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The Journal of Neuroscience, January 15, 1999, 19(2):578-588
Effect of Zolpidem on Miniature IPSCs and Occupancy of
Postsynaptic GABAA Receptors in Central Synapses
David
Perrais and
Nicole
Ropert
Institut Alfred Fessard, Centre National de la Recherche
Scientifique UPR 2212, Gif sur Yvette, France
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ABSTRACT |
GABAA-mediated miniature IPSCs (mIPSCs) were
recorded from layer V pyramidal neurons of the visual cortex using
whole-cell patch-clamp recording in rat brain slices. At room
temperature, the benzodiazepine site agonist zolpidem enhanced both the
amplitude (to 138 ± 26% of control value at 10 µM)
and the duration (163 ± 14%) of mIPSCs. The enhancement of mIPSC
amplitude was not caused by an increase of the single-channel
conductance of the postsynaptic receptors, as determined by peak-scaled
non-stationary fluctuation analysis of mIPSCs. The effect of zolpidem
on fast, synaptic-like (1 msec duration) applications of GABA to
outside-out patches was also investigated. The EC50 for
fast GABA applications was 310 µM. In patches, zolpidem
enhanced the amplitude of currents elicited by subsaturating GABA
applications (100-300 µM) but not by saturating
applications (10 mM). The increase of mIPSC amplitude by
zolpidem provides evidence that the GABAA receptors are not saturated during miniature synaptic transmission in the recorded cells.
By comparing the facilitation induced by 1 µM zolpidem on
outside-out patches and mIPSCs, we estimated the concentration of GABA
seen by the postsynaptic GABAA receptors to be ~300
µM after single vesicle release. We have estimated a
similar degree of receptor occupancy at room and physiological
temperature. However, at 35°C, zolpidem did not enhance the amplitude
of mIPSCs or of subsaturating GABA applications on patches, implying
that, in these neurons, zolpidem cannot be used to probe the degree of receptor occupancy at physiological temperature.
Key words:
benzodiazepines; zolpidem;  -aminobutyric acid type A
receptors; miniature inhibitory postsynaptic currents; synaptic
transmission
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INTRODUCTION |
In the Central Nervous System, fast
inhibitory synaptic transmission is primarily mediated by GABA acting
on GABAA receptors. They bear various modulatory sites,
among them the benzodiazepine (BZD) site (MacDonald and Olsen, 1994 ).
The effects of BZD agonists on currents elicited by GABA to native or
recombinant receptors (for review, see MacDonald and Olsen, 1994;
Lavoie and Twyman, 1996; Mellor and Randall, 1997) are consistent with
a change in the binding kinetics of GABA as well as desensitization
kinetics of the receptor. This makes BZD agonists tools of choice to
study the parameters of GABAA receptor activation during
synaptic transmission (De Koninck and Mody, 1994; Frerking et al.,
1995).
The high concentration of glutamate estimated in the synaptic cleft at
excitatory synapses (Clements et al., 1992) has led to the hypothesis
that receptor saturation occurs during a synaptic event. This
assumption is consistent with the observation of peaky amplitude
distributions of the evoked postsynaptic currents (Edwards et al.,
1990) that result from the summation of elementary events, quanta, with
a low coefficient of variation (CV). Given the low number of receptors
activated during a miniature synaptic event (Edwards et al., 1990;
Ropert et al., 1990), a low CV can be obtained only if the channel open
probability is very high at the peak of the synaptic current (Jonas et
al., 1993), implying that the degree of occupancy of postsynaptic
receptors is high during synaptic transmission.
The view that receptor saturation occurs during single-site release in
central synapses has recently been challenged for non-NMDA glutamate
receptors (Tong and Jahr, 1994; Silver et al., 1996). For NMDA or
GABAA receptors, whose affinity for their endogenous ligand
is higher than for non-NMDA receptors, saturation is thought to occur
during synaptic transmission (Clements, 1996). However, because the
neurotransmitter is present only very briefly in the synaptic cleft,
the relevant parameter is the binding rate of the ligand to its
receptor rather than its affinity; therefore, GABAA and
NMDA receptors may also not be saturated (Holmes, 1995; Frerking and
Wilson, 1996). Consistent with this argument, the variability of
uniquantal synaptic events can be large at inhibitory [Grantyn and
Veselovsky (1997), but see Auger and Marty (1997)] and excitatory
synapses, indicating that receptor occupancy is not maximal (Liu and
Tsien, 1995; Stevens and Wang, 1995; Silver et al., 1996). Finally, it
has been proposed that the amplitude of GABAergic miniature
IPSCs (mIPSCs) depends on transmitter concentration (Frerking et al.,
1995).
In the present study, we examined the issue of GABAA
receptor saturation during miniature synaptic transmission. We have
tested the effect of the BZD agonist zolpidem on mIPSCs in layer V
pyramidal cells in rat visual cortex. At room temperature, the mean
amplitude of mIPSCs was increased by zolpidem, and we established that
this effect reflects the binding and activation of more synaptic
receptors, which implies that GABAA receptors are not
saturated during synaptic transmission.
Some of these results have been published previously in abstract form
(Perrais and Ropert, 1997).
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MATERIALS AND METHODS |
Brain slice preparation. Slices were prepared from
young male Wistar rats (mean 17 d old; range, 15-25 d old). The
animals were anesthetized with sodium pentobarbital and decapitated.
The brain was rapidly removed and submerged in oxygenated (5%
CO2, 95% O2) cold artificial CSF
(ACSF) for dissection of the occipital cortex. Slices (300 µm
thickness) were cut in the sagittal plane using a vibratome (DTK-1000,
DSK) and maintained at a temperature of 35°C for at least 1 hr before recording.
Electrophysiology and data analysis. The neurons were
identified using an upright microscope (Axioskop, Zeiss) with Nomarski optics and an infrared video camera (Newvicon, Hamamatsu) as reported previously (Stuart et al., 1993). Most of the recordings were made at
room temperature (22-25°C) from slices kept under constant (2 ml/min) ACSF perfusion. For the experiments of Figures
8 and 9,
slices were recorded at 35°C. The ACSF was heated before entering the
recording chamber. For outside-out patch recordings at 35°C, the
application pipette was dipped into the bath along 5 mm, and thus the flowing solutions were heated to 35°C (Tong and Jahr, 1994).
The temperature was measured by a thermal probe before and after each experiment.
Recording pipettes were made using cleaned and sterilized borosilicate
glass. Their typical resistance was 1-2 M for whole-cell recordings
and 2-8 M for outside-out somatic patch recordings. The pipettes
were coated with beeswax. Recordings were performed using a patch-clamp
amplifier (Axopatch 200A, Axon Instruments). During recording, the
stability of the series resistance, between 5 and 15 M, was checked
using a +2 or +5 mV voltage step applied every 20 sec, and the
recording was discarded if it increased by >10%. Evoked activity was
stored on a computer online. Spontaneous synaptic activity was filtered
at 2 kHz and stored on digital tape recorder (DTR-1202, 48 kHz sampling
rate, Biologic) for subsequent analysis. The data were acquired using a
Digidata 1200 board (Axon Instruments) and analyzed using programmable
software (Acquis 1, Biologic).
Spontaneous synaptic activity during periods of 1-3 min was digitized
at 20 kHz. Between 200 and 1500 synaptic events per period were
detected using a threshold crossing of the derivative with parameters
set for each cell and kept constant for the whole session. The events
detected were then visually inspected to remove electrical artifacts.
Their peak amplitude and 10-90% rise time were measured. The decay
phase of individual events (with no superimposition) could be fitted by
one or more exponentials. Because the changes observed during zolpidem
application did not consistently affect one component in particular,
the duration of mIPSCs was quantified by calculating an estimation of
the time constant of decay ( e) without any
assumption on the number of decay components:
where I is the current and A is the peak
amplitude of the mIPSC. The integral is taken between the peak of the
IPSC and the return to baseline. If one attempts to fit the decay by a
sum of exponentials I(t)   Aiexp( t/i),
then e  Aiexp(t/i)dt/A = (Aii)/A, which
corresponds to the mean of the decay time constants used for the fit.
The estimation of the duration e (term used in the rest
of this paper) of the mIPSCs using this procedure and a classical fit
with exponential functions gave similar results (see Fig.
1B).
The zolpidem concentration increase of mIPSC duration graphs (see Fig.
3) were fitted with the following equation:
where Z is the concentration of zolpidem,
e,control is the duration of mIPSCs without zolpidem,
Max is the maximal relative increase of the duration,
EC50 is the half-maximal effect concentration, and
h is the Hill coefficient. For each concentration, the
stationarity of the mIPSC parameters was ascertained. Moreover, no
change of the amplitude or the duration of mIPSCs was seen in control
conditions over a period of 30 min, which exceeds the duration of the
recordings necessary to test the effects of zolpidem.
Non-stationary fluctuation analysis (NSFA) was performed on currents
elicited by fast applications of saturating concentrations of GABA on
outside-out patches as described previously (Jonas et al., 1993).
Series of 15-40 applications with stable maximal amplitude and
duration were averaged. For each individual trace, the variance around
the mean, minus the variance of the baseline noise, was computed for
regularly spaced time intervals. For each interval, the corresponding
mean current was measured, and the relation between the mean current
I and the variance  2, minus the variance of
the recording noise basal2, was
drawn. These two parameters can be decomposed as
I(t) = NP(t)i
and 2 basal2 = NP(t)(1 P(t))i2, where
N is the number of channels open at the peak of the current, P(t) is the open probability of channels, and
i is the current carried by a single open channel. From
these expressions a parabolic curve was fitted with the equation
2 basal2 = iI I2/N,
giving i and N. The maximal open probability of
the channels was also calculated with Po,max = 1 (peak2 basal2)/iIpeak
where peak2 and
Ipeak are the variance and the average of the
current at its peak, respectively.
Peak-scaled NSFA was also performed on mIPSCs to estimate i
(Traynelis et al., 1993; De Koninck and Mody, 1994; Silver et al.,
1996). In cells where the mIPSC frequency was low enough, 30-100
mIPSCs were selected, with no overlap with other minis. The
procedure was the same as for NSFA, except that the average of these
mIPSCs was scaled to each individual mini before computing the
variance. Therefore, the relation between I and
2 becomes 2 basal2 = iI I2/Np,
where Np is the number of channels open at the
peak of the current.
All results are given as mean ± SD. The variability was measured
by the CV, which is the ratio of the SD to the mean. The large sample
approximation of the Kolmogorov-Smirnoff test (KS test) was used to
compare the distributions of the mIPSC parameters. The paired or
unpaired Student's two-tailed t test was used to examine
the level of significance of the results.
Solutions. The extracellular standard ACSF contained (in
mM): NaCl 126, KCl 1.5, KH2PO4
1.25, MgSO4 1.5, CaCl2 2, NaHCO3
26, and glucose 10. GABAA-mediated mIPSCs were recorded in
the presence 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM; Tocris) and D()-2-amino-5-phosphonopentanoic acid (APV, 50 µM; Tocris) to block non-NMDA- and NMDA-mediated
glutamatergic synaptic currents, and tetrodotoxin (TTX) to block action
potentials (1 µM; Sigma, Janssen, or Latoxan). The mIPSCs
or the GABA-evoked currents in outside-out patches were blocked by
bicuculline methiodide (10 µM; Sigma) or picrotoxin (100 µM; Sigma). Zolpidem was a gift from Synthelabo
Recherche, and flumazenil (Ro 15-788) was a gift from R. Corradetti
(University of Firenze). Zolpidem was dissolved in water in stock
solutions (5 mM), and flumazenil was dissolved in DMSO. The
final fraction of DMSO was 0.2%, which had no effect on mIPSCs
(n = 2) or on the effect of zolpidem (n = 4). In our preparation, the recovery after an application of zolpidem
was not complete after 30 min wash out; therefore we took a new slice after each zolpidem inflow.
Intrapipette solutions for whole-cell recording contained (in
mM): CsCl 140, HEPES 10, MgCl2 3, EGTA 0.5, pH
7.3, 280 mOsm. For outside-out patches and some whole-cell recordings,
intrapipette solutions contained (in mM): CsCl 120, HEPES
10, ATP 4, GTP 0.5, MgCl2 2, EGTA 10, pH 7.3, 280 mOsm.
Potentials were corrected for a 4 mV junction potential. Because no
differences were seen between the recordings obtained with both
intracellular solutions, the results were pooled together.
Fast application of GABA was performed on outside-out patches as
described previously (Colquhoun et al., 1992). The control solution
contained (in mM): NaCl 140, CaCl2 2, KCl 1.5, MgCl2 1, HEPES 10, adjusted to pH 7.4. In the
GABA-containing solution, we added 30 mM sucrose to
visualize the interface between the control and the GABA solutions, and
10 mM NaCl to measure the 10-90% exchange time between
the control and the agonist solutions after blowing out the patch,
which was typically 0.2 msec (see Fig. 5). Applications were made every
10 sec to avoid desensitization of the GABAA receptors.
Usually, the responses during the first few GABA applications tended to
diminish before reaching a stationary level. This initial amplitude
decrease did not seem to be attributable to cumulative desensitization
of the receptors, because it was not dependent on the application
frequency and was not reversed if the application was stopped. Then the
response could remain stable for up to 20 min. Up to four different
solutions could be applied in each barrel by switching the perfusion
tubes with a valve. The exchange time between the two solutions was
~30 sec. When the effect of bicuculline or zolpidem was tested on the
response to the application of GABA, both control and GABA solutions
contained the modulators.
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RESULTS |
Description of mIPSCs
Spontaneous GABAA-mediated miniature postsynaptic
currents were recorded in layer V cortical pyramidal neurons in
the presence of 10 µM CNQX, 50 µM APV, and
1 µM TTX at a holding potential of 70 mV. The
distributions of time intervals between events were fitted by a single
exponential, as expected for a random Poisson process (see Fig.
2D). The mean amplitudes of mIPSCs were 37.3 ± 9.2 pA (range, 21-57 pA; n = 18). The amplitude
distributions of the mIPSCs recorded in each neuron were highly
variable (CV = 0.56 ± 0.08; range, 0.42-0.69) and not
normally distributed but skewed toward high values (Fig.
1A), as in several
other preparations (Edwards et al., 1990; Ropert et al., 1990; Frerking
et al., 1995; Soltesz et al., 1995; Nusser et al., 1997). The durations
(see calculation in Materials and Methods) and 10-90% rise times of the mIPSCs were also highly variable, and their distributions were also
skewed toward high values (Fig. 1A). Their mean
values were 14.5 ± 2.7 msec (range, 10.7-19.5 msec) and
0.98 ± 0.11 (range, 0.79-1.13 msec), respectively, and their CVs
were 0.49 ± 0.13 and 0.44 ± 0.11, respectively. We saw a
very low positive correlation between duration and rise time of mIPSCs
(correlation coefficient: 0.17 ± 0.13, slope of the linear
regression 0.012 ± 0.009) (Fig. 1C), and no
significant correlation (correlation coefficient < 0.3) between
the amplitude and the rise time or the duration of mIPSCs. This result
indicates that dendritic filtering does not play an important role in
shaping the distribution of mIPSC kinetics (Jonas et al., 1993; Soltesz
et al., 1995).
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Figure 1.
Distribution of miniature IPSCs recorded from a
layer V cortical pyramidal cell. A, Histograms of amplitude
(bin size 2 pA, 457 events), rise time (bin size 0.1 msec, 457 events),
and duration (bin size 1 msec, 170 events) of miniature IPSCs occuring
in 1 min. The mean ± SD of these parameters are 32.4 ± 19.3 pA, 1.00 ± 0.42 msec, and 14.5 ± 7.6 msec,
respectively. In the amplitude histogram, the noise distribution is
also shown (black histogram). B, The average of
mIPSCs with no overlap (taken for duration measurements) is shown for
the same cell. Its decay can be fitted by a single exponential, with
= 13.7 msec. The duration of the same average mIPSC calculated with
the method used for individual events is e = 13.5 msec.
C, Plot of duration versus rise time shows no strong
correlation between these two parameters (correlation coefficient,
0.28; slope of the regression, 0.015).
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Effect of zolpidem on mIPSCs
The effect of the BZD agonist zolpidem (10 µM) on
mIPSCs was studied in layer V pyramidal cells (n = 12).
It did not change the frequency of events (5.7 ± 3.0 Hz in
control vs 6.1 ± 3.5 Hz in zolpidem; p > 0.05, paired t test), consistent with the purely postsynaptic
actions ascribed to this compound (Fig.
2C). Moreover, the input
resistance of the cells and the noise level of the recordings were not
changed by zolpidem. The observed effects are thus presumably caused
exclusively by the binding of zolpidem to postsynaptic
GABAA receptors.
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Figure 2.
Effects of bath application of 10 µM
zolpidem on the mIPSCs recorded in a layer V cortical pyramidal cell.
A, Recordings of miniature activity before and during the
application of 10 µM zolpidem. B, Averages of
mIPSCs in control (thin line, 329 events) and in 10 µM zolpidem (thick line, 313 events).
Inset shows both traces normalized to their peak amplitudes.
C, Cumulative histograms of amplitude, duration, and
interevent interval in control (thin line) and zolpidem
(thick line). The mean mIPSC amplitude is 47 ± 22 pA
in control and 61 ± 29 pA in zolpidem, and the mean mIPSC
duration is 19.5 ± 6.5 msec in control and 32.5 ± 9.6 msec
in zolpidem. The distributions of these two parameters in zolpidem are
significantly different from the control distributions (KS test;
p < 0.001). The dotted lines show the
calculated distributions of uniformly potentiated control values, which
are not significantly different from the distribution in zolpidem (KS
test; p > 0.05). The cumulative histograms of
interevent intervals in control and zolpidem are not significantly
different (p > 0.05).
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Zolpidem applied in the bath enhanced significantly both the duration
(163 ± 14% of control; p < 0.001) and the
amplitude (138 ± 26% of control; p < 0.005) of
events (Fig. 2B). The amplitude distributions in
control and in 10 µM zolpidem were significantly different (KS test; p < 0.001) in 10 of 12 pyramidal
cells, and the distributions of durations were significantly different
in the 12 cells (KS test; p < 0.001). Zolpidem is
among the most selective known BZD agonists: three types of
GABAA receptors with high, intermediate, or low affinity
for zolpidem can be differentiated (Lüddens et al., 1995).
To test whether the zolpidem-induced increases in amplitude and
duration were uniform, we compared the mIPSC distributions with and
without zolpidem. We normalized the control distributions by a scaling
factor equal to the ratio of the amplitude (or duration) in zolpidem
and in control. These two distributions were not significantly
different (KS test; p > 0.05) in the 12 pyramidal
cells tested (Fig. 2C); therefore, the hypothesis of a
non-uniform population of GABAergic synapses with distinct
GABAA receptor subtypes activated in the presence of TTX is
not supported by our data.
Even if all of the postsynaptic receptor clusters, active during TTX
application, are equally affected by zolpidem, not all of the receptors
in a synapse are necessarily equally sensitive to this compound. To
test the intrasynaptic heterogeneity of GABAA receptors
underlying mIPSCs in these cells, we looked at the effect of several
concentrations of zolpidem (Fig. 3). The
potentiation of the duration and amplitude were approximately parallel
at low concentrations (<10 µM), but the effect on the
amplitude decreased at the highest concentration tested (100 µM). In contrast, the dose-response curve for the
duration could be fitted by the logistic equation (see Materials and
Methods), and the calculated EC50, Hill coefficient,
and maximal effect were 5.8 µM, 0.36, and 221% of
control duration, respectively. The effect of zolpidem (1 µM) was reversed by the BZD antagonist flumazenil (10 µM): the mean amplitude of mIPSCs returned to 94 ± 3% of control (n = 5), and their duration returned to
104 ± 5% of control (Fig. 3C).
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Figure 3.
Concentration-dependent effects of zolpidem on
mIPSCs in neocortical pyramidal cells. A,a, Average mIPSCs
recorded in a single cell in control conditions and during the
application of 0.1, 1, 10, and 100 µM zolpidem, from
left to right. b, The traces are
scaled to the control amplitude to show the effect of zolpidem on the
duration of the currents. B, Effect of zolpidem on
(a) mean mIPSC amplitude and (b) mean mIPSC
duration. The values obtained in various concentrations of zolpidem are
plotted as ratios over control values. Each point represents the
mean ± SD of the number of cells given in parentheses.
The parameters of the fitted sigmoidal curve (see Materials and
Methods) are EC50, 5.8 µM;
h, 0.36; Max, 221%. C, Antagonistic
effect of flumazenil (dotted line, 10 µM) on
the effect of zolpidem (thick line, 1 µM)
shown on one cell. Bottom, Summary graph (n = 5) of the effect of zolpidem and flumazenil on the amplitude
(open bars) and duration (black bars) of
mIPSCs.
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It should be noted that the mIPSCs recorded are filtered to some
extent, compared with the synaptic conductance (Llano et al., 1991;
Jonas et al., 1993). Therefore, we examined whether a lengthening of
the synaptic current at its source could induce a significant increase
in the peak amplitude of the recorded current caused by filtering. The
filter (cell and recording system) can be modeled in a first
approximation as a low-pass capacitive filter, with a time constant
T. Because there is no correlation between the amplitude,
rise time, and duration of mIPSCs (Fig. 1C), the events are
presumably filtered to the same extent. We assumed a current source at
the synapse Is(t) with an
instantaneous rise and an exponential decay and calculated how this
current amplitude is changed by filtering. The current source is
Is(t) = I0*exp(t/) for t  0 and I(t) = 0 for t < 0. The
current recorded at the soma is I(t) = I0*(/( T))*(exp(t/) exp(t/T)) for t 0 (Llano et al., 1991). The time-to-peak of the current recorded at the
soma is t0 = (*T/( T))*ln(/T), its amplitude is
I(t0) = I0*exp(t0/),
and its estimated time constant (see Materials and Methods) is
e = /I(t0) = *exp(t0/). The highest rise time and the lowest duration of the recorded mIPSCs give values for events
that are the most sensitive to filtering. They were chosen to estimate
T and . The highest 10-90% rise time of the mIPSCs (1.1 msec) gives a value of t0 equal to 1.5 msec.
From this value and the fastest decay of the mIPSCs (10.8 msec), we
obtain a value of equal to 9 msec and a value of T equal
to 0.5 msec. In these conditions, when is doubled, which is more
than the change in the mIPSC duration observed in 10 µM
zolpidem (163% of the control value), the recorded mIPSC amplitude
would be 107% of the control amplitude, far lower than the value found
(Fig. 3). Thus the increase of the amplitude of the recorded mIPSCs
during zolpidem application is most likely mainly caused by an increase
of the current source amplitude.
A postsynaptic current is attributable to the binding of the
neurotransmitter and the activation of Nb
independent channels (Edmonds et al., 1995). The opening probability of
a channel that has bound the neurotransmitter is a function of time,
termed Po(t), and therefore the
postsynaptic current can be decomposed as I(t) = Nb*Po(t)*i,
where i is the current carried by a single channel. Thus the
increase of the mean mIPSC peak amplitude Ipeak
by zolpidem (Figs. 2, 3) can be attributable to the increase of these
three different terms: Nb,
Po,max, or i. The following
experiments were performed to identify which terms are changed by zolpidem.
Peak-scaled non-stationary variance analysis of mIPSCs
The elementary current i can be derived from
peak-scaled nonstationary variance analysis of postsynaptic currents
(Traynelis et al., 1993; De Koninck and Mody, 1994; Silver et al.,
1996). In six pyramidal cells where the frequency was low enough to
perform such an analysis (Fig. 4), we
found i = 1.85 ± 0.17 pA, which leads, taking a
reversal potential of 0 mV (Fig.
5B), to a single-channel conductance of 26.4 ± 2.4 pS. The mean number of channels open at
the peak (Np) for this sample is
30.9 ± 7.3. When zolpidem (1 or 10 µM) is applied,
the elementary current i remains constant (1.88 ± 0.26 pA, 103 ± 18% of control value; p = 0.8),
whereas Np is enhanced (39.1 ± 7.6, 132 ± 36% of control; p = 0.06). Thus we can
conclude that the enhancement of mIPSC amplitude by zolpidem is not
caused by an increase in the single-channel conductance of postsynaptic
GABAA receptors. However, because the mIPSCs are highly
variable in amplitude (Fig. 1), presumably because the number of
channels at different synapses or the amount of GABA released is
variable, a scaling procedure was used, and thus it could not be
determined by this method whether Nb or
Po,max was enhanced. To answer these questions,
we used a system in which the number of receptors was constant and we
could control the concentration of GABA applied; that is, fast
applications of GABA to outside-out patches.
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Figure 4.
Peak-scaled non-stationary variance analysis of
mIPSCs before and during zolpidem application. A, Individual
mIPSCs are shown (dotted lines) with the average currents
(thick lines) in control and during bath application of 1 µM zolpidem. B, Relationship between the
current and the variance in control and in zolpidem. The curves were
fitted with the equation 2 = iI I2/Np. The values
obtained are 1.75 and 1.82 pA for i, in control and
zolpidem, respectively, and 25.4 and 46.7 for
Np, in control and zolpidem.
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Figure 5.
Fast applications of GABA to an outside-out patch
excised from a pyramidal cell. A, The current elicited by
application of GABA (1 mM) during 1 msec is blocked by
bicuculline (10 µM). The traces represent the averages of
five applications. Bicuculline was added in the control and the GABA
flow. The trace above represents the response once the patch
was blown out, showing the duration of the GABA application.
B, GABA-evoked currents at various membrane potentials.
Traces shown at 70, 50, 30, 10, 0, 10, 30, and 50 mV.
Right, I-V curve of the peak currents. The
reversal potential calculated from the second order polynomial fit is 0 mV. Inset shows the traces at 70 and +50 mV normalized and
superimposed. C, Non-stationary fluctuation analysis of GABA
(10 mM)-evoked currents. a, One individual
current (dotted line) is shown superimposed with the average
current. b, Plot of the current amplitude for successive
applications. c, Plot of the variance-mean current curve,
fitted with the equation 2 = iI I2/N, with i = 1.69 and N = 201. The maximal open probability,
Po,max, is 0.65.
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Fast application of GABA to outside-out patches of layer V
pyramidal neurons
After synaptic release, the neurotransmitter is thought to be
present only very briefly at high concentration in the synaptic cleft
(Clements et al., 1992). This synaptic concentration transient can be
mimicked by short (1 msec) agonist applications to outside-out patches
(Colquhoun et al., 1992; Jones and Westbrook, 1995; Galarreta and
Hestrin, 1997). To test whether zolpidem can enhance the maximal probability of opening (Po,max) of
GABAA receptors in conditions similar to those during
synaptic release, we first determined at which concentration of GABA
the receptors are saturated by a 1 msec application to outside-out patches.
The currents elicited by short pulses (1 msec) of 1 mM GABA
are illustrated in Figure 5. The
GABA currents are blocked by bicuculline (10 µM;
n = 3). Their reversal potential is 0 mV
(n = 7), corresponding to the chloride equilibrium
potential in our recording conditions. On some patches, single-channel
openings could be resolved (Fig.
7D) with an elementary current
of 1.78 ± 0.19 pA (n = 6), leading to a chord
conductance of 25.4 ± 2.7 pS, which is close to the value found
for channels underlying mIPSCs. The decay of these currents can be,
like mIPSCs, well fitted by the sum of two exponentials (Table
1). However, the slowest component is
much greater in patches than for mIPSCs, as reported previously in
various preparations (Tia et al., 1996; Galarreta and Hestrin, 1997;
Mellor and Randall, 1997). The deactivation kinetics of
GABAA receptor-channels are voltage independent (Fig. 5,
Table 1).
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Figure 6.
Concentration-response curve for 1 msec GABA
applications to outside-out patches. A, Responses of a patch
to 100 µM, 300 µM, and 1 mM
GABA. Averages of five traces. The open pipette response is shown
above. B, Concentration-amplitude curve, normalized to the
response to 1 mM GABA. The number of patches used for each
point (mean ± SD) is given in parentheses. The curve
is fitted by a sigmoidal function, whose parameters are
EC50 = 310 µM, h = 1.74, Imax = 1.13.
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Figure 7.
Effects of zolpidem on currents elicited by 1 msec
applications of GABA to an outside-out patch taken from a pyramidal
neuron. A, Averages of five traces before (thin
line) and during (thick line) the application of 1 µM zolpidem. Left, Application of 100 µM GABA. Right, Application of 10 mM GABA. Data were obtained for two different patches.
B, Same traces as in A, normalized and on a
greater time scale. The rise time of the currents is not changed by
zolpidem at the two GABA concentrations. C, Plot of the
change in amplitude induced by zolpidem (1 µM) at
different GABA concentrations. Significant differences between control
and zolpidem for 100 (p < 0.005) and 300 µM GABA (p < 0.05). The
dotted line shows the value found for mIPSCs (see Fig. 3).
D, In this patch, the application of 300 µM
GABA induced the opening of only a few GABAA receptor
channels (filter corner frequency, 1 kHz). Thus single-channel openings
could be clearly resolved, giving an elementary current of 1.9 pA
(conductance, 27 pS). Zolpidem (1 µM) induced a 40%
enhancement of the peak amplitude, as seen at the bottom traces
(averages of 20 responses in each condition), but no change in the
channel conductance, as seen in the single-channel recordings.
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NSFA was performed on three patches (Fig. 5C) with
saturating applications of GABA (10 mM) (see below). The
fits of the variance-mean current gave an elementary current of
1.67 ± 0.02 pA (conductance: 23.9 ± 0.3 pS). This value
is close to the one found for channels underlying mIPSCs and to
measured single channel openings. Po,max could
also be calculated (see Materials and Methods), which gave 0.61 ± 0.06 (n = 3). This value is in good agreement with
previously reported values for GABAA receptors (Jones and
Westbrook, 1997).
We constructed a concentration-response curve by taking the response
to 1 msec pulses of 1 mM GABA as a reference and changing for one or more other concentrations on the same patch (Fig. 6). We
measured an EC50 of 310 µM, a Hill
coefficient of 1.74, and a maximal relative response of 1.13, which is
close to previously reported values for the same cells (Galarreta and
Hestrin, 1997). At a concentration of 10 mM the receptors
are saturated, so we took this concentration of GABA to test the effect
of zolpidem on Po,max.
We also examined the changes in the kinetics of the GABA-evoked
currents with the concentration of agonist applied. The 10-90% rise
time of the currents was reduced as the concentration of GABA increased
(Table 1). We did not see any significant change in the decay kinetics
of the GABA-evoked currents for concentrations of GABA between 300 µM and 10 mM. We saw a significant decrease of the mean with 100 µM GABA, compared with 1 mM, which elicits only 11 ± 9% of the maximal
response, as observed previously (Jones and Westbrook, 1995; Galarreta
and Hestrin, 1997).
Effect of zolpidem on outside-out patches of layer V
pyramidal cells
We tested the effect of 1 µM zolpidem on currents
elicited by various concentrations of GABA for 1 msec on outside-out
patches. When the concentration of GABA was not saturating (100 or 300 µM), the amplitude of the current was significantly
enhanced by zolpidem, whereas it remained constant when 10 mM GABA was applied (Fig. 7). The rise time of the currents
was not affected by zolpidem at all GABA concentrations (Fig.
7B). The effect of zolpidem (1 µM) on the
amplitude of the current was approximately the same when 300 µM GABA was applied during 1 msec (141 ± 22% of
the control response; n = 4) as its effect on mIPSC
amplitude (Fig. 7C). The application of zolpidem did not
change the conductance of the channels when single-channel openings
were examined (n = 2) (Fig. 7D). The
duration of the GABA-evoked currents was also enhanced by zolpidem
(133 ± 26, 122 ± 11, and 134 ± 13% of control;
p < 0.05, for 0.1, 0.3, and 10 mM GABA, respectively).
These data show that zolpidem does not enhance the maximal open
probability of bound GABAA receptors in outside-out
patches. Therefore, it is unlikely that an increased
Po,max could account for the increase of mIPSC
amplitude observed in the presence of zolpidem. The most likely
explanation is that Nb, the number of receptors that have bound the neurotransmitter, is enhanced as a result
of an increase of the affinity of GABA to its receptor.
Activation of GABAA receptors at
physiological temperature
We recorded mIPSCs in layer V pyramidal cells in a more
physiological situation, i.e., at a temperature of 35°C. When
compared with room temperature recordings, the frequency of mIPSCs is
increased (12.8 ± 6.3 Hz; n = 8), and their
kinetics is accelerated: the mean rise time is 0.63 ± 0.16 msec,
and the mean duration is 7.4 ± 1.8 msec. The mean amplitude of
mIPSCs is also increased (49.3 ± 4.5 pA). In five cells, the
elementary current, determined by peak-scaled NSFA, was 2.23 ± 0.13 pA, which gives a chord conductance of 31.9 ± 1.8 pS,
significantly different from the value determined at room temperature
(p < 0.05). On the other hand, the mean number of channels open at the peak of mIPSCs
(Np), calculated as the ratio of the mean
current to the elementary current, was not significantly different
(p > 0.2) at room temperature (20.2 ± 5.0) and at 35°C (22.1 ± 2.0). This suggests that the degree of
occupancy of the receptors is the same at both temperatures.
To examine this latter issue further, we determined the sensitivity of
GABAA receptors to fast applications of GABA on outside-out patches. At 35°C, the current evoked by 10 mM GABA during
1 msec had a rise time of 0.43 ± 0.22 msec (n = 7) and a duration of 16.8 ± 8.0 msec, which was significantly
shorter than at room temperature (p < 0.001).
In two patches, we estimated Po,max (see Materials and Methods). Taking i = 2.2 pA, measured
on single-channel openings in three patches (data not shown), which is
also the value determined by peak-scaled NSFA applied to mIPSCs at
35°C, a value of Po,max equal to 0.8 was found
for both patches. Consequently, we can estimate the number of bound
receptors during an mIPSC (Silver et al., 1996),
Nb = Np/Po,max,
31 ± 8 at room temperature and 28 ± 3 at 35°C, which are
not significantly different (p > 0.2). Assuming
that the number of functional postsynaptic receptors is the same at the
two temperatures, then the degree of occupancy of GABAA
receptors during synaptic transmission is similar. Moreover, the
amplitude of the current evoked by 300 µM GABA, which is
the measured EC50 for such applications at room
temperature, was 30.1 ± 6.9% (n = 7) of
the response to 10 mM GABA (Fig.
8A). At this concentration, the rise time was 0.66 ± 0.18 msec and the
duration was 19.3 ± 6.8 msec (n = 12). If we
assume that the Hill coefficient is the same at room temperature and at
35°C (1.74), we estimate a value of 490 µM for the
EC50 at 35°C, which is higher than the EC50
at 25°C (310 µM).
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Figure 8.
Activation of GABAA receptors at
physiological temperature. A, Peak-scaled NSFA was performed
on 95 mIPSCs recorded at 35°C. The average (thick line)
and three events (dotted line) are shown. The parabolic fit
of the variance-mean current gave i = 2.14 pA and
Np = 37.3. B, Currents (averages of 5 traces) evoked by the application of 300 µM (small
trace) and 10 mM (large trace) GABA to an
outside-out patch during 1 msec at 35°C. The top trace
shows the open pipette response.
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Effect of zolpidem on GABAA receptors at 35°C
The effect of bath-applied zolpidem (1 µM) was
examined on mIPSCs recorded in layer V pyramidal cells at 35°C (Fig.
9A). As seen at room
temperature, zolpidem did not change the frequency of mIPSCs (103 ± 18% of control frequency; n = 6) and enhanced the
duration of events (160 ± 30% of control; p < 0.005). The two duration distributions were significantly different in
the six cells tested (KS test; p < 0.001).
The potentiation of the duration was also uniform, as at room
temperature: when the duration distributions in control were scaled to
the distributions in zolpidem, the resulting distributions were not
significantly different (KS test; p > 0.05) from the
one in zolpidem. However, the amplitude of mIPSCs was much less
enhanced by zolpidem than at room temperature [108 ± 4% of the
control amplitude; when cells are taken individually, a significant
difference between the two distributions (KS test; p < 0.05) was detected in only one of six cells].
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Figure 9.
Action of zolpidem at physiological temperature.
A, Effect of zolpidem on mIPSCs at 35°C. Average mIPSC
before (thin line) and during (thick line) the
application of 1 µM zolpidem. B, Cumulative
histograms of amplitude and duration of mIPSCs before (thin
line) and during (thick line) zolpidem application.
Amplitudes are not significantly different (54.7 ± 38.4 pA in
control, 630 events vs 55.6 ± 40.9 pA in zolpidem, 689 events;
KS test, p > 0.05), whereas durations are different
(7.5 ± 3.1 msec in control, 166 events and 10.2 ± 3.5 msec
in zolpidem, 153 events; KS test, p < 0.001). The
potentiation of durations is uniform: the control distribution scaled
to the distribution in zolpidem (dotted line) and the
zolpidem distribution are not significantly different
(p > 0.05). C, Effect of zolpidem (1 µM) on currents evoked by 300 µM GABA at
35°C. Average of five traces before (thin line) and during
(thick line) zolpidem application. The trace on
top shows the open pipette response. Right, The same
traces are shown on a greater time scale.
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We then examined whether zolpidem could enhance the amplitude of
currents evoked by subsaturating GABA applications. As shown on Figure
9C, the amplitude of the current evoked by 300 µM GABA applications on outside-out patches at 35°C was
not enhanced by zolpidem (104 ± 8% of the control amplitude;
n = 5), whereas its duration was increased (135 ± 22% of control). Therefore, zolpidem does not seem to be able to
reveal the degree of occupancy of GABAA receptors at high temperature.
| |
DISCUSSION |
We have shown that at GABAergic synapses, the BZD agonist zolpidem
enhances, in a concentration-dependent manner, both the duration and
amplitude of mIPSCs recorded at room temperature. The increase in
amplitude is attributable to neither an increase in the conductance of
the channels, as demonstrated by peak-scaled NSFA, nor the enhancement
of the maximal open probability of GABAA channels, as shown
by the effect of zolpidem on currents evoked by fast applications of
saturating doses of GABA to outside-out patches. We therefore propose
that zolpidem, by enhancing the affinity of the receptors for GABA,
increases the number of receptors bound during transmitter release,
indicating that GABAA receptors are not saturated after
release of a single quantum.
Effects of zolpidem on GABAA receptors
Several observations argue for an increase of affinity, and more
specifically, of the binding rate of GABA to its receptor in the
presence of BZD agonists (MacDonald and Olsen, 1994). These compounds
decrease the EC50 for GABA, without changing the maximal current evoked by GABA (Sigel and Baur, 1988), and increase the opening
frequency of GABAA channels, without changing their mean open time and burst duration (Study and Barker, 1981; Rogers et al.,
1994). Moreover, in agreement with Lavoie and Twyman (1996) and Mellor
and Randall (1997), we have shown that at room temperature the
amplitude of GABA-elicited currents can be enhanced by BZD agonists
only when a subsaturating GABA concentration is applied. Moreover, in
agreement with these studies, we have shown that the rise rate of
GABA-evoked current is not enhanced by BZD agonists when a saturating
GABA concentration is tested, consistent with an effect on the binding
rate (Lavoie and Twyman, 1996). However, at 35°C, the amplitude of
currents evoked by a subsaturating GABA application was not enhanced by
zolpidem (Fig. 9C), providing evidence that the binding rate
of GABA is little affected at this temperature. A possible explanation
of these results is that the activation energy necessary for GABA
binding and channel opening, which determines its activation
kinetics (Jones et al., 1998), is lowered by zolpidem at 25°C and is
unchanged at 35°C.
BZD agonists also increase the duration of mIPSCs in several neuron
types (this study; De Koninck and Mody, 1994; Puia et al., 1994; Poncer
et al., 1996; Mellor and Randall, 1997; Nusser et al., 1997), and that
of GABA-elicited currents on outside-out patches [this study; Mellor
and Randall (1997); but see Lavoie and Twyman (1996)]. Moreover,
it has been shown that the duration of the current elicited by a brief
agonist pulse is inversely correlated to the agonist unbinding rate
(Jones and Westbrook, 1995; Jones et al., 1998). Therefore, the
affinity increase of the GABAA receptor for its endogenous
ligand by zolpidem can be attributed to a concomitant increase of the
binding rate and decrease of the unbinding rate.
BZDs have been reported to enhance the conductance of GABAA
channels in dentate gyrus granule cells (Eghbali et al., 1997). However, this effect was seen only when a low concentration (<5 µM) of GABA was applied, and small conductance openings
were observed. During synaptic transmission, however, a high
concentration (>100 µM) of neurotransmitter is thought
to be experienced by the receptors (Clements et al., 1992), so that
this effect would not take place for the mIPSCs. We showed, with
peak-scaled NSFA, that the conductance of channels underlying the
mIPSCs was not changed by zolpidem. Consistent with this observation,
the conductance of channels activated by fast GABA application on
outside-out patches was also unchanged by zolpidem.
The effect of zolpidem on mIPSC amplitude decreased at the highest
concentration tested (100 µM) (Fig. 4). A bell-shaped
dose-response curve of BZD agonists has been reported in several
studies using whole-cell applications of agonist (Sigel and Baur, 1988)
and in single-channel studies (Rogers et al., 1994; Eghbali et al., 1997). The mechanism of such a behavior is unknown but could involve receptor desensitization (Mellor and Randall, 1997).
Use of fast applications to outside-out patches as a
model synapse
The method of fast applications to outside-out patches offers the
best technical approach currently available to mimic the synaptic
release of transmitter. The GABA-elicited currents had fast rise times,
like those of mIPSCs, and the single-channel conductance, determined
directly or by NSFA, is close to that found for mIPSCs. However,
receptors in patches may not behave exactly as in the synapse. The
deactivation kinetics of currents evoked by GABA applications on
outside-out patches is much slower than that of mIPSCs (this
study; Tia et al., 1996; Galarreta and Hestrin, 1997; Jones and
Westbrook, 1997) and is voltage independent, unlike that of mIPSCs in
the same cells (Salin and Prince, 1996). Several explanations can be
proposed: mechanical disturbance of channels during patch excision or
loss of intracellular factors and a different state of phosphorylation
(Jones and Westbrook, 1997) may change the behavior of channels;
receptors in patches may differ from synaptic receptors (Tia et al.,
1996); the concentration in the synaptic cleft may be much lower than
usually thought (Galarreta and Hestrin, 1997); and the duration of GABA
application, which determines partly the deactivation kinetics of
GABAA receptors (Jones and Westbrook, 1995; Mellor and
Randall, 1997), may be longer than the actual timecourse of the
transmitter in the synaptic cleft. In any case, the discrepancy lies
only in the late part of the response; thus the binding and opening
rates are probably the same for synaptic channels and for channels in patches.
Implications for GABAergic synaptic transmission
After observing that zolpidem increased the duration but not the
amplitude of mIPSCs in dentate gyrus granule cells at 35°C, De
Koninck and Mody (1994) concluded that postsynaptic receptors were
saturated by GABA during synaptic transmission. However, Frerking et
al. (1995) have shown in culture at 25°C that diazepam, another BZD
agonist, potentiates mIPSC amplitudes, and they proposed that the mIPSC
amplitude is correlated with the peak concentration of transmitter
released in the synaptic cleft and hence that GABAA receptors are not saturated during synaptic transmission. It has been
shown in various structures that the mIPSC amplitude could be enhanced
at room temperature by BZD agonists (DeFazio and Hablitz, 1997; Mellor
and Randall, 1997; Nusser et al., 1997). In another study (Poncer et
al., 1996), no increase in mIPSC amplitude was seen, suggesting
GABAA receptor saturation in CA3 pyramidal cells. In
cerebellar stellate cells, two populations of GABAergic synapses seem
to have different degrees of receptor occupancy. The smallest mIPSCs
have their amplitude unaffected by the BZD agonist flurazepam, whereas
the amplitude of the largest mIPSCs is enhanced (Nusser et al., 1997).
With a different approach, Auger and Marty (1997) reached a similar
conclusion, showing on the same cells a negative correlation between
the peak open probability of channels at single synapses and the number
of channels, which can be interpreted as a lower degree of occupancy in
larger synapses. In layer V pyramidal cells, we found that the
potentiation of mIPSC amplitude is uniform (Fig. 2), suggesting that
the degree of receptor occupancy is not maximal and is similar for all
of the synapses with miniature activity. An estimate of the
concentration reached by GABA in the synaptic cleft can be made by
matching the enhancement of the mIPSC amplitude with that of the
GABA-elicited currents using 1 µM of zolpidem (Fig.
7C): it gives a concentration of 300 µM, which
is the EC50 found for the GABA dose-response curve (Fig. 6) (Galarreta and Hestrin, 1997). However, it should be noted that the duration of the application to patches (1 msec) is much longer
than estimates of the dwell times of neurotransmitter in the
cleft (Clements et al., 1992; Holmes 1995; Clements, 1996); thus the
peak concentration could be higher to achieve the same degree of occupancy.
For non-NMDA glutamate receptors, receptor occupancy during uniquantal
synaptic transmission is thought to remain the same (Silver et al.,
1996) or diminish as temperature is increased (Tong and Jahr, 1994).
Thus we might expect GABAA receptors not to be saturated at
physiological temperature. Consistent with this prediction, we found
that the EC50 for 1 msec applications is higher at 35°C
than at room temperature, and that the mean number of channels open at
the peak of an mIPSC is not significantly changed between the two
temperatures (also see De Koninck and Mody, 1994). However, we did not
see any significant change in the mIPSC amplitude when zolpidem was
applied at 35°C, as seen in other cell types (De Koninck and Mody,
1994; Soltesz and Mody, 1994; Poisbeau et al., 1997). We have also
shown that zolpidem did not enhance the amplitude of currents evoked by
subsaturating GABA concentration on outside-out patches. Therefore, no
conclusion can be drawn from the effect of zolpidem on mIPSCs regarding
receptor occupancy at pysiological temperature.
Our study shows that the postsynaptic GABAA receptors
expressed by layer V pyramidal cells in visual cortex are not saturated by the release of GABA from a single vesicle, but this may not be the
case in all GABAergic synapses (Auger and Marty, 1997; Nusser et al.,
1997). Moreover, the release of multiple vesicles at a single active
zone could increase receptor occupancy and eventually saturate
postsynaptic receptors (Silver et al., 1996). In this latter case the
variability of the postsynaptic current is very low. Modulation of the
release probability of neurotransmitter could thus regulate both the
strength and the variability of synaptic transmission.
| |
FOOTNOTES |
Received April 30, 1998; revised Oct. 22, 1998; accepted Oct. 29, 1998.
We thank Drs. F. Sladeczek and F. Le Bouffant for their support with
some of the equipment, and G. Sadoc for help with the acquisition and
analysis software. We also thank Drs. B. Barbour and M. Häusser,
and N. Gazères for useful discussions.
Correspondence should be addressed to Dr. Nicole Ropert, Institut
Alfred Fessard, Centre National de la Recherche Scientifique UPR
2212, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France.
| |
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Top
Abstract
Introduction
