the
slow decay of AMPA receptor-mediated EPSCs (Hestrin
1993
), and the presence of subthreshold voltage-activated inward currents (Deisz et al. 1991
; Markram and
Sakmann 1994
; Schwindt and Crill 1995
) and cable
properties (Stafstrom et al. 1984
) explain the
relatively long duration of EPSPs recorded in pyramidal cells. These
characteristics facilitate the temporal integration of the unitary
excitatory inputs in pyramidal neurons. In contrast, the faster decay
of AMPA receptor-mediated EPSCs associated with an almost negligible
activation of NMDA receptors at subthreshold potentials contribute to
the short duration of the EPSPs recorded in FS interneurons and
therefore limit the temporal integration in these cells.
Synaptic responses elicited by two independent inputs were integrated
linearly by FS cells at the resting membrane potential and near the
action potential threshold. This linearity was maintained even when the
amplitude of extracellular stimulus elicited EPSPs were substantially
larger than unitary EPSPs (Fig. 4). This indicates that neither
steady-state nor transient depolarizations modify the integration of
synaptic inputs in FS cells. In the CNS, linear summation of excitatory
inputs has been also reported for spinal chord motoneurons
(Skydsgaard and Hounsgaard 1994), and neocortical (Cash and Yuste 1998
) and CA1 hippocampal pyramidal
cells (Langmoen and Andersen 1983
). Recently,
Urban and Barrionuevo (1998)
described sublinear
summations of synaptic responses elicited on the same apical dendrite
of hippocampal CA3 pyramidal cells. The activation of dendritic A-type
potassium channels would explain the reduction of excitatory synaptic
responses of large amplitude (Cash and Yuste 1998
;
Hoffman et al. 1997
; Urban and Barrionuevo
1998
). Transient A-type currents are also present in
neocortical FS interneurons (Massengill et al. 1997
) and
could in principle affect synaptic integration in these cells. However,
even when synaptic responses of large amplitude were evoked with
extracellular stimulation, we observed linear summation. Our results
thus favor a passive integration of excitatory inputs, independent of
their amplitude, in FS interneurons. However, we do not know the
locations of the tested synaptic inputs on the FS cell dendrites.
Therefore we cannot exclude that we did not sample in our experiments
inputs converging onto the same dendritic compartment and interacting more actively.
The linear summation of the synaptic responses allowed us to estimate
the number of pyramidal cells needed to be activated simultaneously to
drive FS interneurons from 72 mV (near resting membrane potential) to
the action potential threshold (
55 mV). We divided the membrane
potential change necessary to reach the threshold (+17 mV) by the
average EPSP amplitude including failures (2.1 ± 1.3 mV). Given
the present recording conditions and assuming that all connections
studied remained intact in a 300-µm-thick slice, the average number
of pyramidal cells necessary to reach the threshold was 8 ± 5 cells (n = 18). This number is in the range of that
estimated at granule cell to basket cell excitatory connections in the
dentate gyrus (Geiger et al. 1997
). It is worth noting,
however, that, at other excitatory connections onto inhibitory GABAergic interneurons, a single presynaptic neuron could be sufficient to make the postsynaptic cell fire (Ali and Thomson
1998
; Barbour 1993
; Debanne et al.
1995
; Markram et al. 1998
; Miles
1990
).
The low level of temporal summation and the linear summation of
excitatory inputs in FS cells suggest that the synchronous activation
of an average of eight pyramidal cells is necessary to elicit a
discharge in these interneurons. It is difficult to extrapolate this
estimate from our in vitro recordings to in vivo conditions where the
FS interneurons are exposed to a variety of modulators and where the
background synaptic activity of the cortical network strikingly differs
from that observed in slices (Paré et al. 1998).
Higher synaptic activity, however, will decrease the input resistance
of postsynaptic cells (Paré et al. 1998
), leading
to even less temporal integration and favoring the likelihood that FS
interneurons play the role of coincidence detectors.
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ACKNOWLEDGMENTS |
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The authors thank S. B. Ammou for technical assistance and Drs. Afia Ali, Jim T. Porter, and Serge Charpak for helpful comments on the manuscript. This study was supported by Centre National de Recherche Scientifique and by European Union Grants 96-0589 and 96-0382. M. C. Angulo was supported by a fellowship from Instituto Colombiano de Ciencia y Tecnología (Colciencias), Colombia.
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FOOTNOTES |
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Address for reprint requests: E. Audinat, Neurobiologie et Diversité Cellulaire, CNRS UMR 7637, Ecole Supérieure de Physique et de Chimie Industrielles de la ville de Paris, 10 rue Vauquelin, 75231 Paris Cedex 5, France.
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 12 March 1999; accepted in final form 21 April 1999.
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REFERENCES |
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