Department of Physiology, University of Munich, 80336 Munich, Germany
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ABSTRACT |
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Rumpel, Eva and
Jan C. Behrends.
Postsynaptic Receptor Occupancy During Evoked Transmission at
Striatal GABAergic Synapses In Vitro.
J. Neurophysiol. 84: 771-779, 2000.
The effect of benzodiazepines
(BZs) on GABAA-ergic synaptic responses depends
on the control receptor occupancy: the BZ-induced enhancement of
receptor affinity can lead to greater peak amplitudes of quantal
responses only when, under normal conditions, receptors are not fully
saturated at peak. Based on this fact, receptor occupancy at the peak
of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) has
been assessed in various mammalian neuronal preparations. To use the
same principle with compound (or multiquantal), action
potential-evoked IPSCs, complications introduced by quantal asynchrony
in conjunction with the BZ-induced increase in the decay time of the
quantal responses have to be overcome. We used a simple analytic
convolution model to calculate expected changes in the rise time and
amplitude of postsynaptic currents when the decay time constant, but
not the peak amplitude, of the underlying quantal responses is
increased, this being the expected BZ effect at saturated synapses.
Predictions obtained were compared with the effect of the BZ
flunitrazepam on IPSCs recorded in paired pre- and postsynaptic whole
cell voltage-clamp experiments on striatal neurons in cell culture. In
22 pairs, flunitrazepam (500 nM) reliably prolonged the decay of IPSCs
(49 ± 19%, mean ± SE) and in 18 of 22 cases produced an
enhancement in their peak amplitude that varied markedly between 3 and
77% of control (26.0 ± 5.3%). The corresponding change in rise
time, however (+0.38 ± 0.11 ms, range 0.8 to +1.3 ms) was far
smaller than calculated for the observed changes in peak amplitude
assuming fixed quantal size. Because therefore an increase in quantal
size is required to explain our findings, postsynaptic
GABAA receptors were most likely not saturated
during impulse-evoked transmission at these unitary connections. The
peak amplitudes of miniature IPSCs in these neurons were also increased
by flunitrazepam (500 nM, +26.8 ± 6.6%), and their decay time
constant was increased by 26.3 ± 7.3%. Using these values in our
model led to a slight overestimate of the change in compound IPSC
amplitude (+28 to +30%).
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INTRODUCTION |
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Postsynaptic receptor occupancy
during synaptic transmission is an important variable in determining
whether synaptic strength can be increased by the release of more
transmitter molecules into the synaptic cleft, such as may, for
instance, occur with the coordinated fusion of more than one vesicle at
a single or at multiple neighboring release sites (Behrends and
ten Bruggencate 1998; Edwards 1995
;
Frerking and Wilson 1996
; Kirischuk et al. 1999
; Lewis and Faber 1996
; Paulsen and
Heggelund 1996
; Poisbeau et al. 1996
;
Roepstorff and Lambert 1994
; Tong and Jahr
1994
; Vincent and Marty 1996
).
At GABAergic synapses, the action of benzodiazepines to increase
GABAA receptor affinity (Lavoie and Twyman
1996; Rogers et al. 1994
) makes it possible to
test the hypothesis that the amount of transmitter released in a single
quantum is enough to saturate all available postsynaptic receptors
facing a release site. In such experiments, spontaneous miniature
inhibitory postsynaptic currents (mIPSCs) were recorded in the absence
and presence of benzodiazepine agonists. If receptors are saturated by
quantal release, the amplitude distribution of mIPSCs will remain
unchanged despite an increase in binding affinity. Only if under
control conditions some postsynaptic receptors remain free of agonist will the benzodiazepine produce a shift toward larger peak amplitudes.
This kind of experiment has produced differing results at different
GABAergic synapses. For some synapses, receptors were concluded to be
saturated (De Koninck and Mody 1994), whereas at others,
studies have demonstrated increases in quantal size compatible with
absence of saturation either generally (Frerking et al.
1995
; Mellor and Randall 1997
; Perrais
and Ropert 1999
) or at least at a subset of contacts
(DeFazio and Hablitz 1998
; Nusser et al.
1997
). Very recently, using the same approach, Nusser, Mody and
coworkers have shown further evidence that the degree of receptor
occupancy varies between different preparations, and that saturated and
unsaturated synapses may occur on the same postsynaptic neuron
(Nusser et al. 1999
). Thus it appears that at some
inhibitory CNS synapses at least, receptors are not saturated following
the constitutive release of single quanta of GABA. However, the
conclusion that these contacts are equally unsaturated during impulse-evoked transmission does not automatically follow. With evoked
transmission, release of multiple vesicles (see above) or spillover
from neighboring synapses (Destexhe and Sejnowski 1995
;
Isaacson et al. 1993
; Rossi and Hamann
1998
; Vogt and Nicoll 1999
) may produce an
enhancement of cleft transmitter concentration, driving peak receptor
occupancy to saturation (Silver et al. 1996
). In
addition, it is possible that many spontaneous vesicular fusion events
are rapidly reversible (Rahamimoff and Fernandez 1997
), which would reduce the amount of transmitter released into the synaptic
cleft (Clements 1996
). For these reasons, when
transmission is driven by a presynaptic impulse, receptor occupancy
might well be higher than with spontaneous vesicle fusion.
While several studies have shown that benzodiazepines increase the peak
amplitude of IPSCs evoked by selective activation of a single
presynaptic inhibitory neuron or extracellular stimulation of
inhibitory fibers (Roepstorff and Lambert 1994;
Segal and Barker 1984
; Vicini et al.
1986
; Zhang et al. 1993
; but see Williams et al. 1998
), this does not necessarily indicate absence of
saturation of postsynaptic GABAA receptors at
these synapses. Regardless of the absence or presence of an effect on
peak amplitude, benzodiazepines have invariably been shown to prolong
the decay phase of GABAergic mIPSCs (DeFazio and Hablitz
1998
; De Koninck and Mody 1994
; Frerking et al. 1995
; Mellor and Randall 1997
;
Nusser et al. 1997
; Perrais and Ropert
1999
; Puia et al. 1994
), in agreement with their
effect on the decay phase of responses to short pulses of saturating concentrations of GABA applied to outside-out membrane patches (Mellor and Randall 1997
; Perrais and Ropert
1999
; but see Lavoie and Twyman 1996
). Mody and
coworkers have pointed out that, when quanta are not released in
perfect synchrony, a prolongation by benzodiazepines of the decay phase
of the quantal components will produce improved summation. The peak
amplitude of the compound response will thus be enhanced without an
increase in the peak synaptic responses generated at individual contact
sites (De Koninck and Mody 1994
; Mody et al.
1994
).
The present study asks whether, despite this complication arising from quantal asynchrony and changes in quantal kinetics, information about receptor occupancy may still be obtained from the benzodiazepine effect on evoked IPSCs (i.e., compound, as opposed to quantal, responses). We use a relatively simple model calculation to show that, when asynchronous quantal currents are prolonged and therefore sum more effectively, the resulting enhancement in the peak amplitude of the compound IPSC is inevitably linked to an increase in its rise time, the magnitude of which can be estimated. We compare these predictions to the effect of the benzodiazepine flunitrazepam on IPSCs evoked in paired recordings from pre- and postsynaptic striatal neurons in cell culture. We find that the changes in rise times are far too small to explain the observed increases in IPSC peak amplitudes by improved summation of prolonged quanta, and therefore conclude that it must be largely due to an increase in quantal size at nonsaturated contact sites.
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METHODS |
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Cell culture and solutions
Culture methods closely followed those of Gottmann et al.
(1994). Following deep ether anesthesia, pregnant Wistar rats
were decapitated and the uterus dissected out and placed on an ice-cold glass dish. Embryos (embryonic day 17) were removed and
their brains placed into ice-cold culture medium (Eagle's minimal
essential medium). The lateral ganglionic eminences were dissected and
subjected to mechanical dissociation. Cells were plated at low density
to enhance the probability of synaptic connections between neighboring neurons. Cultures were used for recordings from 12 to 19 days in vitro.
Pipettes were filled with a solution composed of (in mM) 110 KCl, 5 MgCl2, 0.6 EGTA, 10 HEPES, and 2 Na-ATP, pH set to 7.3 with KOH, 230 mosmol l
1. In some
experiments KCl was replaced by CsCl, and 5 mM
2(triethylamino)-N-(2,6-dimethylphenyl)acetamide (QX-314)
was added to the postsynaptic pipette solution. The control bath
solution contained (in mM) 125 NaCl, 1 KCl, 2 MgCl2, 20 HEPES, 10 glucose, and 1 CaCl2, pH set to 7.35 with NaOH, 270 mosmol l
1. The low
Ca2+/Mg2+ ratio was used to
prevent the accumulation of frequency-dependent depression of
transmission at these synapses, which depends on release probability
(Jensen et al. 1999
). All constituents were purchased
from Sigma Germany (Munich). A gravity-driven local application system
(Y-tube) was used to switch to flunitrazepam-containing solution.
Flunitrazepam (Sigma Germany, Deisenhofen) was dissolved in dimethyl
sulfoxide (DMSO) and diluted in external solution to 500 nM. Final DMSO
concentration was 0.0025% and without effect on synaptic transmission
(Rumpel and Behrends 1999
).
Electrophysiological recording
In paired recordings, IPSCs were evoked by short (3-5 ms)
depolarizing step commands to the presynaptic neuron in voltage-clamp mode (holding potential near 70 mV) to elicit unclamped breakaway action currents. Presynaptic stimuli were delivered every five seconds.
All experiments were performed at room temperature (23-25°C) under
direct visual control on a Zeiss IM35 inverted microscope. Borosilicate
pipettes with outer tip diameters of 1.5-2 µm and open tip
resistances of 3-5 M
were used. Two EPC-7 patch-clamp amplifiers
(HEKA-Electronics, Darmstadt, Germany) were used for recording without
series resistance compensation (<20 M
). Output signals were
filtered at 3 kHz and digitized on-line at 24 kHz with a National
Instruments (Austin, TX) NB-MIO 16 l 14-bit A/D converter in an
Apple Macintosh PM8100/100 computer.
Data analysis
The means ± SD of the waveform of 10-20 IPSCs were calculated off-line applying software written in LabView (National Instruments). Rise times, peak amplitudes, and decay kinetics of averaged responses were further analyzed using the software IGORPro (Wavemetrics Lake Oswego, OR). Statistical analysis was done using StatView software (Abacus Concepts, Berkeley, CA). Pooled data are expressed as means ± SE.
Modeling compound IPSCs
The time course of multiquantal, evoked, postsynaptic currents
was modeled as a convolution of quantal current waveforms and release
probability. In an initial simplified approach, we assumed instantaneously rising quantal currents M(t) with
a monoexponential decay obeying the equation
M(t) = M0 · emt. The time course of
release probability G(t) was approximated by an
exponentially decaying function G(t) = G0 · e
gt. A probability density
function has to fulfill the requirement that
0
G(t)dt = 1, i.e., the probability that
a given quantal event will occur between time 0 and infinity
is 100%.
0
G0e
gtdt = 1 only if G0 = g, leading
to the final form of the release probability function
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(1) |
Convolving M(t) and G(t)
results in a difference of two exponentials
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(2) |
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(3) |
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(4) |
However, in the above calculation, the fact that quantal events
themselves have a finite rise time is disregarded. In a second approach, therefore quantal responses were modeled by a difference of
two exponentials
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(5) |
Calculating the maximum of the above function leads to an expression
for the quantal rise time ttpq
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(6) |
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(7) |
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(8) |
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RESULTS |
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Modeling multiquantal IPSCs: an increase in peak amplitude due to a reduced quantal decay rate is coupled to a prolongation of time-to-peak
We used a simple convolution model, as described in
(METHODS), to calculate the dependence of rise time and
peak amplitude on the quantal decay rate m. This value was
chosen to be 0.05 ms1.
This corresponds to a decay time constant of 20 ms, close to the mean
value previously determined in this preparation (Behrends and
ten Bruggencate 1998
). The parameter g represents
the decay rate of the release function and therefore the degree of quantal synchrony. To produce PSC waveforms with rise times in the
experimentally observed range (4.5 ± 0.3 ms, mean ± SE; see RESULTS) (see also Rumpel
and Behrends 1999
), g = 0.5 ms
1 and g = 1 ms
1 were chosen as
boundary values for a first working estimate, resulting in
times-to-peak of 3.15 and 5.12 ms, respectively.
Figure 1 provides a
summary of the modeling results. Analytic results from Eqs.
3 and 4, disregarding the rise time of the quantal
responses (see METHODS), are shown by continuous lines, whereas symbols represent the values obtained graphically from PSC
waveforms calculated according to Eq. 8. Here, a rise
time of the quantal events of 2 ms was assumed (Behrends and ten
Bruggencate 1998). As shown in Fig. 1A, the
time-to-peak of a compound IPSC increases with decreasing quantal decay
rate. This confirms the intuition that with longer duration of quantal
events, more later quanta can overlap with the earliest responses and
add to the rising phase of the compound PSC. With rapidly decaying
quantal events, only the very first quanta will contribute to the peak amplitude, thus keeping the rise time low.
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As expected, inclusion of the rise time of quanta shifts the curves
toward larger times-to-peak (symbols). However, the point of reference
given by the experimentally determined mean time-to-peak of the
compound IPSCs (4.5 ms) with an assumed quantal decay rate of 0.05 ms1 is straddled by the
estimates using g = 1 ms
1 and g = 0.5 ms
1, irrespective
of whether or not the quantal rise time is taken into account.
Similarly, Fig. 1B shows the change in PSC peak amplitude
resulting from a slowing of quantal decay. PSC peak amplitude is given
as a fraction of the maximal amplitude attained when quanta are
released in perfect synchrony (g
) or become step
functions with m
0. An isolated decrease in quantal
decay rate, as expected when receptors are saturated at peak (see
INTRODUCTION), does indeed result in an enhancement of PSC
peak amplitude. The slope of the relationship obtained from Eq. 4 (continuous lines) depends on g, such that with
more synchronous release, peak amplitude is affected less by changes in
quantal decay. This is still obvious when accounting for the quantal
rise time, as shown by symbols. However, quanta with a finite rise time
superimpose more effectively than when they rise instantaneously. This
effect shifts the relation toward larger relative peak amplitudes and
decreases its slope. Therefore the effect on peak PSC amplitude of a
change in quantal decay rate depends both on the synchrony of release
and on the quantal rise time.
Figure 1C plots the increase in time-to-peak against the
increase in peak amplitude of the compound response, both as a
consequence of decreasing quantal decay rate. Depending on the degree
of quantal asynchrony (g), the analytic results (continuous
curves) predict that time-to-peak increases by between 1.8 and 2.3 ms, for an increase in peak amplitude by 15%. Note that, for this to
occur, the quantal decay rate must decrease from 0.05 ms1 to 0.01644 ms
1 (g = 0.5 ms
1) or 0.00305 ms
1 (g = 1 ms
1), corresponding to
a change from a quantal
decay of 20 ms
to 61 or 328 ms. For amplitude increases >15%, the slope of the
curves increases rapidly, leading to improbably high values for the
increase in time-to-peak.
When the rise time of quanta is accounted for, this relationship is shifted to the left, and the steep increase in time-to-peak occurs even earlier (symbols). From our calculations, therefore we can conclude that, with a pure change in quantal decay rate, for an increase in peak amplitude >10-15%, time-to-peak must increase by at least >1-2 ms.
Benzodiazepine agonist flunitrazepam causes a robust increase in IPSC decay time and a variable enhancement of peak amplitude
Application of 500 nM flunitrazepam typically resulted in an
increase in duration as well as in amplitude of IPSCs (Fig.
2A). The IPSC decay was fitted
by a bi-exponential function: I(t) = Af · e(t/
f) + As · e(
t/
s).
In 22 experiments, 500 nM flunitrazepam increased the fast decay time
f (26.7 ± 2.5 ms in control, 40.7 ± 5.8 ms in flunitrazepam, P < 0.01) as well as the
slow decay time
s (97 ± 10 ms in
control, 137 ± 13 ms in flunitrazepam, P < 0.01). For a more comprehensive measure we calculated a weighted global
time constant of IPSC decay (Rumpel and Behrends 1999
).
Control
glob was 55.8 ± 4.1 ms and
increased to 83.0 ± 4.9 ms in flunitrazepam (paired
t-test: P < 0.0001).
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The enhancement of IPSC peak amplitudes was highly variable. Whereas in
18 of 22 experiments flunitrazepam evoked an increase in peak amplitude
by up to 77%, in 4 experiments a small decrease was observed
(4.8 ± 0.2%). Averages of 10-20 traces immediately before and
after flunitrazepam application revealed a mean increase in peak
amplitude of 20.4 ± 5.1% (n = 22, P < 0.001, paired t-test). Because the
effects of flunitrazepam on IPSC amplitude and decay were not readily
reversible, a new dish of cells was used for each experiment.
In cells where flunitrazepam enhanced IPSC amplitudes (n = 18), the mean increase in peak amplitude was by 26.0 ± 5.3%. As illustrated in Fig. 2B, the relative increases in peak amplitude and in global decay time correlate significantly (P = 0.012, R = 0.52, correlation z-test, n = 22).
Enhancement by flunitrazepam of IPSC peak amplitudes is not accompanied by the expected increases in time-to-peak
To test the prediction of our model calculations, all experiments
where flunitrazepam enhanced the IPSC peak amplitude (n = 18) were assessed for changes in time-to-peak. IPSCs showed a mean
rise time (0-100%) of 4.5 ± 0.3 ms in control and of 4.9 ± 0.4 ms in 500 nM flunitrazepam. The mean change in time-to-peak was
small (+0.38 ± 0.11 ms; range: 0.8 to +1.3 ms) but significant (P < 0.01, paired t-test). Figure
3 plots the increase in time-to-peak against the fractional increase in peak IPSC amplitude. The
experimental data and the theoretical results from Fig. 1C
are shown superimposed. It is clear that most of the data points lie
outside our predictions, i.e., the fractional increase in amplitude is
far too large to be solely accounted for by enhanced superposition of
quantal events because of a prolonged falling phase.
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Flunitrazepam increases both peak amplitude and decay time of mIPSCs
mIPSCs were detected in the presence of TTX (1 µM). KCl (15 mM)
was applied to increase their frequency. More than or equal to 100 events per cell were aligned by their rising phase and averaged. In
control, the mean peak amplitude of mIPSCs was 28.57 ± 3.25 pA
and the mean decay rate 0.0447 ± 0.0050 ms1 (n = 5). In 500 nM flunitrazepam, mIPSC peak amplitudes increased to a mean
of 36.23 ± 3.11 pA. In all cases, the amplitude distributions before and after flunitrazepam exposure were significantly different with P < 0.001 (Kolmogorov-Smirnov test), and the mean
decay rate of mIPSCs was reduced to 0.0354 ± 0.0034 ms
1 (P < 0.05, paired t-test). The mean increase in peak amplitude was by 26.8 ± 6.6%, and the decay time constant increased by
26.3 ± 7.3%. The times-to-peak of mIPSCs before and after
application of flunitrazepam were not significantly different
(1.96 ± 0.25 vs. 2.05 ± 0.26 ms). Experimental results from
one cell are shown in Fig. 4.
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We can use these data to predict the enhancement of peak amplitude of
the compound IPSC by the kinetic and amplitude changes observed in the
miniature responses. Figure 5A
shows a number of PSC waveforms (thin lines) calculated according to
Eq. 8 for various values of quantal decay rate,
m: 0.1, 0.0666 ... , 0.05, 0.0333 ... , 0.02, 0.01 ms1; corresponding to
quantal decay time constants of 10, 15, 20, 30, 50, and 100 ms. The
decay rate of release, g, was fixed at 1 ms
1 and the quantal
amplitude was kept constant. The two dashed lines show the two boundary
conditions that will give the maximal PSC peak amplitude (here set to
1) for a given quantal size: g
and
m
0. The two bold lines are PSC waveforms calculated using the mean values for m observed before and after
application of flunitrazepam but neglecting the change in quantal size.
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The time-to-peak of the calculated PSC changed from 4.03 to 4.24 ms (by
0.21 ms) while the amplitude increased from 0.940 to 0.949 (1%). Even
a decrease of m to 0.01 ms1
(
decay of 100 ms) results only in a 4%
enhancement of peak amplitude. At the same time, the time-to-peak would
have to increase by 1.43-5.46 ms.
With more asynchronous release (g = 0.5 ms1) the measured
flunitrazepam-induced prolongation of quantal decay alone would produce
a 2.1% increase in peak amplitude with an increase in time-to-peak by
0.38 ms (not shown).
In contrast, as shown in Fig. 5B, including the observed increase in quantal size by multiplying with a factor of 1.27 results in an increase in the peak response by 28% at g = 1. At g = 0.5 this increase would be by 30%.
We can therefore conclude that modeling the compound response with the values obtained from mIPSC recordings slightly overestimates the increase in peak amplitude.
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DISCUSSION |
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Because of their action to increase agonist binding affinity at
the GABAA receptor, benzodiazepines are a unique
pharmacological tool for probing receptor occupancy at
GABAA-ergic synapses. While from the first such
experiments receptor saturation was concluded (De Koninck and
Mody 1994; Mody et al. 1994
), newer studies
suggest that, at some synapses at least, postsynaptic receptors are not invariably saturated following quantal transmitter release
(DeFazio and Hablitz 1998
; Frerking and Wilson
1996
; Frerking et al. 1995
; Mellor and
Randall 1997
; Nusser et al. 1997
,
1999
).
A recent report suggests that benzodiazepines might also increase the
single channel conductance of the GABAA receptor
(Eghbali et al. 1997), which would invalidate any
conclusions regarding receptor occupancy derived from their effects.
However, in agreement with most other studies, flunitrazepam increased
the open probability without a change in unitary conductance of
GABAA receptor-gated Cl
channels in outside-out patches activated by 1-3 µM GABA in our preparation (data not shown). Therefore the fact that benzodiazepines potentiate mIPSC amplitude indicates a lack of receptor saturation.
As outlined in the INTRODUCTION, however, it is not
self-evident that spontaneous quantal transmission is a good estimate for what happens at a single contact during impulse-evoked
transmission. In the present work, therefore we attempted to adapt this
kind of experiment to multiquantal or compound (as opposed to quantal) IPSCs. The problem, here, was that an increase in compound IPSC amplitude may be produced without a change in the number of channels open at the peak of quantal responses, but can be solely due to more
efficient temporal summation because of a slower decay of quantal
currents (De Koninck and Mody 1994; Mody et al.
1994
). By mathematically predicting the increase in
time-to-peak that will result from enhanced summation, we have shown
here that it is possible to distinguish between this effect and a true
increase in quantal size underlying an increased peak amplitude of the compound response. Comparison of these predictions with our
experimental results obtained from striatal neurons in cell culture
(cf. Fig. 3) indicates that an increase in quantal size must have
occurred and therefore suggests that receptors were not saturated at
individual contact sites during evoked IPSCs.
Model predictions: assumptions and sources of error
Deconvolution of a compound PSC waveform with that of a miniature
synaptic response has often been used to extract the time course of
transmitter release (Dempster 1986; Diamond and
Jahr 1995
; Van der Kloot 1988a
,b
; Van der
Kloot and Molgo 1994
). Conversely, convolution may be used to
estimate the waveform of a compound PSC given the average underlying
quantal waveform and the release function. In the present context, it
was our aim to understand the effect of changes in quantal decay rate
on the peak amplitude and the time-to-peak of the compound response. By
extracting, from the simplest form of this convolution, equations that
can be solved for these variables, we were able to obtain a full, continuous description of this effect, including its dependence on the
degree of asynchrony of release (cf. Fig. 1). To account for quantal
rise time, values of peak amplitude and time-to-peak had to be obtained
graphically from PSC waveforms calculated from a more complex
expression (Eq. 8). This second approach results in an in
even faster increase of time-to-peak with peak amplitude (Fig.
1C). This can be explained by the additional time available for summation.
It should be noted that, for all of these calculations, the release
function was assumed to rise instantaneously followed by an exponential
decline. For the problem under discussion, it is the effective duration
of the phasic release function that is important, but not its precise
shape. This effective duration defines quantal synchrony and the
time-to-peak of the compound response (given a constant quantal decay
rate m). The values chosen for the decay rate of the release
function (g) resulted in realistic times to peak and were
therefore reasonable estimates of the effective duration of phasic
release. Using a function of different shape, such as the
gamma-function (Barrett and Stevens 1972; Geiger
et al. 1997
; Isaacson and Walmsley 1995
), will
not affect our results, provided that its parameters are chosen to give
the same control time-to-peak of the compound PSC and therefore the
same degree of quantal synchrony.
The release function can, in suitable preparations, be measured
directly from the latency of single quanta at a low probability of
release (Barrett and Stevens 1972; Isaacson and
Walmsley 1995
). However, in our preparation as in many others,
release sites are distributed at various terminals of branching axons
rather than at a single, large ending as at the neuromuscular junction
or the endbulb of Held. Therefore the quantal latency distribution will
be strongly dependent on axonal conduction, which has been shown to be
affected by the interventions necessary to lower release probability,
such as very low Ca2+ concentrations or
application of Cd2+ (Lüscher et al.
1994
). Therefore it is doubtful whether, in this situation,
release kinetics can be inferred by this method.
Deconvolution estimates of the decay rate of the release function at
other small mammalian CNS synapses range from >3
ms1 (Geiger et al.
1997
; Williams et al. 1998
) in slice
preparations at temperatures >30°C, to 0.26 ms
1 (Diamond and
Jahr 1995
) in cell culture at 20-22°C. The boundary values
we estimated from the control time-to-peak (1 and 0.5 ms
1) are in good
agreement with those findings. More precisely, the mean control
time-to-peak of IPSCs in this study was 4.5 ± 0.3 ms, which,
according to Eq. 8, with m = 0.0447 ms
1 would correspond to a
g of 0.81 ms
1.
However, measurements of time-to-peak are susceptible to error,
particularly in the presence of noise, when filtering is used and when
uncompensated series resistance and electrotonic distance slows the
response of the voltage clamp. All three conditions must be considered
for our recordings, and therefore our estimate of g probably
represents a lower limit. Furthermore, the changes in time-to-peak we
observed following flunitrazepam application were almost certainly too
small to be accurately measured, despite the fact that they reached
statistical significance. Nonetheless, the changes in time-to-peak
predicted if the enhancement of IPSC amplitude was exclusively due to
slower quantal decays should have been detectable (cf. Fig. 3): in 13 of 18 cases, IPSC amplitude was increased by 10%, which would have
necessitated an increase in time-to-peak of at least 1 ms.
Voltage-clamp errors may also have affected our measurements of changes in IPSC amplitude, but will lead to an underestimation rather than an overestimation of amplitude increase. Therefore our main conclusion, that the observed increases in peak amplitude must be due to larger quantal size would be unaffected.
Finally, although such an effect has not been described, a strong
synchronization of release by flunitrazepam might explain some of the
increase in IPSC peak amplitude. However, as shown in Fig.
5A, even total synchronization (i.e., g
)
would only increase peak amplitude by <10%. Also, such an effect
would strongly reduce the time-to-peak of the IPSC, which we did not observe.
Conditions under which slower quantal decays can increase compound PSC amplitude
Which are the conditions that would enable an isolated decrease in
quantal decay rate to produce significant changes in the peak amplitude
of compound responses? In their original demonstration of this effect,
De Koninck and Mody (1994) used summation of mIPSCs recorded from dentate granule cells before and after application of the
BZ1-selective agonist zolpidem. These mIPSCs were
unchanged in amplitude, but their decay time constant was strongly
slowed from a control value of about 5 ms. Quanta were summed with
delays between their occurrence governed by an exponential function
that had a time constant of 5 ms. This provides an exact match of our model of the release function, the time constant corresponding to
g = 0.2 ms
1. Equation 8 predicts that, indeed, with this relatively asynchronous release, compound peak amplitude is very sensitive to changes in
quantal decay constant alone, changing by 28.3 and 56.0% for an
increase in quantal decay time constant from 5 to 10 and 20 ms,
respectively. At the same time, the time-to-peak of the compound responses increases by 1.93 and 4.24 ms, respectively. In this case,
the assumption of asynchronous release (but see Williams et al.
1998
) together with a rapid decay of control quanta explains the vigorous effect on peak amplitude. A small but significant contribution also results from the rapid rising phase of mIPSCs in
dentate granules (<1 ms), which was accounted for in the above calculations. Thus strong increases in the peak amplitude of compound responses can occur as a result of changes in quantal decay rate when
the decay time constant of the release function is not fast with
respect to the kinetics of the quantal components. However, under these
conditions as well, the time-to-peak of the compound response increases
in a predictable fashion, making it possible to separate effects due to
changes in quantal kinetics from a possible additional change in
quantal sizes.
Variability of flunitrazepam action
One of the most prominent features of flunitrazepam action in the
present study was the high variability of changes in IPSC peak
amplitude. The fact that the amplitude changes produced by flunitrazepam covary positively and significantly with the changes in
decay time constant (Fig. 2B) might be taken to suggest that some of this variance is due to a heterogeneity in the flunitrazepam sensitivity of GABAA receptors involved in
different unitary connections. We do not have direct information about
the subunit composition of the GABAA receptors
subserving inhibitory transmission in the cultured neurons studied
here. It is possible that striatal neurons express several combinations
of GABAA receptor subunits, which may differ in
their response to benzodiazepines. Flunitrazepam, unlike zolpidem, does
not discriminate between BZ1 and
BZ2 binding sites, and therefore the response to
the agonist used here should be independent on the subtype of the
-subunit expressed, which, in rat striatum is primarily of the
2
variant (Fritschy and Mohler 1995
; Fujiyama et
al. 2000
). On the other hand, it is not impossible that
combinations of subunits exist at these contacts that make benzodiazepine-insensitive receptors, such as those containing the
-
or
-subunits instead of the
-subunit. The
-subunit is known to
be expressed at moderate levels in normal rat striatum, while the
2-subunit is expressed abundantly (Fritschy and Mohler 1995
).
An alternative explanation for the variability of flunitrazepam action
is that the less benzodiazepine-responsive IPSCs are primarily mediated
by saturated contacts, whereas unsaturated synapses contribute more to
those where flunitrazepam has a strong effect. Higher receptor
occupancy at a subset of contacts may result from lower numbers of
postsynaptic receptors (Auger and Marty 1997;
Nusser et al. 1997
) or from the presence of subunit combinations with particularly high affinity for GABA (Auger and Marty 1997
). Alternatively, a high peak cleft concentration of the transmitter may also increase receptor occupancy. Both high transmitter concentrations and high receptor affinity might be associated with longer IPSC decays because of longer presence of GABA
or slower unbinding. In both cases, IPSCs with longer decay times in
control would show less relative change in peak amplitude with
flunitrazepam application. A significant negative correlation between
these values is, indeed, present in our dataset (n = 22, R = 0.541, P < 0.01, correlation
z-test).
In 4 of 22 experiments, we observed a small decrease in peak amplitude
after application of flunitrazepam. The most likely reason for this
decrease is tonic GABAA receptor desensitization by subliminal concentrations of tonically released GABA
(Brickley et al. 1996; Gaspary et al.
1998
; Overstreet et al. 1999
; Wall and
Usowicz 1997
). This effect would be enhanced when the binding affinity of the GABAA receptors is increased by
flunitrazepam. At synapses where tonic release of GABA is strong, it
may outweigh the positive effect on responses to phasic GABA release.
However, we cannot exclude the possibility that some
GABAA receptors can display an inverse response
to flunitrazepam.
The increase in peak amplitude of the compound response calculated from the flunitrazepam-induced change in peak amplitude and kinetics observed with mIPSCs slightly overestimated the experimentally observed changes in compound IPSC amplitude, suggesting that, on average, more contacts are saturated with evoked transmission than with spontaneous release. However, in 3 of 22 cases, the increase in compound IPSC amplitude was several times larger than the mean increase in mIPSC size (cf. Fig. 3). In these cases, it is possible that some contacts were involved in generating these IPSCs that had extremely low receptor occupancy. Responses arising at such contacts might be too small under control conditions to be detected in mIPSC recordings. The strong change in amplitude likely to arise from flunitrazepam treatment at such contacts would then not contribute to the change in mIPSC amplitude distribution, because they did not contribute to the control sample. In fact, following flunitrazepam application, they would probably still be smaller than the control average, in effect subtracting from the measured change in mean mIPSC amplitude.
In summary, we have used modeling of compound synaptic responses to distinguish between the contribution of a change in quantal size and quantal kinetics to benzodiazepine-induced increases in the peak amplitude of IPSCs in rat striatal neurons. In our preparation, we conclude that only an increase in quantal size can explain our experimental findings, strongly suggesting that during impulse-evoked transmission postsynaptic receptors are not uniformly saturated. The approach used here may be useful for similar studies at other synapses where different conditions may prevail.
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ACKNOWLEDGMENTS |
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This work was done in the laboratory of Prof. Gerrit ten Bruggencate, whom we thank for continuing support and encouragement. We thank N. Fertig (Dept. of Physics, Univ. of Munich) and Prof. F. Kolb for verifying the mathematics, and L. Kargl and A. Grünewald for valuable technical assistance.
This work was supported by Deutsche Forschungsgemeinschaft Grant Be 1739-2 and the Friedrich Baur-Stiftung.
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FOOTNOTES |
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Address for reprint requests: J. C. Behrends, Physiologisches Institut, Pettenkoferstr. 12, 80336 Munich, Germany (E-mail: j.behrends{at}lrz.uni-muenchen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 December 1999; accepted in final form 20 April 2000.
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REFERENCES |
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