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The Journal of Neuroscience, August 1, 2000, 20(15):5594-5607
Efficacy and Stability of Quantal GABA Release at a Hippocampal
Interneuron-Principal Neuron Synapse
Udo
Kraushaar and
Peter
Jonas
Physiologisches Institut der Universität Freiburg, D-79104
Freiburg, Germany
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ABSTRACT |
We have examined factors that determine the strength and dynamics
of GABAergic synapses between interneurons [dentate gyrus basket cells
(BCs)] and principal neurons [dentate gyrus granule cells (GCs)]
using paired recordings in rat hippocampal slices at 34°C. Unitary
IPSCs recorded from BC-GC pairs in high intracellular Cl concentration showed a fast rise and a
biexponential decay, with mean time constants of 2 and 9 msec. The mean
quantal conductance change, determined directly at reduced
extracellular Ca2+/Mg2+
concentration ratios, was 1.7 nS. Quantal release at the BC-GC synapse
occurred with short delay and was highly synchronized. Analysis of IPSC
peak amplitudes and numbers of failures by multiple probability
compound binomial analysis indicated that synaptic transmission at the
BC-GC synapse involves three to seven release sites, each of which
releases transmitter with high probability (~0.5 in 2 mM
Ca2+/1 mM Mg2+).
Unitary BC-GC IPSCs showed paired-pulse depression (PPD); maximal depression, measured for 10 msec intervals, was 37%, and recovery from
depression occurred with a time constant of 2 sec. Paired-pulse depression was mainly presynaptic in origin but appeared to be independent of previous release. Synaptic transmission at the BC-GC
synapse showed frequency-dependent depression, with half-maximal decrease at 5 Hz after a series of 1000 presynaptic action potentials. The relative stability of transmission at the BC-GC synapse is consistent with a model in which an activity-dependent gating mechanism
reduces release probability and thereby prevents depletion of the
releasable pool of synaptic vesicles. Thus several mechanisms converge
on the generation of powerful and sustained transmission at
interneuron-principal neuron synapses in hippocampal circuits.
Key words:
GABAergic interneurons; basket cells; dentate gyrus; unitary IPSCs; paired-pulse depression; release probability; functional
release sites; vesicular pools; paired recording; multiple probability
compound binomial analysis
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INTRODUCTION |
GABAergic interneurons are the main
presynaptic source of inhibitory synaptic transmission in the mammalian
CNS (for review, see Freund and Buzsáki, 1996 ). Although
interneurons numerically represent only ~10% of the neuronal
population, they control the activity of the entire neuronal network.
In the hippocampus, several interneuron types have been identified that
form synapses on different domains of their postsynaptic target cells
(Han et al., 1993; Freund and Buzsáki, 1996). Interneurons that
innervate the perisomatic domain of principal neurons, referred to as
basket cells, mediate a particularly powerful form of inhibition.
Activation of a single basket-type interneuron can suppress repetitive
discharge and delay spike initiation in principal neurons (Miles et
al., 1996). Furthermore, activation of a single interneuron can entrain
spiking of target neurons and synchronize the activity of large
neuronal ensembles (Cobb et al., 1995). The impact of basket
cell-mediated inhibition may be explained by the anatomical location of
synaptic contacts in the perisomatic region of their postsynaptic
target cells. Alternatively, the efficiency could be caused by specific functional properties of basket cell output synapses, such as the
number of functional release sites (Edwards et al., 1990; Tamás
et al., 1997), the number of GABA molecules released from a single
vesicle (Frerking et al., 1995), the number of postsynaptic GABAA receptors (Nusser et al., 1997, 1998), and
the receptor occupancy after release (Edwards et al., 1990; Frerking et
al., 1995).
Unlike principal neurons, basket-type interneurons are able to generate
action potentials with high frequency during sustained current
injection in vitro (Han et al., 1993; Martina et al., 1998)
and in vivo (Penttonen et al., 1998; Csicsvari et al.,
1999). This suggests the possibility that the dynamic properties of
output synapses of interneurons are adapted to high-frequency activity. In the neocortex, GABA release from interneurons is more stable than
glutamate release from principal cells during high-frequency stimulation (Galarreta and Hestrin, 1998; Varela et al., 1999); however, the mechanisms underlying this stability are unknown. Stability of inhibition during high-frequency stimulation could be
generated by a larger readily releasable pool of synaptic vesicles in
comparison with excitatory synapses (Stevens and Tsujimoto, 1995).
Alternatively, stability of inhibition could be conferred by a
reduction of release probability during repetitive stimulation (Betz,
1970; Wu and Borst, 1999) or activity-dependent replenishment of the
releasable pool of synaptic vesicles (Kusano and Landau, 1975; Dittman
and Regehr, 1998).
A rigorous analysis of the factors determining the strength and
dynamics of GABAergic synapses requires the selective stimulation of
identified presynaptic interneurons in the paired recording configuration (Miles and Poncer, 1996). We have therefore examined unitary IPSCs at the basket cell (BC)-granule cell (GC) synapse in the
dentate gyrus of hippocampal brain slices. Our results indicate that
transmission at the BC-GC synapse is very efficient, mainly because of
a large quantal size and a high release probability. An
activity-dependent gating mechanism that reduces release probability may help to preserve the releasable pool of synaptic vesicles.
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MATERIALS AND METHODS |
Paired recording. Transverse hippocampal slices (300 µm thickness) were cut from brains of 18- to 25-d-old Wistar rats
using a vibratome (DTK-1000, Dosaka). Animals were killed by
decapitation, in agreement with national and institutional guidelines.
Patch pipettes were pulled from thick-walled borosilicate glass tubing (2 mm outer diameter, 0.5 mm wall thickness); when filled with intracellular solution, the resistance was 3-5 M for presynaptic recordings and 2-4 M for postsynaptic recordings. Simultaneous recordings from synaptically connected BCs and GCs in the dentate gyrus
were obtained under visual control using infrared differential interference contrast videomicroscopy (Edwards et al., 1989; Stuart et al., 1993; Koh et al., 1995). A tight-seal (>2 G) whole-cell recording was first established in a putative BC. Selected cells had
somata located at the granule cell layer-hilus border and generated
>200 action potentials during 1 sec depolarizing current pulses
(1.4-2 nA). Subsequently, whole-cell recordings were made from GCs;
their somata were typically located in the outer half of the granule
cell layer within 100 µm distance from the BC soma. Under ideal
conditions, the probability of inhibitory synaptic coupling between the
BC and the GC was up to 50%. Based on the morphological appearance of
presynaptic neurons, fast spiking on sustained current injection (Koh
et al., 1995), and fast rise time of evoked IPSCs in postsynaptic
granule cells (see Fig. 3C), we denote the presynaptic
neurons as putative basket cells, although we cannot exclude the
possibility that they were axo-axonic cells or hilar interneurons with
axon coaligned with the commissural-associational pathway (HICAP cells)
in some cases (Freund and Buzsáki, 1996). Pairs with initial
resting potentials more positive than  60 mV and more positive
than 70 mV (BC and GC, respectively) or pairs in which evoked IPSCs
triggered unclamped action potentials in the GC were discarded. The
recording temperature was 34 ± 2°C.
Two Axopatch 200A amplifiers (Axon Instruments) were used for current-
and voltage-clamp recording. The presynaptic neuron was held in the
current-clamp mode and stimulated at a frequency of 0.25 Hz, unless
specified differently. Action potentials were elicited by brief current
pulses (duration 1 msec, amplitude 1.4-2.4 nA). The postsynaptic cell
was held in either the current-clamp or voltage-clamp mode, using
series resistance (RS) compensation (nominally 85-95%, lag ~100 µsec;
RS before compensation 5-20 M). The constancy of the series resistance in the postsynaptic GC
was assessed from the amplitude of the capacitive current in response
to a 5 mV pulse, and the compensation was readjusted during the
experiment when necessary. Presynaptic action potentials and IPSPs or
IPSCs were filtered at 5 kHz using the four-pole low-pass Bessel filter
of the amplifiers and digitized at 10 kHz using a 1401plus
laboratory interface (Cambridge Electronic Design) connected to a
Pentium-PC. Commercial programs from Cambridge Electronic Design were
used for stimulus generation and data acquisition.
Solutions. The physiological extracellular solution
contained (in mM): 125 NaCl, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2. In some
experiments, the Ca2+ and
Mg2+ concentrations were varied to alter
the release probability. The intracellular solution contained (in
mM): 145 KCl, 0.1 EGTA, 2 MgCl2, 2 Na2ATP, and 10 HEPES (KCl intracellular
solution); the pH was adjusted to 7.2 with KOH. In some experiments, a
solution containing (in mM): 140 K-methylsulfate, 2 KCl, 10 EGTA, 2 MgCl2, 2 Na2ATP,
and 10 HEPES (K-methylsulfate intracellular solution, pH adjusted with
KOH; see Fig. 1C,D) or a solution with (in
mM): 145 CsCl, 0.1 EGTA, 2 MgCl2, 2 Na2ATP, and 10 HEPES (CsCl intracellular solution, pH adjusted with CsOH; see Fig.
1E) was used for the postsynaptic GC. For
perforated-patch recordings, the intracellular solution contained (in
mM): 78 KCl, 78 K-gluconate, 0.1 EGTA, 2 MgCl2, 2 Na2ATP, 10 HEPES,
and 18 µg/ml gramicidin; for tip filling, the same
solution without gramicidin was used. Bicuculline methiodide was from Sigma (stock solution prepared in distilled water),
CGP55845A was from Novartis (stock solution in dimethylsulfoxide), K-methylsulfate was from ICN, and other chemicals were from Merck, Sigma, Riedel-de Haen, or Gerbu.
Data analysis. Evoked IPSPs and IPSCs were analyzed
using programs written in Pascal (Borland, version 7.0). The rise
time was determined as the time interval between the points
corresponding to 20 and 80% of the peak amplitude, respectively. The
peak current was determined as the maximum within a window of 2 msec
duration after the presynaptic action potential. The mean frequency of spontaneous events was 8.2 ± 1.2 Hz (range: 0.9-17 Hz; 18 GCs), indicating that contaminating spontaneous events with onsets in this
window may occur in 1.6% of traces. The synaptic latency was
determined as the time interval between the maximum of the first
derivative of the presynaptic action potential and the onset of the
first subsequent IPSC; the onset point was determined from the
intersection of a line through the 20 and 80% points with the
baseline. The decay phase of the IPSCs was fitted with the sum of two
exponentials and a constant using a nonlinear least-squares fit
algorithm; the best-fit value of the constant was close to zero in all
cases. Amplitude ratios given refer to the time of the peak current. A
trace was classified as a failure when the amplitude was less than
three times the SD of the preceding baseline. Average IPSCs were
obtained from single IPSCs aligned to the steepest point in the rise of
the presynaptic action potential, unless specified differently.
The time course of quantal release was determined from the first
latency histogram using the method of Barrett and Stevens (1972). To
examine the validity of the approach, quantal IPSCs were aligned at
their rising phase and averaged. The release probability distribution
was reconvolved with the average quantal IPSCs by multiplying the
respective discrete Fourier transforms with each other (Geiger et al.,
1997). Coefficients of variation (CV, SD/mean) of unitary IPSC peak
amplitudes were calculated from traces during stationary periods; the
number of traces included was 20-50. CV values were not corrected for
baseline noise (unless specified differently), because the influence of
a correction was very small. To determine the locus of paired-pulse
depression (PPD; see Fig. 7C) and its possible dependence on
previous release (see Fig. 8), an interpulse interval of  100 msec was
used, which allowed the first IPSC to decay completely to baseline.
Values are given as mean ± SEM. Error bars in the Figures also
indicate SEMs, unless specified differently. Membrane potentials
reported in the text were not corrected for junction potentials.
Significance of differences was assessed by two-tailed Student's
t test at the significance level (p) indicated.
Multiple probability compound binomial analysis. To
determine the number of functional release sites and the release
probability, we used a hybrid approach [termed multiple probability
compound binomial analysis (MP-CBA)] that combines elements of
amplitude distribution fitting (Edwards et al., 1990; Jonas et al.,
1993) and multiple probability variance analysis (Silver et al., 1998). Amplitude distributions and numbers of failures for two to four different
Ca2+/Mg2+
concentration ratios obtained consecutively from the same pair were
fitted with a compound binomial model of release (Redman, 1990). The
number of traces recorded in each condition was >100. Data in each
condition were tested for stability of series resistance and
stationariness of peak amplitudes. Data in conditions of low release probability were also tested for randomness using runs analysis
of failures and events (Swed and Eisenhart, 1943) (our Table 2; see
footnote 1 for results).
The compound binomial release model assumed that quantal currents were
normally distributed and accounted for nonuniformity of both quantal
size and release probability. Alternative models based on skewed gamma
distributions or models assuming uniformity of quantal size and release
probability were also explored, but they generally gave worse fits to
the data. The model had the following free parameters: mean quantal
size <q>, intrasite (type 1) coefficient of variation
CV1, intersite (type 2) coefficient of variation
CV2 (Jack et al., 1994; Walmsley, 1995), mean
release probabilities <p>, and shape factor
 p describing the nonuniformity of individual
release probabilities. <q>, CV1, and
CV2, and p were assumed
to be the same, whereas <p> was specified separately for
each condition. The quantal current generated at the i-th site was
assumed to follow the distribution
ND(qi,
 2, x), where ND is a normal
distribution with mean qi and variance 2 = (CV1
<q>)2 (the mean of all
qi values is <q>), and x
is the current amplitude. If the release probability at the i-th site
is denoted as pi (the mean of all
pi values is <p> ), then
the compound binomial model for three release sites would be stated as follows.
The probability to observe a failure is:
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(1)
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The amplitude distribution generated by the activation of one of
three sites is:
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(2)
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where 0 is the SD of the baseline. The
amplitude distribution generated by the simultaneous activation of two
of three sites is:
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(3)
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Finally, the amplitude distribution generated by the
simultaneous activation of all three sites is:
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(4)
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and the total IPSC amplitude distribution can be obtained
as:
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(5)
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For Nr > 3 release sites, the
model was extended analogously.
Site-to-site variation of quantal amplitudes
qi was generated using a normal
distribution (with mean <q> and coefficient of variation
CV2). Site-to-site variation of release
probabilities pi was implemented using
a beta distribution [with mean <p> and shape factor
p (Silver et al., 1998)]. The
qi and
pi values were obtained by dividing
the area under the probability density function into equal portions
(the respective areas would be
1/(2Nr),
1/(2Nr) + 1/Nr,
1/(2Nr) + 2/Nr,... . , 1 1/(2Nr) for
Nr release sites). Positive and
negative correlations between quantal size and release probability were
generated by ranking qi and
pi values in identical or reverse
order, respectively, and the model that gave the better fit was
adopted. 0 was determined from the region of
the baseline preceding the IPSC, with the same settings as those used
for determining the peak current.
Estimates of the free parameters of the compound binomial model were
obtained by fitting unbinned data by a maximum-likelihood method. The
negative logarithm of the likelihood was minimized using a Simplex
algorithm (Caceci and Cacheris, 1984) implemented in Pascal, running on
350-600 MHz Pentium PCs. Ranges were defined for each parameter
(<q>: 30-500 pA; CV1 and
CV2: 0.001-2; <p>: 0.01-0.85;
p: 0.01-100), and the negative log-likelihood
was increased when these ranges were exceeded. The final fit results were within the defined parameter space, with the exception of p, which was frequently at the upper border
(100). The criterion for convergence was a relative difference
<1012 or
1014 in the
log-likelihood between best and worst vertex in 10 consecutive iterations (for two and four release conditions, respectively). Within
a twofold range of starting values, the final results were relatively
insensitive to the initial values. Independent fits were made for
different numbers of functional release sites
Nr (range: 3-12).
Nr was accepted as the best-fit value
when the corresponding log-likelihood was larger than that for
Nr1 and Nr+1, respectively.
Confidence intervals of parameter estimates (see Table 2) were obtained
by bootstrap methods. To obtain balanced resampling (Davison et al.,
1986; Efron and Tibshirani, 1998), 100 copies of the original data set
(size n) were concatenated, and a random permutation of all
100*n elements was generated. Subsequently, 100 bootstrap
replications were read off as successive blocks of length n
in the permutated data and were refitted, using the best-fit values for
the original data set as initial values (Stricker et al., 1994, 1996).
Errors were then estimated from percentile intervals (Efron and
Tibshirani, 1998).
Models of vesicular pool dynamics. Synaptic depression
during trains of pulses was described by models with two pools
(releasable/available and unavailable pool) of synaptic vesicles (Liu
and Tsien, 1995a,b; Weis et al., 1999; Matveev and Wang,
2000):
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(6)
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where
pR(Nv)
is the release probability, k is the rate of refilling of
the releasable pool, f is the stimulation frequency, and
Nv is the number of vesicles in the
releasable pool at any point in time, with initial value and upper
limit Nv0 (the "capacity" of the
releasable pool). The stochastic implementation of the model assumed a
univesicular release constraint in response to a presynaptic action
potential (Korn et al., 1982). The release probability was
pR(Nv) = 1 exp(-v
Nv), where v
denotes the time-integrated fusion rate for a single vesicle (Dobrunz
and Stevens, 1997; Matveev and Wang, 2000). The probability of
refilling of each vacancy in the releasable pool in the time interval
 t = 1/f between two consecutive stimuli
was pRefill = 1 exp(k t) (Matveev and Wang, 2000).
pR during and after high-frequency stimulation was computed by averaging the results from 1000 Monte-Carlo simulations, using programs written in Pascal.
Activity-dependent decrease of release probability and increase of
refilling rate were modeled as the activation of a two-state modification process:
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(7)
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where and  denote forward and backward rates of the
modification. The fractional occupancy a(t) of
the activated state was calculated from the equations:
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(8)
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where a0 and
a indicate initial and final values
of a(t) during the stimulation and recovery
period. Activity-dependent decreases of release probability and
increases in refilling rate were implemented as:
![k′=k[1+a<SUB><UP>max</UP></SUB>a(t)], <UP>with </UP>a<SUB><UP>max</UP></SUB>≥0,](/content/vol20/issue15/fulltext/5594/img017.gif)
|
(9)
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where amax indicates the
maximal modification. Thus the activity-dependent pool models had the
following free parameters: pool capacity
Nv0, initial release probability
pR, refilling rate k, maximal
modification amax, and rates of
activity-dependent modification and . Parameters of the pool
model were specified arbitrarily (see Fig. 10). Alternatively,
estimates of the parameters of the pool model were obtained using a
least-squares method, minimizing the sum of squares of differences
between experimental observations and model predictions with a Simplex
algorithm. Weights were chosen arbitrarily according to inverse of
variance of data points, numbers of experiments, and number of data
points in each set (see Fig.
9C-E,H).
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RESULTS |
Unitary IPSPs and IPSCs at the BC-GC synapse
We examined inhibitory synaptic transmission between pairs of
monosynaptically connected BCs and GCs in slices from mature rats
(18-25 d old) at physiological temperatures (34°C). This synapse has
several technical advantages (Fig. 1).
First, interneurons and principal neurons in this circuit can be
distinguished by morphological criteria (Fig. 1A) and
by the ability of the presynaptic interneuron to generate
high-frequency trains of action potentials during sustained current
injection (Fig. 1B). Second, paired recordings from
synaptically connected interneurons and principal neurons can be
obtained with relatively high probability, because the axonal
arborization of BCs is extensive (Fig. 1A) (Freund
and Buzsáki, 1996; Geiger et al., 1997). Third, although unitary IPSPs and IPSCs have small amplitudes at physiological intracellular Cl concentrations (Fig.
1C,D), the amplitude of unitary IPSCs with high
intracellular Cl concentration (149 mM) is substantially larger, with excellent signal-to-noise ratio under these conditions (Fig.
1E). Fourth, the voltage-clamp conditions of the
unitary IPSCs are ideal, because of the mainly perisomatic location of
synaptic contacts and the favorable electrotonic properties of
postsynaptic granule cells. Finally, synaptic transmission in the
paired recording configuration is stationary over long periods of time,
with a stimulation frequency of  0.25 Hz (Fig.
1F).
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Figure 1.
Unitary IPSPs and IPSCs at the BC-GC synapse in
the paired recording configuration. A, Schematic
illustration of the BC-GC microcircuit in the dentate gyrus.
B, High-frequency train of action potentials evoked in a
putative BC by a current pulse (200 msec, 1.4 nA). C,
Unitary IPSPs recorded from a BC-GC pair at 30 mV with 6 mM intracellular Cl concentration.
Single presynaptic action potential evoked by a depolarizing current
pulse in the BC is shown on top, single IPSPs are shown
superimposed in the center, and average IPSP is depicted
at the bottom. D, Unitary IPSCs in a
BC-GC pair with 6 mM intracellular Cl
concentration. Single presynaptic action potential is shown on
top, single IPSCs at 100 and 50 mV are shown
superimposed in the center, and average IPSCs at 100
to 50 mV (10 mV increment) are depicted at the bottom.
E, Unitary IPSCs in a BC-GC pair with 149 mM intracellular Cl concentration.
Single presynaptic action potential is shown on top,
single IPSCs at 100 and +30 mV are shown superimposed in the
center, and average IPSCs at 70 to +30 mV (20 mV
increment) are depicted at the bottom. F,
Unitary IPSC peak amplitude plotted against recording time. Note that
the amplitude was stationary (correlation coefficient
r = 0.05, p > 0.1). Averages
are from 30-100 single synaptic events. Data in
C-F are from different pairs. Pair shown
in F is pair #1 in Table 2.
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Figure 2 shows the properties of unitary
IPSCs in high intracellular Cl in an
individual pair, and Figure 3 summarizes
the results from 78 BC-GC pairs. The mean synaptic latency, measured
from the steepest point in the rising phase of the presynaptic action
potential to the onset of the IPSC, was 1.1 msec. The rise of the
synaptic events was almost instantaneous (average 20-80% rise time
0.26 msec). The mean unitary IPSC peak amplitude (including failures of
transmission) was 504 pA at 70 mV. Because the mean reversal potential of unitary IPSCs is +4.2 mV (Table
1), this corresponds to a peak
conductance change of 6.8 nS. The decay of the unitary IPSCs was better
fitted with the sum of two exponentials than with a single exponential
in the majority of pairs; the average values of the time constants were
1.9 msec (38% amplitude contribution) and 9.4 msec, respectively.
Synaptic transmission at the BC-GC synapse was very reliable; the mean
percentage of failures was 6.5%. Thus BC-mediated inhibition shows
shortlatency, rapid onset, large peak-conductance change, long
duration, and high reliability (Table 1).
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Figure 2.
Amplitude and time course of the unitary
postsynaptic conductance change at the BC-GC synapse. Data are from a
single pair. A, Presynaptic action potential
(top), single unitary IPSCs (9 sweeps superimposed),
average IPSC (from 60 sweeps), and sum of two exponentials fitted to
the average IPSC (bottom, with individual components)
are depicted. B, Latency, measured from the steepest
point in the rising phase of the presynaptic action potential to the
onset of the first IPSC in a trace. C, Rise time
(20-80%) of unitary IPSCs. D, Peak amplitude of
unitary IPSCs. Thirty-one failures are not displayed. E,
Decay time constants of unitary IPSCs: fast decay time constant
 1 (open bars); slow decay time constant
2 (filled bars). F,
Amplitude contribution of the fast component of decay
(A1) obtained by biexponential fit.
Extracellular Ca2+ and
Mg2+ concentrations were 2 and 1 mM, respectively. Holding potential was 70 mV;
intracellular Cl concentration in the postsynaptic
GC was 149 mM. All data are from the same BC-GC
pair.
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Figure 3.
Amplitude and time course of the unitary
postsynaptic conductance change at the BC-GC synapse. Summary graphs
of data from 78 pairs. A, Percentage of failures of
transmission. B, Mean first latency. C,
Mean 20-80% rise time. D, Mean peak amplitude of
average IPSCs (including failures). E, Mean decay time
constants: fast decay time constant 1 (open
bars); slow decay time constant 2
(filled bars). F, Mean amplitude
contribution of the fast component of decay
(A1). Extracellular
Ca2+ and Mg2+ concentrations were 2 and
1 mM, respectively. Holding potential was 70 mV;
intracellular Cl concentration in the postsynaptic
granule cells was 149 mM in all experiments.
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Direct recording of quantal IPSCs at the BC-GC synapse
Rigorous analysis of the transmitter release process requires
direct recording of quantal currents (Katz, 1969; Isaacson and Walmsley, 1995). We therefore examined quantal BC-GC IPSCs in conditions of low release probability with a reduced
Ca2+/Mg2+
concentration ratio in the bath solution (Fig.
4). Figure 4, A and
B, shows unitary IPSCs in a pair at two different
Ca2+/Mg2+
concentrations. When the
Ca2+/Mg2+
concentration ratio was reduced, the amplitude of the unitary IPSC
decreased, and the number of failures increased substantially, to
>80% with Ca2+ concentrations <0.5
mM (Fig. 4C). Under these conditions,
the amplitude of the successful unitary IPSCs excluding failures
reached an asymptotic value corresponding to the quantal size (Fig.
4D). The mean peak amplitude of these putative
quantal IPSCs was 129 ± 19 pA at 70 mV. With the mean reversal
potential of unitary IPSCs of +4.2 mV, this corresponds to an apparent
quantal conductance change of 1.7 ± 0.3 nS. The mean CV of the
putative quantal IPSC in conditions of low release probability,
including both intrasite and intersite components, was 33.7 ± 1.0% (five pairs at Ca2+ concentrations
of 0.1-0.3 mM, corrected for baseline noise). Thus direct recording of quantal IPSCs at the BC-GC synapse reveals a
large quantal size and a moderate variability of the quantal amplitude.
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Figure 4.
Direct recording of quantal IPSCs at the BC-GC
synapse. A, B, Unitary IPSCs at
physiological Ca2+/Mg2+
concentrations (A) (2 mM
Ca2+, 1 mM Mg2+) and
after reduction of release probability (B) (0.5 mM Ca2+, 2.5 mM
Mg2+). Six traces are shown superimposed on
top; average IPSCs including failures are shown at the
bottom. C, Percentage of failures plotted
against the extracellular Ca2+ concentration.
D, Mean peak amplitude of successful IPSCs
(excluding failures) plotted against extracellular
Ca2+ concentration. Error bars indicate SD of IPSC
amplitudes. Open circles indicate experiments in which
the Mg2+ concentration was kept constant (3 or 4 mM); filled circles represent experiments in
which the sum of Ca2+ and Mg2+
concentrations was maintained (3 mM). As the
Ca2+ concentration was reduced, the number of
failures increased, but the amplitude of the successful IPSCs
approached asymptotically a minimal value. This suggests that IPSCs
at Ca2+ concentrations below 0.5 mM are
mainly quantal IPSCs. Data in C and D are
from 11 pairs; data obtained from the same pair were connected by
dashed lines.
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The time course of quantal release
Asynchrony of transmitter release can provide a significant
contribution to the time course of the average postsynaptic conductance change at various synapses (Diamond and Jahr, 1995; Isaacson and Walmsley, 1995; Geiger et al., 1997). To determine the time course of
quantal release at the BC-GC synapse, we used the approach of first
latency measurements (Barrett and Stevens, 1972) (Fig. 5). The time course of quantal release
(open bars), determined from the distribution of first
latencies (filled bars) in conditions of reduced
Ca2+/Mg2+
concentration ratio, rose and decayed within a time window of ~1
msec, indicating that GABA release was highly synchronized (Fig.
5A). To test the validity of the first latency approach, single quantal events in low
Ca2+/Mg2+
concentration ratio were aligned to their onset, averaged, and reconvolved with the time course of quantal release in the same pair.
The time course of the reconvolved IPSC was almost indistinguishable from that of the averaged unitary IPSC (four pairs), suggesting that
quantal contributions superimposed independently (Fig. 5B). The logarithmic plot of the mean time course of release from four pairs
illustrates that the release period decayed approximately exponentially, with a time constant of 0.23 msec (Fig. 5C).
Thus quantal release at the BC-GC synapse showed high synchrony,
comparable to that of fast excitatory synapses (Isaacson and Walmsley,
1995; Geiger et al., 1997).
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Figure 5.
Time course of quantal release at the BC-GC
synapse. A, First latency distribution
(filled bars) and time course of quantal release
(open bars) in a BC-GC pair, calculated from first
latencies using the correction method of Barrett and Stevens (1972).
Data were obtained with 0.5 mM Ca2+ and
2.5 mM Mg2+ in the bath; data are from
299 IPSCs (401 failures). B, Average unitary IPSC in 0.5 mM Ca2+, 2.5 mM
Mg2+, average quantal IPSC (obtained after aligning
single events on their rising phase), and a simulated IPSC generated by
reconvolution of the time course of quantal release with the time
course of the quantal conductance change are shown superimposed. The
three traces were normalized to the same peak value.
C, Mean time course of quantal release. Histograms of
the time course of release were aligned to the bin with the
maximal number of events (nmax, which
is represented as time 0 in the graph). Numbers
of events were normalized by nmax and
plotted logarithmically. The line represents the results
of linear regression of the decay, yielding a decay time constant of
0.23 msec. Data are from four pairs. D, Average unitary
IPSCs in 2 mM Ca2+/1 mM
Mg2+ and 0.5 mM
Ca2+/2.5 mM Mg2+,
normalized to the same peak amplitude value, are shown superimposed.
The absolute peak amplitudes were 612 and 164 pA, respectively.
E, Plot of the decay time constants against
extracellular Ca2+ concentration: fast decay time
constant 1 (open symbols), slow decay
time constant 2 (filled symbols);
different symbol shape indicates different pairs (12 total). The
graph illustrates that the decay time course of the
IPSCs is only weakly dependent on extracellular Ca2+
concentration. F, Plot of the decay time constants (mean
value of the two time constant values weighted with the respective
amplitude contribution) of single unitary IPSCs in four pairs against
the peak amplitudes. Both weighted time constant and amplitude were
normalized to the mean value in the recorded ensemble.
Line represents the results of linear regression. No
significant correlation between time constant and amplitude was
apparent (p > 0.1). Data in
A, B, and D are from the
same pair.
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To further examine the possibility of cross talk (Barbour and
Häusser, 1997) or multivesicular release (Auger et al., 1998) at
higher release probabilities, we tested whether the decay time course
of average unitary IPSCs became slower as the
Ca2+/Mg2+
concentration ratio was increased. IPSCs in conditions of normal and
reduced
Ca2+/Mg2+
concentration ratio, normalized to their respective peak current amplitudes, showed almost identical time courses (Fig. 5D).
The mean decay time constant was very similar for
Ca2+ concentrations between 0.1 and 2 mM, with only a slight prolongation at higher
concentrations (Fig. 5E; 2-12 pairs per
Ca2+ concentration). Furthermore, as
indicated by the scatter plot from individual evoked IPSCs, the decay
time constant was independent of the IPSCs amplitude (Fig.
5F; four pairs). Thus the analysis of the decay time course
of IPSCs provides no evidence for cross talk or increase in the number
of fused vesicles with increasing Ca2+
concentration at this synapse.
Number of functional release sites and release probability
Because a decrease of the
Ca2+/Mg2+
concentration ratio reduced the amplitude of the IPSCs excluding
failures (Fig. 4), it appeared likely that the BC-GC synapse comprises
multiple functional release sites. To determine the number of
functional release sites and the probability of release at individual
sites quantitatively, we fitted IPSC peak amplitude distributions at
different
Ca2+/Mg2+
concentration ratios with compound binomial models of release (MP-CBA;
see Materials and Methods) (Fig. 6)
(Redman, 1990). Failures were also included in the fit because (1) they
could be unequivocally distinguished from successful IPSCs because of
the favorable recording conditions and (2) they appeared to be entirely
failures of synaptic transmission (stimulation failures were not
present in the paired recording configuration, and conduction failures
appeared unlikely; see Discussion).
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Figure 6.
Estimation of the number of functional release
sites and the release probability using multiple probability compound
binomial analysis. A, Peak amplitude distributions from
a pair in 2 mM Ca2+/1 mM
Mg2+ (a) and 0.5 mM Ca2+/2.5 mM
Mg2+ (b). The thick
curve represents the total probability density function ( Pi(x)); the
thin curves represent individual components
(P1(x) P7(x)) as obtained by
maximum-likelihood fit. For model parameters, see Table 2, pair #1.
Failures are not depicted; measured numbers of failures were 1 (a) and 430 (b), and
predicted numbers of failures were 1 (a) and 429 (b). B, Similar analysis for a
different pair (#3) in 2 mM Ca2+/1
mM Mg2+ (a) and
0.5 mM Ca2+/2.5 mM
Mg2+ (b). Measured numbers of
failures were 13 (a) and 731 (b), and predicted numbers of failures were 8 (a) and 729 (b).
C, Results of bootstrap analysis for the number of
functional release sites (left) and the release
probabilities in the two conditions (right) for pair #1
(a) and pair #3 (b).
Bootstrap replications (100) of the original data set were fitted in a
manner identical to the original data set, and the distributions of
estimated number of release sites and release probabilities (in 2 mM Ca2+/1 mM
Mg2+ and 0.5 mM
Ca2+/2.5 mM Mg2+,
respectively) were plotted. For details, see Materials and Methods.
D, Plot of estimated mean release probability
<p> against extracellular Ca2+
concentration for the five pairs shown in Table 2. Data were fitted
with a Hill equation f(c) = pmax [1 + (EC50/c)n]1,
with maximal release probability pmax = 0.79, EC50 = 1.5 mM, and apparent Hill
coefficient n = 2.4, where c denotes
the extracellular Ca2+ concentration.
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Two examples are shown in Figure 6, with amplitude distributions in 2 mM Ca2+/1 mM
Mg2+ on top and in 0.5 mM
Ca2+/2.5 mM
Mg2+ at the bottom. The estimated number
of functional release sites was seven and five, and the mean release
probability in 2 mM Ca2+/1
mM Mg2+ was 0.61 and 0.57, respectively. Bootstrap analysis further indicated that the errors in
the estimates of number of functional release sites and release
probability were small for both pairs (Fig. 6C, Table
2).
In five pairs in which IPSC amplitudes, rise times, and series
resistance were stationary and the total number of traces was >700,
the number of functional release sites estimated by MP-CBA ranged from
three to seven, and the mean release probability in physiological
divalent concentrations was between 0.41 and 0.63 (Table 2). In all
pairs both intrasite (Liu and Tsien, 1995a) and intersite (Nusser et
al., 1997) components of quantal variability appeared to be present.
Although the compound binomial model accounted for inhomogeneity of
release probability in principle, the high value of the shape parameter
p (101.4 102) (Table 2) indicated that the release
probability was relatively uniform among sites (Silver et al.,
1998).
MP-CBA further allowed us to investigate the dependence of the
estimated mean release probability on extracellular
Ca2+ concentration, as shown in Figure
6D. Fitting the data with a Hill equation revealed a
half-maximal release probability at a Ca2+
concentration of 1.5 mM, an apparent Hill
coefficient of 2.4, and an extrapolated maximal release probability of
0.79. Fitting the data at the low-concentration limit
(1mM) in double logarithmic representation gave
similar results (apparent Hill coefficient 2.5; fit results not
illustrated). In conclusion, single action potentials in the
presynaptic BC trigger powerful inhibition of the postsynaptic GC,
attributable to a large quantal size, a high release probability at
physiological Ca2+ concentrations, and the
presence of multiple functional release sites.
Depression induced by paired-pulse stimulation
In the intact hippocampal network, BCs can generate high-frequency
trains of action potentials (Penttonen et al., 1998; Csicsvari et al.,
1999). This raises the question of whether dynamic changes of
transmission occur at BC-GC synapses during repetitive stimulation. We
first examined paired-pulse modulation (Fig.
7). When two action potentials were
elicited in the BC, separated by intervals of variable duration, the
amplitude of the second IPSC was smaller than that of the first (Fig.
7A). The maximal paired-pulse depression, measured for 10 msec interpulse intervals, was 37 ± 6%. Recovery from PPD was
complete after 5 sec; when fitted with a single exponential function,
the time constant of recovery was 1.97 sec (Fig. 7B) (3-11
pairs).
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Figure 7.
Properties of PPD of IPSCs at the BC-GC
synapse. A, IPSCs evoked by pairs of action potentials
in the presynaptic BC, separated by intervals of variable duration.
Traces shown are averages of 30 unitary IPSCs and were
normalized to the same amplitude for the first average IPSC (absolute
values of A1 were 1393, 1180, and 1216 pA, respectively). B, Time course of recovery from PPD.
The ratio of amplitudes of the second
(A2) and the first
(A1) unitary IPSC, both measured from
their respective baselines as indicated in A, was
plotted against the interpulse interval. The curve
represents a fitted exponential function with a time constant of 1.97 sec. Number of pairs is indicated in parentheses above
the data points. C, Coefficient of variation analysis
suggests a presynaptic locus of PPD. The inverse of the square of the
coefficient of variation of A2
(CV2) was
plotted against the mean peak amplitude; data were normalized by the
CV2 and mean,
respectively, of A1. Data are from 10 pairs. Intervals between presynaptic action potentials were 100 msec
( ), 500 msec ( ), 1 sec ( ), 2 sec ( ), and 3 sec ( ).
Curve a represents the prediction of Equation 8 of Silver et
al. (1998) for a pure change in release probability p
superimposed on the data points (number of release sites = 5, release probability = 0.53, CV1 = 0.18, CV2 = 0.34; no variation in p).
Curve e represents the prediction of a pure change in
quantal size q, and curves
b-d show predictions for mixed changes (75, 50, and 25% contribution of changes in p, with
p = xa
and q = x1-a, where a is the
fractional contribution of the change in p and
x is the normalized mean). D, PPD appears
to be independent of presynaptic GABAB receptor activation.
Left, Average IPSCs in control conditions
(top) and in the presence of 5 µM
CGP55845A in the bath solution (center) are depicted,
together with a superposition of both traces (bottom).
Right, Summary bar graph of mean
A2/A1 in
control conditions and in the presence of CGP55845A. Extracellular
Ca2+ and Mg2+ concentrations were 2 and 1 mM, respectively. Interpulse interval, 100 msec. Failures
included in all averages. Number of pairs indicated in
parentheses above the bars.
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To determine whether the depression was presynaptic or postsynaptic in
origin, we examined the variation of peak current amplitudes during the
first and second IPSC for interpulse intervals of 100 msec. Figure
7C shows a summary plot of the inverse of the square of the
CV (CV2)
against the mean amplitudes of the second IPSC (nine pairs); both
CV2 and
mean were normalized to the values of the first IPSC (Malinow and
Tsien, 1990). The data points were superimposed with the predictions of
a model with variable presynaptic and postsynaptic contributions to
PPD, using Equation 8 of Silver et al. (1998) and mean values for
Nr, <p>,
CV1, and CV2 as obtained by
MP-CBA (Table 2). A comparison of data points and model curves suggests
that the results were more consistent with a reduction in
<p> than a reduction in <q>, implying that
PPD was mainly presynaptic in origin.
If PPD was caused by the activation of presynaptic
GABAB receptors (Deisz and Prince, 1989; Lambert
and Wilson, 1994) after release of GABA from a single BC, it should be
blocked by the selective, high-affinity GABAB
receptor antagonist CGP55845A (Kaupmann et al., 1997); however, PPD for
100 msec intervals was not significantly different in the absence and
presence, respectively, of 5 µM CGP55845A (Fig.
7D) (p > 0.2), indicating that PPD
is independent of presynaptic GABAB receptor activation.
If PPD was caused by depletion of the releasable pool of synaptic
vesicles (Stevens and Tsujimoto, 1995; Debanne et al., 1996; Dobrunz
and Stevens, 1997), its extent should be dependent on the average
release probability. In contrast to this prediction, the extent of PPD
at the BC-GC synapse was not significantly different in various
Ca2+/Mg2+
concentration ratios (Fig.
8A)
(p > 0.2). Furthermore, if PPD was caused by
depletion, the peak amplitude of the second IPSC evoked by paired
stimulation should be inversely related to that of the first (Debanne
et al., 1996); however, amplitudes of IPSCs evoked by paired stimuli
were not significantly correlated at the BC-GC synapse (Fig.
8B). Thus PPD at the BC-GC synapse was presynaptic
in origin, but unexpectedly appeared to be independent of both
extracellular Ca2+ concentration and
previous release.
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Figure 8.
PPD at the BC-GC synapse appears to be
independent of release probability and previous exocytosis.
A, PPD is independent of extracellular
Ca2+ concentration. Left, Average
IPSCs in 4 mM Ca2+/0.5 mM
Mg2+ (top) or 0.5 mM
Ca2+/4 mM Mg2+
(center) are depicted (failures included), together with
a superposition of both traces after normalization to give the same
peak amplitude for the first average IPSC (bottom).
Right, Mean A2/A1
(open bars) and percentage of failures
(filled bars) for 0.5 and 3 mM Ca2+/3
mM Mg2+. Interpulse interval, 100 msec. Number of pairs indicated in parentheses
on top. B, PPD appears to be independent of previous
release. Plot of A2 against
A1 for individual events is shown.
Amplitudes were normalized to the mean A1 in
the recorded ensemble. Line represents the results of
linear regression. No significant correlation between IPSC peak
amplitudes was apparent (p > 0.05);
interpulse interval, 100 msec. Data were from 10 pairs; different pairs
are represented by different symbols.
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Depression induced by multiple-pulse stimulation
We then examined dynamic changes of transmission at the BC-GC
synapse during 1-50 Hz trains of 900-1000 action potentials (Fig.
9). Evoked IPSCs at the BC-GC synapse
showed a marked depression during high-frequency stimulation, as
reported previously for inhibitory synapses in the neocortex (Galarreta
and Hestrin, 1998; Varela et al., 1999). The onset of depression was
biexponential, with time constants of 61 msec and 17.6 sec for a
stimulation frequency of 20 Hz (Fig. 9C) (23 pairs). The
steady-state depression increased with the frequency of stimulation;
half-maximal depression occurred at 5.0 Hz (Fig. 9D). After
a 20 Hz train of action potentials, recovery from depression was
biexponential, with time constants of 1.3 and 31.7 sec (Fig.
9E) (23 pairs).
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Figure 9.
Depression of IPSCs at the BC-GC synapse during
multiple-pulse stimulation. A, Unitary IPSCs during a 20 Hz train of 1000 action potentials; stimulation frequency before and
after the train was 0.25 Hz. Presynaptic action potentials
(top) were truncated. B, Unitary IPSCs at
an expanded time scale from the same pair as shown in A
in control conditions (top), at the end of the 20 Hz
train (center), and after recovery
(bottom). C, Onset of depression during a
20 Hz train. Each data point represents the mean IPSC peak amplitude in
23 pairs, normalized to the mean peak amplitude at 0.25 Hz before the
train. D, Plot of mean unitary IPSC peak amplitudes
(action potentials 851-900, failures included) against stimulation
frequency, normalized to the control value (at 0.25 Hz). Number of
pairs indicated in parentheses above the data points.
E, Recovery from depression induced by a 20 Hz train.
Each data point represents the mean peak amplitude from one (first
point) or three (all following points) consecutive IPSCs in 23 pairs,
normalized to the mean peak amplitude at 0.25 Hz after complete
recovery from depression (which, on average, was 1.05-fold larger than
that before the train, indicating a slight post-tetanic potentiation).
F, Coefficient of variation analysis of short-term
depression. The inverse of the square of the CV of the peak amplitude
of unitary IPSCs (action potentials 851-900) was plotted against the
mean peak amplitude for different frequencies; data were normalized by
the CV2 and mean, respectively,
of IPSCs evoked at 0.25 Hz. Stimulation frequency was 1 Hz ( ), 2 Hz
( ), 10 Hz (), 20 Hz (), 40 Hz (), and 50 Hz ().
Curve a represents the prediction of Equation 8 of
Silver et al. (1998) for a pure change in p superimposed
on the data points (with same parameters as in Fig. 7). Curve
e represents the prediction of a pure change in
q, and curves b-d show predictions for
mixed changes (75, 50, and 25% contribution of changes in
p). G, Onset of depression during a 20 Hz
train in the presence of 5 µM CGP55845A. Each data point
represents the mean peak amplitude in seven pairs, normalized to the
mean peak amplitude at 0.25 Hz before the train. H, The
slow component of depression appears to be dependent on release
probability. Onset of depression during 20 Hz trains in 0.5 mM Ca2+/2.5 mM
Mg2+ (9 pairs, ) and 2 mM
Ca2+/1 mM Mg2+ ().
Each data point represents the mean peak amplitude, normalized to the
mean peak amplitudes at 0.25 Hz before the train. I,
Correlation of peak amplitudes of consecutive IPSCs in the late portion
of 20 Hz trains (last 500 action potentials) in 2 mM
Ca2+/1 mM Mg2+. Peak
amplitudes An+1 of IPSCs (or failures) were
plotted against the amplitudes An of the
directly preceding IPSCs (or failures), both normalized to the mean
amplitude of the data set. Eleven of 11500 points are located outside
the plot range. Line represents the results of linear
regression. A slight but significant negative correlation between IPSC
peak amplitudes was apparent (slope 0.054; p < 0.001). Extracellular Ca2+ and Mg2+
concentrations were 2 and 1 mM, respectively, in all cases
except H. Time 0 in C, E,
G, and H indicates the time of change in
frequency. SEMs in C, G, and
H were not shown for clarity. Continuous
curves in C-E and H
represent the predictions of a two-pool model with activity-dependent
reduction in release probability fitted to the data points (Fig.
10).
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To determine whether the depression was presynaptic or postsynaptic in
origin, CV2
was plotted against the mean unitary IPSC in steady-state conditions at
a given frequency; both
CV2 and
mean were normalized to the control values at 0.25 Hz (Fig. 9F). The data points were superimposed with the
predictions of a model with variable presynaptic and postsynaptic
contributions to depression (Fig. 7C). Similar to PPD,
comparison of data and predictions suggests that multiple-pulse
depression was mainly presynaptic in origin. In the presence of 5 µM CGP55845A, the time course of depression was
qualitatively similar, but the steady-state IPSC amplitude was
significantly larger (33 ± 6% vs 21 ± 2%,
p < 0.01) (Fig. 9G) (seven pairs),
suggesting a small contribution of GABAB
receptors to multiple-pulse depression.
Frequency-dependent depression is often interpreted as a depletion of
the releasable pool of synaptic vesicles (Liley and North, 1953;
Stevens and Tsujimoto, 1995). If this were the case, its extent would
depend on the average release probability (Dittman and Regehr, 1998),
and amplitudes of consecutive IPSCs in the train may be correlated
(Matveev and Wang, 2000). We therefore examined depression during 20 Hz
trains at different
Ca2+/Mg2+
concentration ratios (Fig. 9H). In conditions of
reduced release probability (0.5 mM
Ca2+), the fast component of depression
was comparable, but the slow component showed a reduced amplitude and a
slower time course (Fig. 9H, open symbols). The
ratio of currents at the end of the train to those before the train was
significantly larger in 0.5 mM
Ca2+ (0.55 ± 0.08) than in 2 mM Ca2+ (0.27 ± 0.03; p < 0.001). Furthermore, amplitudes of
subsequent IPSCs in the second portion of the train were negatively
correlated (Fig. 9I). These results are consistent
with the hypothesis that the slow component of depression is caused by
depletion of the vesicular pool (Matveev and Wang, 2000).
To understand the interactions between fast and slow depression, we
examined the quantitative predictions of pool models with different
forms of activity dependence (Betz, 1970; Kusano and Landau, 1975).
Figure 10 illustrates the predictions
of a two-pool model with constant release probability and replenishment
(Fig. 10, model a) and two alternative models with either
activity-dependent reduction in release probability (model
b) or activity-dependent increase in replenishment (model
c). In the absence of evidence for multivesicular release
(Fig. 5), we implemented a univesicular release constraint (Korn et
al., 1982; Matveev and Wang, 2000). Among the alternatives tested, the
model with the activity-dependent reduction in release probability
would be most consistent with the experimental observations. Unlike the
other models, it predicts the biexponential time course of
multiple-pulse depression (Fig. 10B) very closely.
Furthermore, the model predicts the approximate extent of PPD and the
independence of PPD on release probability (Fig. 10, legend). If this
model is used to fit the depression data in Figure 9,
C-E and H, with release probabilities
constrained to the mean values obtained by MP-CBA (Fig.
6D, Table 2), we estimate that the capacity of the
releasable pool is 51 vesicles per release site at the BC-GC synapse
(see Discussion and continuous curves in Fig. 9 for quantitative
predictions).
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Figure 10.
Activity-dependent gating of release may
prevent depletion of the releasable pool of synaptic vesicles.
A, Schematic illustration of different vesicular pool
models. Univesicular release constraint. The capacity of the releasable
pool (Nv0) was assumed as 50, the
initial release probability (pR at
time 0) as 0.5, and the refilling rate as k = 0.01 sec1. a,
Two-pool model with constant rates. b, Two-pool model
with activity-dependent reduction in release probability (Betz, 1970).
Activity-dependent reduction of pR was
defined by amax = 1, |
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ABSTRACT
INTRODUCTION
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