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The Journal of Neuroscience, November 1, 1998, 18(21):8863-8874
Silent Synapses in the Developing Rat Visual Cortex: Evidence for
Postsynaptic Expression of Synaptic Plasticity
Simon
Rumpel,
Hanns
Hatt, and
Kurt
Gottmann
Lehrstuhl für Zellphysiologie, Ruhr-Universität Bochum,
D-44870 Bochum, Germany
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ABSTRACT |
In the developing visual cortex activity-dependent refinement of
synaptic connectivity is thought to involve synaptic plasticity processes analogous to long-term potentiation (LTP). The recently described conversion of so-called silent synapses to functional ones
might underlie some forms of LTP. Using whole-cell recording and
minimal stimulation procedures in immature pyramidal neurons, we
demonstrate here the existence of functionally silent synapses, i.e.,
glutamatergic synapses that show only NMDA receptor-mediated transmission, in the neonatal rat visual cortex. The incidence of
silent synapses strongly decreased during early postnatal development. After pairing presynaptic stimulation with postsynaptic depolarization, silent synapses were converted to functional ones in an LTP-like manner, as indicated by the long-lasting induction of AMPA
receptor-mediated synaptic transmission. This conversion was dependent
on the activation of NMDA receptors during the pairing protocol.
The selective activation of NMDA receptors at silent synapses could be
explained presynaptically by assuming a lower glutamate concentration
compared with functional ones. However, we found no differences in
glutamate concentration-dependent properties of NMDA receptor-mediated
PSCs, suggesting that synaptic glutamate concentration is similar in
silent and functional synapses. Our results thus support a postsynaptic
mechanism underlying silent synapses, i.e., that they do not contain
functional AMPA receptors. Synaptic plasticity at silent synapses might
be expressed postsynaptically by modification of nonfunctional AMPA
receptors or rapid membrane insertion of AMPA receptors. This
conversion of silent synapses to functional ones might play a major
role in activity-dependent synaptic refinement during development of
the visual cortex.
Key words:
visual cortex; synapse development; synaptic plasticity; silent synapses; AMPA receptor; NMDA receptor; glutamate
concentration
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INTRODUCTION |
Activity-dependent refinement of
synaptic connectivity during the early postnatal development of the
visual cortex is thought to underlie the emergence of the adult pattern
of cortical neuronal circuits (Goodman and Shatz, 1993 ; Singer, 1995;
Katz and Shatz, 1996). The cellular and molecular mechanisms involved
in the competition of presynaptic axons for their postsynaptic targets
have been proposed to be analogous to synaptic plasticity phenomena
such as long-term potentiation (LTP) and long-term depression (Artola and Singer, 1987; Bear et al., 1987, 1992; Tsumoto, 1992; Fox and Daw,
1993; Crair and Malenka, 1995; Kirkwood et al., 1995; Singer 1995; Katz
and Shatz, 1996).
Recently, a novel mechanism leading to LTP at glutamatergic synapses,
i.e., the conversion of silent synapses to functional ones, has been
described in the hippocampus and in the somatosensory cortex (Isaac et
al., 1995, 1997; Liao et al., 1995; Durand et al., 1996). Silent
synapses exhibit exclusively NMDA receptor-mediated synaptic
transmission and are functionally silent at the resting membrane
potential because of the strong, voltage-dependent
Mg2+ block of NMDA receptors (McBain and Mayer,
1994). A conversion of silent synapses to functional ones can be
achieved by pairing presynaptic stimulation with postsynaptic
depolarization, and the conversion is dependent on the activation of
NMDA receptors (Isaac et al., 1995, 1997; Liao et al., 1995; Durand et
al., 1996).
There are two major alternative explanations regarding the molecular
mechanisms underlying silent synapses (Kullmann and Siegelbaum, 1995;
Malenka and Nicoll, 1997; Kullmann and Asztely, 1998). First, silent
synapses might be postsynaptically silent; i.e., they might contain
either nonfunctional or no AMPA receptors (Isaac et al., 1995; Liao et
al., 1995; Durand et al., 1996). Second, silent synapses might be
presynaptically silent (Kimura et al., 1997); i.e., they might release
much less glutamate than functional synapses, or they might not release
glutamate at all. Because NMDA receptors exhibit a much higher affinity
to glutamate than do AMPA receptors (Patneau and Mayer, 1990; Hestrin,
1992), NMDA receptors might be activated by low concentrations of
glutamate that are not sufficient to elicit a detectable AMPA
receptor-mediated PSC. Spillover of glutamate from a functional synapse
adjacent to a silent synapse releasing no glutamate might also provide
a low concentration of glutamate that would activate only NMDA
receptors (Kullmann et al., 1996; Asztely et al., 1997; Barbour and
Häusser, 1997; Kullmann and Asztely, 1998).
In the present study, we demonstrate the existence of silent synapses
in the neonatal rat visual cortex. We further address whether the
glutamate concentration is lower at silent synapses compared with
functional ones by comparing the rise times of NMDA PSCs, which
strongly depend on glutamate concentration (Clements and Westbrook,
1991). Furthermore we studied glutamate concentration-dependent block
of NMDA PSCs by the rapidly unbinding NMDA receptor antagonist D-aminoadipate (D-AA) (Clements et al., 1992;
Clements, 1996). We found no difference between silent and functional
synapses in these glutamate concentration-dependent properties of NMDA PSCs. Our results do not support the hypothesis that the glutamate concentration is lower at silent synapses and are thus consistent with
the idea that a postsynaptic mechanism underlies synaptic plasticity at
silent synapses.
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MATERIALS AND METHODS |
Electrophysiology. Coronal slices (300 µm
thick) of the visual cortex from 3- to 11-d-old Wistar rats were cut
with a vibratome. Slices were incubated in artificial CSF (in
mM: 125 NaCl, 3 KCl, 1.25 NaH2PO4, 25 NaHCO3,
20 D-glucose, 2.5 CaCl2, and 1.5 MgCl2, saturated with 95%
O2/5% CO2, pH 7.3) and allowed
to recover for at least 1 hr at room temperature. All recordings were
made at 28-30°C in a submerged slice chamber perfused with
artificial CSF with added picrotoxin (100 µM).
Whole-cell patch-clamp recordings were obtained in layers V/VI of the
visual cortex using the blind-patch technique (Blanton et al., 1989).
Recording electrodes (7-10 M ) were filled with intracellular
solution I (in mM: 135 K-gluconate, 20 KCl, 2 MgCl2, 10 HEPES, 1 EGTA, 4 Na2-ATP, and
0.5 Na2-GTP, and 0.3% Lucifer yellow, adjusted to pH 7.3 with KOH) in current-clamp experiments. In voltage-clamp experiments
intracellular solution II (in mM: 135 CsCl, 20 TEA-Cl, 2 MgCl2, 10 HEPES, 4 Na2-ATP, 0.5 Na2-GTP, and 1 or 10 EGTA, adjusted to pH 7.3 with CsOH)
was used. In experiments designed to investigate synaptic plasticity,
intracellular solution II contained 1 mM EGTA, otherwise 10 mM. PSCs were evoked at a stimulation frequency of 0.2 Hz
with a glass monopolar electrode placed in the visual cortex in
different layers within 100-800 µm of the recording electrode. The
vast majority of synaptic responses were evoked by stimulation in
layers V/VI. The stimulation frequency was not changed during the
experiments. The input resistance (1-0.5 G) was continuously
monitored by delivering a voltage step command, and cells
were discarded when it changed by >20%. Recordings were made using a
HEKA (Lambrecht/Pfalz, Germany) EPC-7 patch-clamp amplifier, filtered
at 3 kHz, and sampled at 20 kHz ( 80 mV holding potential) or 2 kHz
(+40 mV holding potential). Sampling was done with a TL-1 interface
using pClamp 5.5.1 Software (Axon Instruments, Foster City, CA), and
data were stored on computer disk.
Data analysis. Off-line analysis of PSCs was done using
AUTESP software (H. Zucker, Max-Planck-Institut für
Psychatrie, Martinsried, Germany). Briefly, response amplitudes at 80
mV holding potential were measured as the difference between the
amplitude during a 0.8 msec window including the peak of the PSC and
the amplitude during a window 2 msec before the stimulus. To avoid
contamination of measurements by the stimulus artifact, averaged traces
of visually identified failures were subtracted before analysis. Noise
amplitudes were measured as the difference between the amplitude during
the window 2 msec before the stimulus and a third window 6 msec before the stimulus. PSC amplitudes at +40 mV holding potential were measured
analogously, but subtraction of failures was omitted, and the size of
the windows used was 8 msec. To estimate the amplitude of the NMDA
receptor-mediated component of the PSC, the window was positioned 30 msec after the stimulus, assuming complete decline of the AMPA
receptor-mediated component within 30 msec.
For each trace the SD of the baseline noise before the stimulus
was calculated. Response amplitudes smaller than twofold SD of the
noise were considered failures. In the experiments designed to estimate
the proportion of silent synapses by comparing failure rates at 80
and +40 mV holding potential the fraction of silent synapses was
calculated according to the method of Wu et al. (1996). Data are
expressed as mean ± SEM. For statistical tests of significance, a
two-tailed Student's t test was applied. Statistical
comparison of response or noise amplitude distributions was done using
a Kolmogorov-Smirnov test.
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RESULTS |
Silent synapses in the neonatal visual cortex
To study silent synapses in the developing rat visual cortex,
whole-cell recordings were obtained from layer V/VI neurons in acute
brain slices from rat pups [postnatal day 3 (P3)-P12] using the
blind-patch technique. To characterize the recorded neurons
morphologically, 48 cells were filled with Lucifer yellow. Most neurons
(88%) had the morphology typical of immature pyramidal neurons (Kasper
et al., 1994b; Fig. 1). At P > 5 the pattern of action potential discharge in these immature pyramidal
cells was similar to that in nonbursting cells (Kasper et al., 1994a).
Pyramidal neurons in slices obtained from younger animals (P3-P4)
exhibited a pattern of action potential firing that was even more
immature, typically showing only one action potential after current
injection (Fig. 1).
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Figure 1.
Morphology and action potential discharge pattern
of immature pyramidal neurons in layers V/VI. A,
Reconstruction of a Lucifer yellow-filled pyramidal neuron at P3.
Orientation to the pial surface is indicated by the
arrow. At P3 pyramidal neurons typically respond with a
single action potential to current injection. Amplitude of injected
current is indicated at the corresponding membrane voltage record.
B, Reconstruction of a Lucifer yellow-filled pyramidal
neuron at P10. These more differentiated pyramidal neurons showed a
train of action potentials after current injection.
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To examine the properties of glutamatergic synapses, PSCs were evoked
by intracortical extracellular stimulation using a patch pipette that
was located within 100-800 µm of the recorded neuron. GABAA receptor-mediated PSCs were blocked by addition of
100 µM picrotoxin to the extracellular solution. In the
initial experiments, a small stimulation strength near minimal
stimulation (Stevens and Wang, 1994) was used. At 80 mV holding
potential and in the presence of 1.5 mM
Mg2+, PSCs were completely and reversibly blocked
(94 ± 1% of peak amplitude; n = 5) by the
non-NMDA receptor antagonist DNQX (10 µM), demonstrating
that they were mediated by AMPA receptors. At a holding potential of
+40 mV the PSCs were dominated by NMDA receptor-mediated components, as
shown by reversible blockade (85 ± 4% of peak amplitude;
n = 4) by the NMDA receptor antagonist D-AP-5 (25 µM). In the presence of
D-AP-5 a small, rapidly declining AMPA receptor-mediated
component remained at +40 mV holding potential. However, 30 msec after
the stimulus, the PSCs at +40 mV holding potential were exclusively
mediated by NMDA receptors as demonstrated by the complete blockade
(97 ± 1%) by D-AP-5.
With minimal stimulation a small number of synapses are activated, and
synaptic failures are observed in addition to PSCs (Fig.
2A; Malinow 1991;
Stevens and Wang, 1994). Activation of silent synapses in the presence
of Mg2+ results in failures at hyperpolarized
membrane potential and in NMDA PSCs, in addition to failures, at
depolarized membrane potential, because silent synapses exhibit only
NMDA receptor-mediated synaptic transmission (Isaac et al., 1995, 1997;
Liao et al., 1995; Durand et al., 1996; Wu et al., 1996). Thus, silent
synapses are indicated by a significant difference in failure rates at hyperpolarized and depolarized membrane potentials in the presence of
Mg2+. Furthermore, the incidence of silent synapses
can be estimated from this difference in failure rates (Liao et al.,
1995; Wu et al., 1996; Isaac et al., 1997), assuming that the average
release probability is similar at functional and silent synapses. At
P3-P5 the mean failure rate at +40 mV holding potential in the
presence of 1.5 mM Mg2+ was 0.48 ± 0.05, whereas the mean failure rate at 80 mV holding potential was
significantly (p < 0.01) higher (0.72 ± 0.06) (Fig. 2B). From these data the percentage of
silent synapses was calculated to be 55% (Fig. 2C). During
the first postnatal week the mean failure rate at 80 mV membrane
potential significantly (p < 0.01) decreased,
whereas the mean failure rate at +40 mV membrane potential did not
change significantly. At P9-P11 no significant difference in mean
failure rates (80 mV, 0.38 ± 0.04; +40 mV, 0.35 ± 0.04) was observed, suggesting a strong developmental decline in the incidence of silent synapses. The mean amplitude of AMPA PSCs (P3-P4,
15 ± 3 pA; P9-P11, 27 ± 3 at 80 mV holding potential) and that of PSCs recorded at +40 mV holding potential (P3-P4, 26 ± 3 pA; P9-P11, 28 ± 3 pA) did not change significantly. Our results suggest that silent synapses are present in the neonatal visual
cortex and that their incidence strongly decreases during early
postnatal development, similar to the developing hippocampus (Durand et
al., 1996) and the neonatal somatosensory cortex (Isaac et al.,
1997).
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Figure 2.
Different failure rates of glutamatergic PSCs at
hyperpolarized and depolarized membrane potentials indicate the
existence of silent synapses. A, PSCs evoked by
intracortical stimulation at 80 mV holding potential
(a) and at +40 mV holding potential
(b) in the presence of 1.5 mM
Mg2+. At +40 mV holding potential, PSCs are
dominated by an NMDA receptor-mediated component, which is blocked by
Mg2+ at 80 mV holding potential, leading to purely
AMPA receptor-mediated PSCs. Failures are indicated by
asterisks. Note the reduced failure rate after changing
the holding potential to +40 mV. B, Mean failure rates
at 80 mV holding potential (open bars) and at +40 mV
holding potential (filled bars) at different
stages of early postnatal development. Postnatal age (P) and number of
experiments (n) are indicated below the bars.
Significant differences in failure rates are indicated by
asterisks. Error bars represent SEM. Note the large
difference in failure rates at P3-P5 that disappeared during postnatal
development. C, Percentage of silent synapses calculated
from the mean failure rates at 80 mV holding potential and at +40 mV
holding potential according to Wu et al. (1996) (see Materials and
Methods).
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To demonstrate the existence of silent synapses more directly,
stimulation strength was reduced until no AMPA PSCs were observed for
10 min (112 stimuli) at 80 mV holding potential in the presence of
1.5 mM Mg2+ (Fig.
3A,B). Accordingly, no
significant difference (Kolmogorov-Smirnov test) was observed between
the amplitude distribution of the recording noise and the distribution
of response amplitudes (Fig. 3C). However, after changing
the holding potential to +40 mV and stimulating at the same strength
and rate (0.2 Hz), in addition to failures, NMDA PSCs were frequently
observed. As is typical for silent synapses, only NMDA
receptor-mediated synaptic transmission was observed. The released
glutamate failed to activate AMPA receptors.
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Figure 3.
Example of a silent synapse at P4 showing
exclusively NMDA receptor-mediated synaptic transmission.
A, Individual response amplitudes recorded during the
experiment at 80 mV holding potential and at +40 mV holding
potential. B, Eight superimposed individual traces
(left) and average currents (right) from
the experiment shown in A at 80 mV holding potential
(a) and at +40 mV holding potential
(b). Note the absence of AMPA receptor-mediated
PSCs. C, Amplitude histograms (bin width, 2 pA) of
response amplitudes (thick line) and noise amplitudes
(open bars) at 80 mV holding potential
(a) and at +40 mV holding potential
(b) from the experiment shown in
A.
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NMDA receptor-dependent conversion of silent synapses to
functional synapses
Conversion of silent synapses to functional synapses using a
pairing protocol that consisted of postsynaptic depolarization and
presynaptic stimulation has been described (Isaac et al., 1995, 1997;
Liao et al., 1995; Durand et al., 1996). To address whether such a
conversion can also occur in the developing visual cortex, we used a
similar pairing protocol. First, the stimulation strength was reduced
to a failure rate >90% at 80 mV holding potential in the presence
of Mg2+ (Fig.
4A-C). After changing
the holding potential to +40 mV to demonstrate the presence of NMDA
PSCs, the postsynaptic neuron was depolarized to 10 mV for 4 min (48 stimuli), and presynaptic stimulation was continued at the same
frequency (0.2 Hz) to prevent any changes in axon excitability. Within
a few minutes after returning to 80 mV holding potential, the failure
rate was strongly reduced, and PSCs were frequently detected. In four
of seven cells, induction of functional synapses was indicated by a
significant (Kolmogorov-Smirnov test, p < 0.01)
difference in the distributions of response amplitudes before and
20-30 min after the pairing protocol (Fig. 4D,E).
The PSCs induced by the pairing protocol were completely blocked by DNQX (10 µM; n = 2) and thus mediated by
AMPA receptors. In the remaining three cells no change in the failure
rate at 80 mV holding potential and no significant change in the
distribution of response amplitudes was observed after the pairing
protocol. In the developing hippocampus, NMDA receptor activation
during the pairing protocol is necessary for conversion of silent
synapses to functional synapses (Isaac et al., 1995; Durand et al.,
1996). To examine this point, we blocked NMDA receptors by
D-AP-5 (25 µM) during the pairing protocol.
This treatment abolished NMDA PSCs at +40 mV holding potential
(Fig. 5). In all eight cells tested, no
change in the failure rate at 80 mV holding potential and no
significant change in the distribution of response amplitudes was found
after the pairing protocol in the presence of D-AP-5.
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Figure 4.
Conversion of silent synapses in functional ones
by a pairing protocol. A, Individual response amplitudes
recorded during the course of the experiment. Response amplitudes were
recorded at 80 mV holding potential and at +40 mV holding potential
before and after pairing of presynaptic stimulation and postsynaptic
depolarization. Note the appearance of AMPA PSCs at 80 mV holding
potential after the pairing protocol. B, Eight
superimposed individual current traces recorded at the positions
indicated in A at 80 mV holding potential (a,
c, d) and at +40 mV holding potential (b, e).
C, Average currents corresponding to the individual
traces shown in B. D, Amplitude histogram
(bin width, 2 pA) of response amplitudes (thick line)
and noise amplitudes (open bars) at 80 mV holding
potential before pairing from the experiment shown in A.
E, Amplitude histogram (bin width, 2 pA) of response
amplitudes (thick line) and noise amplitudes
(open bars) at 80 mV holding potential 20-30 min
after pairing from the experiment shown in A.
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Figure 5.
Conversion of silent synapses in functional ones
is dependent on NMDA receptor activation. A, Individual
response amplitudes recorded during the course of the experiment.
D-AP-5 was applied after recording response amplitudes at
+40 mV and at 80 mV holding potential. Blockade of NMDA receptors was
confirmed by recording response amplitudes at +40 mV holding potential
before pairing. After pairing no AMPA PSCs were observed at 80 mV
holding potential. B, Eight superimposed individual
current traces recorded at the positions indicated in A
at +40 mV holding potential (a, c ,e) and at-80 mV
holding potential (b, d). C, Average
currents corresponding to the individual traces shown in
B. D, Amplitude histogram (bin width, 2 pA) of response amplitudes (thick line) and noise
amplitudes (open bars) at 80 mV holding potential
before pairing from the experiment shown in A.
E, Amplitude histogram (bin width, 2 pA) of response
amplitudes (thick line) and noise amplitudes
(open bars) at 80 mV holding potential 20-30 min
after pairing from the experiment shown in A.
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A summary of the experiments involving a pairing protocol is shown in
Figure 6. For experiments in which the
pairing protocol was performed in the absence of D-AP-5,
only those cells were included that showed a significant change in the
distribution of response amplitudes at 80 mV holding potential after
the pairing protocol. In these four cells the mean success rate (1 failure rate) at 80 mV holding potential was significantly
(p < 0.01) increased from 0.07 ± 0.01 to
0.54 ± 0.11 20-30 min after the end of the pairing protocol. The
mean success rate at +40 mV holding potential did not change
significantly (0.77 ± 0.04 before pairing and 0.67 ± 0.21 after pairing). At 80 mV holding potential a slight increase
(p < 0.05) occurred in the mean success
amplitudes (7 ± 2 pA before pairing and 10 ± 2 pA 20-30
min after pairing), whereas at +40 mV holding potential no significant
change occurred (21 ± 4 pA before pairing and 34 ± 9 pA
after pairing) during the experiments. This increase can be explained
solely by the fact that with a higher number of functional synapses the
probability that two or more quanta are released synchronously
increases (experiment, 144% increase; calculated, 139% increase).
However, an increase in quantal size in addition to an increase in the
number of release sites cannot be excluded. In the presence of
D-AP-5 during the pairing protocol the increase in mean
success rate at 80 mV holding potential was blocked (0.05 ± 0.01 before pairing and 0.06 ± 0.02 20-30 min after pairing). In
conclusion, our results are consistent with the induction of functional
transmission by AMPA receptors at hyperpolarized membrane potentials at
previously silent synapses after pairing of postsynaptic depolarization
and presynaptic stimulation. Functional synapse induction required the
activation of NMDA receptors.
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Figure 6.
Summary of experiments demonstrating NMDA
receptor-dependent conversion of silent synapses in functional ones.
A, Mean success rates at 80 mV holding potential
(open bars) before (10-0 min) and 0-10 min, 10-20
min, and 20-30 min after pairing. The asterisk
indicates a significant increase in success rate of AMPA PSCs after
pairing. Filled bars represent mean success rates at +40
mV holding potential before and after pairing. Error bars represent
SEM. B, Mean success rates at 80 mV holding potential
(open bars) before and after pairing in the presence of
25 µM D-AP-5. No increase in mean success
rate of AMPA PSCs was observed. Filled bars represent
mean success rates at +40 mV holding potential before and after pairing
in the presence of D-AP-5. C, Mean success
amplitudes at 80 mV holding potential (open bars)
before and after pairing. The asterisk indicates a
significant increase in mean success amplitude of AMPA PSCs after
pairing. Filled bars represent mean success amplitudes
at +40 mV holding potential before and after pairing. The slight
increase was not significant. Error bars represent SEM.
D, Mean success amplitudes at 80 mV holding potential
(open bars) before and after pairing in the presence of
25 µM D-AP-5. No increase in mean success
amplitude of AMPA PSCs was observed. Filled bars
represent mean success rates at +40 mV holding potential before and
after pairing in the presence of D-AP-5.
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Comparison of glutamate concentration-dependent properties of NMDA
PSCs at silent and functional synapses
There are two alternative ways to explain why silent synapses are
nonfunctional at hyperpolarized membrane potential (Kullmann and
Siegelbaum, 1995; Malenka and Nicoll, 1997; Kullmann and Asztely, 1998). First, silent synapses might contain only functional NMDA receptors. AMPA receptors would be either nonfunctional or not present
in the postsynaptic membrane. Because of the strong voltage-dependent Mg2+ block of NMDA receptors, such synapses would be
silent at hyperpolarized membrane potential (Isaac et al., 1995; Liao
et al., 1995; Durand et al., 1996; Wu et al., 1996). Alternatively, the
glutamate concentration in the synaptic cleft might be smaller in
silent synapses. Because NMDA receptors have a higher affinity for
glutamate than do AMPA receptors, a low concentration of glutamate
might selectively activate NMDA receptors (Kullmann et al., 1996;
Asztely et al., 1997; Malenka and Nicoll, 1997). Thus, these
alternative explanations differ markedly relative to the postulated
concentration of glutamate in the synaptic cleft.
To address the mechanisms underlying silent synapses, we attempted to
compare the glutamate concentrations in silent and functional synapses.
First, we examined whether the mean rise time (10-90%) of NMDA PSCs
is dependent on the percentage of silent synapses present in a given
recording (Fig. 7). As demonstrated in
outside-out patches (Clements and Westbrook, 1991), the rise time of
NMDA receptor-mediated currents is strongly dependent on the glutamate concentration (at concentrations  50 µM). The percentage
of silent synapses was estimated for each recording from the failure
rates (112 stimuli at 0.2 Hz) obtained at 80 mV and at +40 mV holding potential in the presence of Mg2+. In some
experiments (n = 8) the mean 10-90% rise time of NMDA PSCs at +40 mV holding potential was determined after addition of DNQX
(10 µM) to the extracellular solution. Addition of DNQX did not significantly affect the 10-90% rise times of PSCs at +40 mV
holding potential, because these PSCs are largely mediated by NMDA
receptors (Fig. 7D). Therefore we also included the mean 10-90% rise times of PSCs at +40 mV holding potential, which were obtained without blocking AMPA receptors by DNQX (n = 25). As shown in Figure 7E, the 10-90% rise time of NMDA
PSCs was independent of the percentage of silent synapses present in a
given experiment. For recordings containing 0-10% silent synapses,
the mean rise time of NMDA PSCs was 9.9 ± 0.6 msec
(n = 5) and did not significantly differ from the mean
rise time of NMDA PSCs in recordings containing 90-100% silent
synapses (9.8 ± 0.4 msec, n = 6). These results thus do not support that the glutamate concentration is lower in the
synaptic cleft of silent synapses.
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Figure 7.
NMDA PSC rise times do not differ between silent
and functional synapses. A, Individual response
amplitudes recorded at 80 mV holding potential and at +40 mV holding
potential. The failure rates obtained from this part of the experiment
were used to calculate the proportion of silent synapses present. After
application of DNQX, block of AMPA receptors was confirmed at 80 mV
holding potential, and NMDA PSCs were recorded at +40 mV holding
potential. B, Eight superimposed individual traces
recorded at the positions indicated in A at 80 mV
holding potential (a, c) and at +40 mV holding potential
(b, d). C, Average currents corresponding
to the individual traces shown in B. D,
Application of 10 µM DNQX did not significantly change
the rise time of PSCs recorded at +40 mV holding potential.
E, The rise times (10-90%) of PSCs recorded at +40 mV
holding potential in the absence (circles) or presence
(asterisks) of DNQX did not depend on the percentage of
silent synapses present in a given experiment.
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Furthermore, we used the rapidly dissociating NMDA receptor antagonist
D-AA to compare glutamate concentrations in silent and
functional synapses. D-AA has been used to estimate the
glutamate concentration in the synaptic cleft of functional hippocampal synapses (1.1 mM; Clements et al., 1992). D-AA
is displaced at functional synapses by presynaptically released
glutamate in a glutamate concentration-dependent manner. At its
KD (30 µM) D-AA blocks
NMDA PSCs of functional synapses by ~50%, whereas a 75% block would
be expected if no displacement of D-AA occurs (at glutamate
concentrations 100 µM; Clements et al., 1992). Assuming a reduced glutamate concentration (100 µM) in the
synaptic cleft of silent synapses, an increased block of NMDA PSCs by
D-AA compared with functional synapses would be expected.
Again, we first estimated the percentage of silent synapses present for
each recording from the failure rates (112 stimuli at 0.2 Hz) obtained
at 80 mV and at +40 mV holding potential in the presence of
Mg2+ at P3-P4 (Fig.
8). Then, 30 µm D-AA was
applied at +40 mV holding potential, and the resulting block of the
NMDA receptor-mediated component of the PSC (amplitude 30 msec after
stimulus) was determined. As shown in Figure 8D, we
found no correlation between the amount of block by D-AA
and the percentage of silent synapses present. In recordings containing
20-40% silent synapses the mean block of the NMDA receptor-mediated
component of the PSCs by D-AA was 49 ± 6%
(n = 4) and did not significantly differ from the mean block in recordings containing 80-100% silent synapses (54 ± 4%, n = 8). These results again do not support a lower
glutamate concentration in the synaptic cleft of silent synapses.
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Figure 8.
Block of NMDA PSCs by D-AA does not
differ between silent and functional synapses. A,
Individual response amplitudes recorded at 80 mV holding potential
and at +40 mV holding potential. The failure rates obtained from this
part of the experiment were used to calculate the proportion of silent
synapses present (68%). After application of D-AA the
amount of blockade of PSCs at +40 mV holding potential was determined.
B, Eight superimposed individual traces (top
panel) and corresponding average currents (bottom
panel) recorded at the positions indicated in
A at 80 mV holding potential (a)
and at +40 mV holding potential (b, c, d).
C, Superimposed average PSCs recorded at +40 mV holding
potential before application of D-AA
(Control), during application
(DAA), and after washout of the antagonist
(Wash). D, The mean amount of blockade of
PSCs at +40 mV holding potential by D-AA was not dependent
on the percentage of silent synapses. Error bars represent SEM;
n is indicated below mean values.
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In conclusion, we present evidence for the existence of silent synapses
in the neonatal visual cortex, which seem to disappear rapidly during
postnatal development. These silent synapses appear to be
postsynaptically silent, i.e., contain no or nonfunctional AMPA
receptors, because no difference in glutamate concentration was
observed compared with functional synapses. Conversion of silent
synapses to functional ones thus suggests a postsynaptic expression of
synaptic plasticity.
| |
DISCUSSION |
In the present study we demonstrate the existence of so-called
silent synapses in the neonatal rat visual cortex. The properties of
silent synapses have been studied mainly in the developing hippocampus
(Isaac et al., 1995; Liao et al., 1995; Durand et al., 1996). In the
presence of Mg2+ silent synapses typically show
exclusively NMDA receptor-mediated transmission at depolarized membrane
potential, whereas AMPA receptor-mediated transmission is not
detectable at hyperpolarized membrane potential. Thus, they are
functionally silent at resting membrane potential because of the
voltage-dependent blockade of NMDA receptors by Mg2+. In the neonatal visual cortex we found
strikingly higher failure rates of glutamatergic synapses at
hyperpolarized compared with depolarized membrane potentials. Assuming
a colocalization of AMPA and NMDA receptors at functional synapses
(Bekkers and Stevens, 1989), failure rates at hyperpolarized and
depolarized membrane potentials should be equal. Thus, the above result
suggests the presence of silent synapses exhibiting only NMDA
receptor-mediated transmission (Liao et al., 1995; Wu et al., 1996;
Isaac et al., 1997). Furthermore the existence of silent synapses in
the visual cortex was directly confirmed by observing synapses that
showed purely NMDA receptor-mediated synaptic transmission at
depolarized membrane potential and no AMPA receptor-mediated
transmission at hyperpolarized potentials (see Fig. 3). In the immature
somatosensory cortex Isaac et al. (1997) studied the properties of
thalamocortical synapses and also demonstrated the presence of silent
synapses in the neocortex. However, in layer V/VI pyramidal neurons of the visual cortex the vast majority of glutamatergic synapses arises
from intracortical connections (DeFelipe and Fariñas, 1992;
Kageyama and Robertson, 1993), suggesting that the majority of synapses
studied in this report represent intracortical synapses.
The molecular mechanisms underlying silent synapses are debated
controversially (Kullmann and Siegelbaum, 1995; Malenka and Nicoll,
1997; Kullmann and Asztely, 1998). The phenomenon of exclusively NMDA
receptor-mediated transmission can be explained entirely postsynaptically. At silent synapses AMPA receptors either might be in
a nonfunctional state or might not occur in the postsynaptic membrane
(Isaac et al., 1995, 1997; Liao et al., 1995; Durand et al., 1996; Wu
et al., 1996). Alternatively, presynaptic reasons can account for the
existence of silent synapses. Because NMDA receptors show a much higher
affinity for glutamate than AMPA receptors (Patneau and Mayer, 1990;
Hestrin, 1992), a low concentration of glutamate in the synaptic cleft
might selectively activate NMDA receptors without eliciting a
detectable AMPA receptor-mediated response. At silent synapses vesicles
might release a much smaller amount of glutamate, thus leading to a
much lower glutamate concentration compared with functional synapses. A
low concentration of glutamate could also be provided by spillover of
glutamate from adjacent functional synapses on a neighboring neuron
(Kullmann et al., 1996; Asztely et al., 1997; Barbour and
Häusser, 1997; Kullmann and Asztely, 1998). These alternative
hypotheses differ markedly in the postulated concentration of glutamate
in the synaptic cleft and could be distinguished by examining the
synaptic glutamate concentration at silent and functional synapses.
Direct measurements of synaptic glutamate concentrations are impeded by
the small size of the synaptic cleft. However, glutamate concentrations
can be estimated from the properties of NMDA PSCs. We examined two
properties of NMDA PSCs that are dependent on glutamate concentration.
First, the rise times of NMDA receptor-mediated currents are strongly
dependent on the concentration of glutamate at concentrations <50
µM (Clements and Westbrook, 1991). Besides the agonist
concentration theoretically only  could also be a rate-limiting
step; however, all calculated or measured -values and all
mathematical models exclude as a factor that determines the rise
time of the synaptic current independent of agonist concentration (Dudel et al., 1992; Heckmann et al., 1996; Colquhoun et al., 1997).
Thus, we compared the rise times of NMDA PSCs at functional and silent
synapses. We found no significant differences in NMDA PSC rise times,
suggesting that the glutamate concentration at silent synapses reaches
at least 50 µM. Second, the selective NMDA receptor
antagonist D-AA can be used to estimate the synaptic glutamate concentration (Clements et al., 1992; Clements, 1996). Because of its fast unbinding rate, D-AA is displaced from
the NMDA receptors by synaptically released glutamate in a glutamate concentration-dependent manner. Thus, application of D-AA
results in a glutamate concentration-dependent blockade of NMDA PSCs. Assuming two binding sites for D-AA at NMDA receptors, at
its KD (30 µM) D-AA
should block NMDA PSCs by 75% if no displacement of D-AA
occurs. At glutamate concentrations >100 µM,
displacement of D-AA leads to a reduction of
D-AA block of NMDA PSCs. From the considerable displacement
of D-AA leading to only ~50% blockade of NMDA PSCs at
functional synapses, Clements et al. (1992) estimated the concentration
of glutamate to reach 1.1 mM. Thus, at low glutamate concentrations 30 µM D-AA is expected to
block NMDA PSCs by 75%, whereas a significantly weaker blockade is
expected at higher concentrations. We found a blockade of NMDA PSCs of
~50% that was independent on the fraction of silent synapses
present, thus suggesting that glutamate concentrations do not differ
between functional and silent synapses. Using a similar experimental
approach, Tong and Jahr (1994) demonstrated by studying evoked autaptic NMDA PSCs in cultured hippocampal neurons that drugs that alter presynaptic release lead to a significant change in the inhibition of
NMDA PSCs by a fast unbinding NMDA receptor antagonist.
The above results suggest that synaptic glutamate concentrations do not
differ between silent and functional synapses, supporting that silent
synapses contain either no or nonfunctional AMPA receptors. Alternative
explanations assuming a lower glutamate concentration at silent
synapses are not supported by our findings. However, even glutamate
concentrations of up to 200 µM may be insufficient to
elicit detectable AMPA receptor-mediated PSCs. At 200 µM
~20% of the AMPA receptors mediating the AMPA PSC at functional
synapses (at 1.1 mM glutamate) might be activated, as
estimated from the dose-response relationship of AMPA receptors
(Hestrin, 1992). Thus, a small window of glutamate concentrations
(100-200 µM) remains at which the properties of NMDA
PSCs would not differ compared with functional synapses and at which no
detectable AMPA PSC would be elicited. However, spillover of glutamate
from adjacent functional synapses should lead to a wide range of
glutamate concentrations (down to 5 µM) that activate
NMDA receptors sufficiently to detect a NMDA PSC. Thus, if spillover
underlies silent synapses at least in a part of the silent synapses,
the glutamate concentration-dependent properties of NMDA PSCs should
differ from those of functional synapses.
A typical property of silent synapses is that they can be converted to
functional synapses. Such a conversion is indicated by the appearance
of AMPA PSCs after an LTP induction protocol (Isaac et al., 1995, 1997;
Liao et al., 1995; Durand et al., 1996). In the visual cortex we could
convert silent synapses to functional ones by pairing presynaptic
stimulation and postsynaptic depolarization. This conversion was
strictly dependent on NMDA receptor activation. After the pairing
protocol we observed a large increase in the success rate of AMPA PSCs,
whereas the success rate of NMDA PSCs did not change. As discussed
above, silent synapses appear to be postsynaptically silent; i.e., they
do not contain functional AMPA receptors. Thus, this selective increase
in the success rates of AMPA PSCs suggests a postsynaptic expression of
synaptic plasticity, i.e., a modification of AMPA receptors making them
functional. This could occur by phosphorylation of
membrane-incorporated AMPA receptors, leading to changes in their
biophysical properties. There is some evidence that activation of
calcium-calmodulin-dependent protein kinase II (CaMKII) by NMDA
receptor-mediated Ca2+ influx might mediate AMPA
receptor modification (Shirke and Malinow, 1997). Expression of
constitutively active CaMKII in frog optic tectum neurons led to an
acceleration of the developmental disappearance of silent synapses (Wu
et al., 1996). Moreover, phosphorylation of AMPA receptors by CaMKII
after LTP induction has been demonstrated by Barria et al. (1997).
Alternatively, other postsynaptic mechanisms of AMPA receptor
modification are conceivable, e.g., a fast insertion of a cluster of
AMPA receptors into the postsynaptic membrane (Malinow, 1994) or a fast
accumulation of extrasynaptic AMPA receptors in the subsynaptic
membrane.
In the visual cortex we observed a strong developmental decline in the
incidence of silent synapses during the first two postnatal weeks, as
estimated from differences in failure rates of AMPA and NMDA PSCs.
Because silent synapses are observed during the early period of
synaptogenesis (Blue and Parnavelas, 1983) it may be suggested that
after the initial formation of glutamatergic synapses a considerable
portion exhibits only NMDA receptor-mediated transmission. During
further development silent synapses might be converted in functional
ones, if activation of NMDA receptors by synchronous presynaptic and
postsynaptic activity occurs.
In the visual system it has been proposed that mechanisms analogous to
NMDA receptor-dependent LTP underlie the modification of synaptic
circuitry by experience during the critical period (Goodman and Shatz,
1993; Singer, 1995; Katz and Shatz, 1996). The conversion of silent
synapses in functional ones appears to be a major mechanism of LTP-like
synaptic plasticity in the neonatal neocortex, as suggested by the
close correlation of the disappearance of silent synapses and the loss
of the ability to generate LTP (Crair and Malenka, 1995; Isaac 1997).
Thus, the NMDA receptor-dependent conversion of silent synapses to
functional ones might be one cellular mechanism involved in
activity-dependent refinement of synaptic connections. Developmental
refinement processes might occur much more effectively by a process
that can rapidly add new functional elements than by a process that
slowly changes the efficacy of existing synapses.
| |
FOOTNOTES |
Received May 4, 1998; revised Aug. 21, 1998; accepted Aug. 21, 1998.
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB
509/C1. We thank Dr. B. Ache for helpful comments on this manuscript
and H. Jung and H. Bartel for excellent technical assistance.
Correspondence should be addressed to Dr. Kurt Gottmann at the above
address.
| |
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Top
Abstract
Introduction
