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The Journal of Neuroscience, August 15, 1998, 18(16):6147-6162
Calcium Dependence and Recovery Kinetics of Presynaptic
Depression at the Climbing Fiber to Purkinje Cell Synapse
Jeremy S.
Dittman and
Wade G.
Regehr
Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts 02115
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ABSTRACT |
Short-term depression is a widespread form of use-dependent
plasticity found in the peripheral and central nervous systems of
invertebrates and vertebrates. The mechanism behind this transient decrease in synaptic strength is thought to be primarily the result of
presynaptic "depletion" of a readily releasable neurotransmitter pool, which typically recovers with a time constant of a few seconds. We studied the mechanism and dynamics of recovery from depression at
the climbing fiber to Purkinje cell synapse, where marked presynaptic depression has been described previously. Climbing fibers are well
suited to studies of recovery from depression because they display
little, if any, facilitation (even under conditions of low-release
probability), which can obscure rapid recovery from depression for
hundreds of milliseconds after release. We found that recovery from
depression occurred in three kinetic phases. The fast and intermediate
components could be approximated by exponentials with time constants of
100 msec and 3 sec at 24° C. A much slower recovery phase was also
present, but it was only prominent during prolonged stimulus trains.
The fast component was enhanced by raising extracellular calcium and
was eliminated by lowering presynaptic calcium, suggesting that, on
short time scales, recovery from depression is driven by residual
calcium. During regular and Poisson stimulus trains, recovery from
depression was dramatically accelerated by accumulation of presynaptic
residual calcium, maintaining synaptic efficacy under conditions that
would otherwise deplete the available transmitter pool. This represents a novel form of presynaptic plasticity in that high levels of activity
modulate the rate of recovery as well as the magnitude of
depression.
Key words:
presynaptic depression; paired-pulse depression; Purkinje
cell; climbing fiber; inferior olive neuron; Poisson stimulus
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INTRODUCTION |
Many synapses exhibit decreased
efficacy with repeated use that lasts for seconds to minutes (Del
Castillo and Katz, 1954 ; Betz, 1970; Kusano and Landau, 1975; Thomson
et al., 1993; Varela et al., 1997). Such synaptic depression was
described nearly 60 years ago (Eccles et al., 1941; Feng, 1941), but
the potential significance of this phenomenon is only now being
appreciated. It has been suggested that synaptic depression can allow a
neuron to detect synchronous rate changes in populations of synaptic inputs (Abbott et al., 1997) and can serve as a form of synaptic gain
control (Markram and Tsodyks, 1996; O'Donovan and Rinzel, 1997; Varela
et al., 1997). As we become aware of the potential utility of synaptic
depression, there remains a need to understand the mechanisms
underlying depression and how the magnitude and recovery kinetics of
depression can be modulated in a use-dependent manner.
One of the first and simplest presynaptic models maintains that, after
exocytosis, release sites require a finite recovery time and that
depression reflects an activity-dependent "depletion" of available
release sites (Takeuchi, 1958; Elmqvist and Quastel, 1965; Betz, 1970).
Although this model accounts for many features of depression at a
variety of synapses, it greatly overestimates the amount of depression
during repetitive trains. This observation gave rise to the hypothesis
that recovery from depression might be more rapid during stimulus
trains (Kusano and Landau, 1975; Byrne, 1982). However, the presence of
prominent facilitation made it difficult to test this hypothesis, so
the dynamics of synaptic depression remained poorly understood. An
additional complication is that a variety of other pre- and
postsynaptic factors may contribute to short-term synaptic depression,
including presynaptic calcium channel inactivation (Gingrich and Byrne, 1985; Patil et al., 1998), failure of action potential initiation or
conduction failure (Hatt and Smith, 1976; Smith and Hatt, 1976), negative feedback through autoinhibitory metabotropic receptors (Deisz
and Prince, 1989; Davies et al., 1993; von Gersdorff et al., 1997), and
receptor desensitization (Trussell and Fischbach, 1989; Trussell et
al., 1993).
The synapse between inferior olivary neurons and Purkinje cells is well
suited to studies of paired-pulse depression (PPD) and to clarification
of the factors governing recovery from depression. Axonal terminals of
inferior olivary neurons are known as climbing fibers because they
appear to climb along the Purkinje cell dendrites where they form
extensive synaptic contacts (Ramon y Cajal, 1911). They can be
activated in an all-or-none manner triggering a series of action
potentials followed by a long-lived afterhyperpolarization (Eccles et
al., 1964). Elimination of active conductances reveals pronounced PPD
of synaptic responses, as first described in vivo by Eccles
and colleagues (Eccles et al., 1966a). More recently, this synapse has
been studied in brain slices (Konnerth et al., 1990; Perkel et al.,
1990; Takahashi et al., 1995; Hashimoto and Kano, 1998), where it has
been shown that neither presynaptic metabotropic receptors sensitive to
glutamate, GABA, or adenosine nor postsynaptic receptor desensitization
contribute to PPD (Hashimoto and Kano, 1998). Moreover, manipulations
that decreased the release probability also decreased PPD, indicating
that for climbing fibers PPD is primarily a consequence of presynaptic
mechanisms (Hashimoto and Kano, 1998), as seems to be the case at other
synapses (Otsuka et al., 1962; Betz, 1970; von Gersdorff and Matthews,
1997; von Gersdorff et al., 1997).
Here we investigate depression at the climbing fiber to Purkinje cell
synapse. The magnitude of PPD correlated with release probability, as
expected from simple depletion models (Lundberg and Quilisch, 1953;
Dobrunz and Stevens, 1997). In addition, recovery from depression had
three distinct kinetic components, which we refer to as the fast,
intermediate, and slow phases. We found that the fast and intermediate
components of recovery account for the majority of depression during
brief stimulus trains, whereas prolonged stimulation reveals a
pronounced slow component. In this study we focus on the intermediate
and fast phases, which are well approximated by exponentials with time
constants of 3 sec and 100 msec. Finally, we show that fast recovery is
driven by residual calcium. This calcium-dependent recovery plays a
major role in determining synaptic strength during periods of
repetitive activation, when presynaptic calcium reaches high levels and
greatly accelerates recovery from depression.
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MATERIALS AND METHODS |
Synaptic physiology. Transverse slices (300 µm
thick) were cut from the cerebellar vermis of 9- to 13-d-old Sprague
Dawley rats. Slices were superfused with an external solution
containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4,
and 25 glucose, bubbled with 95% O2/5%
CO2. Flow rates were 1-2 ml/min at 24°C and 5-8 ml/min
at 34°C. Bicuculline (20 µM) was added to the external
solution to suppress synaptic currents mediated by GABAA
receptors; 4 Cae corresponds to 4 mM
CaCl2 and 0 mM MgCl2 in the
external solution, 2 Cae refers to 2 mM
CaCl2 and 1 mM MgCl2 and 1 Cae refers to 1 mM CaCl2 and 2 mM MgCl2.
Whole-cell recordings of Purkinje cells were obtained as described
previously (Mintz et al., 1995) with an internal solution of (in
mM): 35 CsF, 100 CsCl, 10 EGTA, 10 HEPES, and 0.2 D600, adjusted to pH 7.2 with CsOH. Synaptic currents were
monitored at a holding potential of  40 mV to inactivate voltage-gated
Na channels, and D600 was included to block voltage-gated calcium channels. The access resistance and leak current (20 to 200 pA
holding at 40 mV) were monitored continuously. Experiments were
rejected if either access resistance or leak current increased significantly during recording.
Recording and isolation of the climbing fiber response. Two
glass electrodes (tip diameter, 10-12 µm) filled with external saline solution were placed in the granular cell layer near the Purkinje cell soma. The pipettes were connected to a stimulus current
generator in a bipolar configuration. After establishing a whole-cell
voltage-clamp recording from the Purkinje cell, brief pulses (200-400
µsec) of current (1-10 µA) were passed between the electrodes.
They were repositioned until a climbing fiber EPSC (CF-EPSC) was
activated. A typical CF-EPSC recorded at a holding potential of 40 mV
is shown in Figure 1 Peak CF-EPSC magnitudes typically ranged from 6 to 12 nA. Because of the large size
and rapid time course of the CF-EPSC, we used low-resistance recording
electrodes (resistance was 0.8-1.2 M with a tip diameter >3 µm)
and maximal series resistance compensation to minimize voltage-clamp
errors. In addition, a small amount (0.5-2 µM) of the
competitive AMPA/kainate receptor antagonist CNQX was added to the
superfusate to reduce the magnitude of the CF-EPSC. Figure 1A demonstrates that an eightfold reduction in
CF-EPSC size after addition of 3 µM CNQX had only minor
effects on the time course of the CF-EPSC. At 24°C, there was
variability in the time course of the EPSCs in well-clamped cells, with
decay time constants of the EPSCs ranging from 4 to 8 msec.
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Figure 1.
Stimulation of climbing fiber synaptic currents.
A, Evoked climbing fiber EPSC before (thin
line) and during (thick line) bath application
of 3 µM CNQX. The synaptic response in CNQX was scaled to
the control response (dotted line) for comparison of the
EPSC waveform. Traces are averages of 10 trials each.
B, Consecutive traces taken during
perithreshold stimulation of a climbing fiber demonstrating the
all-or-none behavior of CF-EPSCs. The inset is taken
from the boxed region indicated. The
arrow indicates the climbing fiber failure.
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In addition to activating CF-EPSC, stimulation within the granule cell
layer can activate glutamatergic parallel fiber EPSCs (PF-EPSC).
Contamination with PF-EPSCs, which exhibit a large paired-pulse
facilitation (PPF), can result in an underestimate of the true climbing
fiber PPD. This problem is accentuated in conditions of low external
calcium, in which climbing fiber PPD is small, but parallel fiber PPF
is pronounced (Atluri and Regehr, 1996); even a small parallel fiber
contamination can make a CF-EPSC appear facilitated in low external
calcium. With this potential complication in mind, we minimized
parallel fiber contamination by adjusting stimulus strength to
threshold and analyzing failures by the presence of PF-EPSCs.
Figure 1B shows two consecutive stimuli. The first
elicited a CF-EPSC; the second failed to drive the CF, and no
underlying PF-EPSC was apparent. In all experiments, PF-EPSCs were
<0.5% of the CF-EPSC. In addition, cells with more than one CF were
rejected.
Cyclothiazide experiments. Cyclothiazide (CTZ) was used in
some experiments to explore the contributions of postsynaptic receptor desensitization to PPD. In the presence of 20-60 µM CTZ,
CF-EPSC decay times were significantly prolonged, and peak synaptic
currents were somewhat reduced (see Fig. 5A,
left). In contrast, when CNQX was included in the
superfusate to reduce CF-EPSC amplitude, application of CTZ
consistently increased the peak synaptic currents as well as the time
constant of decay (see Fig. 5A, right). A
possible explanation for this increase is that CTZ either competes with CNQX for a binding site on the AMPA receptor or allosterically decreases the affinity of CNQX, thereby reducing steady-state blockade
(Yamada and Rothman, 1992; Yamada and Turetsky, 1996).
Data acquisition and analysis. Outputs of the Axopatch 200A
were filtered at 1 kHz and digitized at 5 kHz with a 16-bit
digital-to-analog converter (Instrutech, Great Neck, NY) using Pulse
Control software (Herrington and Bookman, 1995). Random train stimuli
were generated off-line and sent through the DAC to the stimulus
isolation unit in 10 sec epochs, whereas the holding potential was
maintained at 40 mV. Both on- and off-line analysis as well as
computer simulations were done using Igor Pro software (Wavemetrics,
Lake Oswego, OR).
Calcium-dependent model of presynaptic depression. According
to Scheme II (see Results) and assuming that this scheme accounts for
all possible states of the release apparatus, R + T + N = No,
where R sites are in a refractory state, T sites
are in a transitional state, N sites are release ready, and
there are a total of No release sites.
Calcium dynamics must also be considered to account for recovery from
depression according to Scheme II. After a presynaptic action potential
arrives, free calcium rapidly jumps from its resting value
(Carest)) to an initial concentration (Cao + Carest) and decays
exponentially with time constant  c back to
Carest (Neher and Augustine, 1992; Tank et al., 1995). The
assumption of a single exponential decay was made for simplicity, but
at some presynaptic terminals, calcium transients are better
approximated by a double exponential decay (Atluri and Regehr, 1996).
The postulates about the recovery dynamics of the climbing fiber
presynaptic terminal can be expressed quantitatively in the following
manner:
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(1)
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(2)
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(3)
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where an action potential arrives at the presynaptic terminal at
time to. Rapid equilibration with calcium
is assumed, and in analogy with the Michaelis-Menten reaction scheme,
T is assumed to be in steady state, with
dT/dt = 0 (Schulz, 1994). Here, the kinetic
constant KN = (k + kmax)/k+.
Equations 1 and 2 can be solved analytically for the response to a
single action potential, but the solution can be simplified enormously
with one further approximation. If resting calcium is very low relative
to the total calcium influx after an action potential
(Carest  Cao), then recovery can
be divided into two separate phases: a rapid recovery occurring when Ca
is near Cao, and a slower phase occurring when
Ca has decayed to low levels that we call the intermediate recovery
phase. Under this assumption, Equations 1 and 3 can be approximated
by:
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(4)
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(5)
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where calcium jumps from 0 to Cao and
decays back to 0, whereas krecov can range
between a minimal rate ko and the same
maximal rate kmax as in Equation 3. This
simplification can be interpreted in two ways. First, resting calcium
may not contribute to the intermediate recovery kinetics observed at
this synapse, so ko would represent the
recovery rate of a separate, calcium-independent pathway used when
residual calcium falls to a low concentration. Alternatively, both the
fast and intermediate phases of recovery may depend on calcium, as
indicated in Scheme II, and ko would correspond to the recovery rate when Ca = Carest
[from Equation 3, ko = kmax (1 + KN/Carest)1].
Our data did not distinguish between these two interpretations because
we had no means of altering or eliminating resting calcium. After this
simplification, equations 2, 4, and 5 can be solved for the response to
a single action potential, yielding:
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(6)
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The paired-pulse depression recovery curves (see Fig. 9) were
fit to Equation 6 with three free parameters:
KN/Cao, kmax, and c.
Note that ko and
po were constrained by the data because
PPD at early times gives a rough estimate of po and residual calcium has decayed to
baseline levels within the first few seconds so the recovery rate is
essentially ko. When external calcium was
altered, po and Cao
were changed accordingly, while KN,
kmax, and c
were fixed. When EGTA-AM was added, c was
decreased by a factor comparable with that seen in parallel fibers
(Atluri and Regehr, 1996), and po was
decreased slightly, consistent with the observed effect of EGTA-AM on
the evoked EPSC (see Results).
For trains of stimuli, Equation 4 can be solved explicitly for the
value of residual calcium immediately after the ith pulse (Regehr et al., 1994):
![<UP>Ca</UP><SUB>i</SUB>=<UP>Ca</UP><SUB>i<UP>−</UP>1</SUB> · <UP>exp</UP>[<UP>−</UP>(t<SUB>i</SUB>−t<SUB>i<UP>−</UP>1</SUB>)/&tgr;<SUB>c</SUB>],](/content/vol18/issue16/fulltext/6147/img007.gif)
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(7)
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where ti1 and
ti are the (i-1)th and
ith stimulus times, respectively. In the special case of
evenly spaced stimuli given at rate r, calcium just after
the ith pulse is given by:
![<UP>Ca</UP><SUB>i</SUB>=<UP>Ca</UP><SUB>∞</SUB>[1−<UP>exp</UP>(<UP>−</UP>i/r&tgr;<SUB>c</SUB>)],](/content/vol18/issue16/fulltext/6147/img008.gif)
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(8)
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where  is the steady-state value of calcium reached after a
large number of stimuli:
![<UP>Ca<SUB>∞</SUB>=Ca</UP><SUB>o</SUB>[1−<UP>exp</UP>(<UP>−</UP>1/r&tgr;<SUB>c</SUB>)]<SUP>−1</SUP>.](/content/vol18/issue16/fulltext/6147/img009.gif)
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(9)
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After each stimulus, the number of available release sites decreases by
a fraction proportional to the release probability (i.e.,
 Ni = Nipo), and
each release site recovers with a rate that depends on the value of
residual calcium. Using Equations 2 and 5:
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(10)
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where:
and Ni is the number of available
sites immediately before the ith stimulus. Equation 10 was
used to calculate depression during trains (see Figs. 7, 8, 10-12 with
the parameters given in Fig. 9). After a sufficient number of stimuli,
the number of available sites reaches a steady-state value given
by:
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(11)
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In the case of calcium-independent recovery (or in the limit
KN  so   1),
equation (11) reduces to:
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(12)
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Equations (11) and (12) were used to calculate steady-state
attenuation as a function of frequency for the calcium-dependent (Scheme II) and calcium-independent recovery models (Scheme I), respectively (see Fig. 12B).
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RESULTS |
Paired-pulse depression and release probability
Experiments were performed to determine the dependence of PPD,
defined as (EPSC1 EPSC2)/EPSC1, on presynaptic
calcium influx and release probability (Fig.
2). Under control conditions, with 2 mM external calcium (Cae) and 1 mM external magnesium (Mge), there is
~50% depression for pulses separated by 30 msec. When Cae was lowered to 0.5 mM and Mge
raised to 2.5 mM, both the peak EPSC and PPD decreased
significantly (Fig. 2A). Although the EPSC was
reduced by approximately a factor of three, no underlying facilitation
was revealed (see Materials and Methods). At intermediate Cae (1 mM Cae and 2 mM
Mge), peak EPSCs and PPD decreased moderately (Fig.
2B), whereas high Cae (4 mM
Cae and 0 mM Mge) slightly
raised the magnitude of PPD and had little effect on peak EPSCs (Fig. 2C).
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Figure 2.
Dependence of release probability and PPD
on external Ca. A, Top left, Peak EPSC
time course for a pair of EPSCs recorded with a 30 msec interstimulus
interval in 2 Cae. EPSC1 is indicated by
open circles, and EPSC2 is indicated by
filled circles. Low Ca external solution (0.5 mM Ca; 2.5 mM Mg) was applied during the time
indicated by the thick horizontal line. Bottom
left, Paired-pulse depression plotted for each data point.
Top right, Average of 10 traces taken
before (thin lines) and during (thick
lines) exposure to low Cae. Bottom
right, Traces replotted normalized to the peak
of the first EPSC. B, Application of 1 Cae.
C, Application of 4 Cae.
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The relationship between Cae and peak EPSC suggests that
the climbing fiber to Purkinje cell synapse operates at a high baseline release probability. Although raising Cae from 0.5 to 1 mM greatly enhanced synaptic strength, further increases in
Cae had little effect on synaptic strength. This behavior
is a hallmark of a synapse that operates near saturation and is in
sharp contrast to nonsaturated synapses, where doubling Cae
can enhance synaptic strength 10-fold (Zucker, 1989).
It is interesting to consider these findings in terms of the depletion
model, for which the magnitude of PPD at very brief interpulse
intervals approximates the probability of release (see Materials and
Methods). With our definition of PPD, if the second stimulus occurs
when there has not been sufficient time for recovery, then PPD  po, where po
is defined as the probability that a site releases its transmitter
given that it is release ready (see Discussion and subsequent sections
of Results for a more detailed discussion). Based on the depletion
model and with the assumption that very little recovery has occurred at
30 msec, the probabilities of release are estimated to be 0.04, 0.3, 0.5, and 0.7 in 0.5, 1, 2, and 4 mM Cae,
respectively. Thus, the data in Figure 2 are qualitatively consistent
with this model to the extent that there is less PPD when
po is reduced in low Cae
(Lundberg and Quilisch, 1953; Thies, 1965).
Kinetics of recovery from paired-pulse depression
In addition to the degree of depression, another important factor
is the time course of PPD. Our estimates of
po may underestimate the true release
probability if some recovery has occurred within 30 msec. To examine
recovery from depression, we calculated the time course of PPD for a
variety of different interpulse intervals in the presence of different
concentrations of Cae (Fig.
3). Under all conditions tested, recovery
from depression had a phase that persisted for many seconds (termed
intermediate recovery phase). An additional fast recovery phase was
apparent in 2 mM Cae (Fig. 3B) and
was more pronounced in 4 mM Cae (Fig.
3C). Averages of multiple experiments of this type revealed
similar trends in recovery kinetics. Under conditions of low-release
probability and small presynaptic calcium influx (1 mM
Cae), PPD is well approximated by a single
exponential with a time constant = 3.6 sec and amplitude A = 36% (Fig. 4A,
top). In 2 mM Cae, recovery
from depression is approximated by a double exponential: a fast
component with Afast = 21% and
fast = 100 msec and an intermediate component with
amplitude Ainter = 40% and inter = 3.2 sec (Fig. 4A, middle). In 4 mM Cae, PPD decay is also described by a
sum of two exponentials, with Afast = 44%,
fast = 87 msec, Ainter = 38%,
and inter = 2.5 sec (Fig. 4A,
bottom). Superposition of all three PPD curves illustrates
the similarity of the intermediate recovery phase (Fig.
4B) and the systematic increase in amplitude of the
fast recovery phase (Fig. 4B, inset).
Depression at interpulse intervals of <10 msec was not explored
because climbing fiber activation was unreliable, and we had no
independent means of confirming that an action potential successfully
reached the presynaptic terminals after the second stimulus. Under all
conditions, a small degree of depression (<5%) was still present
after 10 sec. This residual depression may have reflected a slow
recovery phase that became prominent during long stimulus trains.
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Figure 3.
Kinetics of recovery from paired-pulse depression.
Representative experiments showing recovery from depression in 1 Cae (A), 2 Cae
(B), and 4 Cae
(C). Insets are single
traces for paired-pulse intervals of 10, 15, 20, 30, 40, and 50 msec after control stimulation. %PPD = 100 (EPSC1 EPSC2)/EPSC1, where
EPSC1 and EPSC2 are, respectively, the
amplitudes of the control and depressed EPSCs.
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Figure 4.
Dependence of recovery kinetics on external
calcium. A, Average recovery of depression in 1 Cae (top; n = 9), 2 Cae (middle; n = 11),
and 4 Cae (bottom; n = 12). Error bars indicate SEM. In 1 Cae, data were
fit to %PPD = Aet/ , with
A = 36%, and = 3.6 sec. In 2 and 4 Cae, data were fit to %PPD = Afastet/fast + Ainteret/inter.
In 2 Cae, Afast = 21%,
Ainter = 40%, fast = 99 msec, and inter = 3.2 sec. In 4 Cae,
Afast = 44%,
Ainter = 38%, fast = 87 msec, and inter = 2.5 sec. B,
Superimposed PPD curves for the three external calcium conditions.
Inset, Early time points for the rapid phase of recovery
from depression in 1 Cae (filled
circles), 2 Cae (open circles), and
4 Cae (filled diamonds).
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The major features of depression found at 24°C were also present at
34°C, although recovery was accelerated significantly. At 34°C in 2 Cae, Afast = 15% and
fast = 44 msec, and an intermediate component exists
with amplitude Ainter = 36% and
inter = 1.2 sec (n = 5; data not shown).
Similarly, in vivo measurements of PPD at this synapse in
adult cats showed two components of recovery from depression with
Afast of ~20% and fast of
~20 msec and with Ainter of ~30% and
inter of ~0.5 sec (Eccles et al., 1966a,b).
Postsynaptic contributions to the rapid recovery phase
One possible explanation for the rapid recovery phase of PPD is
that it reflects recovery from postsynaptic receptor desensitization. Consistent with this hypothesis is the observation that it takes tens
of milliseconds for AMPA receptors to recover from desensitization, and
desensitization can be more pronounced under conditions of enhanced
neurotransmitter release (Trussell et al., 1993). To test the
contribution of desensitization to PPD at the climbing fiber to
Purkinje cell synapse, we determined the effect of the benzodiathiazide
diuretic CTZ on PPD. CTZ slows the rate of desensitization for
AMPA receptors (Yamada and Rothman, 1992) and has a number of
additional effects (Diamond and Jahr, 1995). If the rapidly recovering
component of PPD is attributable to recovery from desensitization, then
it should be greatly reduced or eliminated by CTZ, and the magnitude of
PPD should be reduced for short interpulse intervals.
CTZ (20-60 µM) consistently prolonged EPSC decay times
with or without CNQX (Fig.
5A). However, CTZ had little
effect on the magnitude of PPD present for brief interpulse intervals
(Fig. 5B). Figure 5, C and D, shows
averages of multiple PPD time-course experiments performed with 40 µM CTZ under control conditions (2 mM
Cae) and in high Cae (4 mM).
In all experiments (n = 7), CTZ did not attenuate the
fast recovery component relative to that in control conditions (39%
fast component in 2 Cae; 50% fast component in 4 Cae); in fact, the fast component was more
pronounced after treatment with CTZ in both 2 and 4 Cae.
These results suggest that postsynaptic receptor desensitization does
not contribute significantly to the rapid recovery phase of
paired-pulse depression for intervals >10 msec. Furthermore, the
change in PPD suggests that CTZ acts presynaptically to enhance
transmitter release. Similar presynaptic effects have been reported
at other synapses (Diamond and Jahr, 1995).
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Figure 5.
Effects of cyclothiazide on the rapid phase of
recovery from depression. A, Top,
Synaptic currents recorded in the absence (left) and
presence (right) of 1 µM CNQX before
(thick line) and during (thin line)
exposure to 40 µM CTZ. CTZ slowed the decay times from 4 to 9 msec (left) and from 6 to 11 msec
(right). Bottom, Traces
normalized for comparison. The effect of CTZ on the amplitude of the
EPSC is discussed in Materials and Methods. Traces are
averages of 10 trials. B, Left, Effect of
40 µM CTZ on the amplitude of EPSC1
(filled circles), EPSC2 (open
circles), and %PPD (bottom;
t = 15 msec). Right, Averages of
10 traces in control (thin lines) and 40 µM CTZ (thick lines). Lower
trace, Same traces scaled to the peak of the first EPSC.
C, Average time course of PPD in control conditions
(open circles) and in 2 Cae and 40 µM CTZ (filled circles; four
experiments). The CTZ recovery curve was fit to a double exponential
with Afast = 38%,
Ainter = 39%, fast = 25 msec, and inter = 2.4 sec. D, Average
time course of PPD in 4 Cae without (open
circles) and with 40 µM CTZ (filled
circles; four experiments). The CTZ recovery curve was fit to a
double exponential decay with amplitudes
Afast = 50%,
Ainter = 52%, fast = 23 msec, and inter = 1.7 sec.
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Dependence of rapid recovery on presynaptic calcium dynamics
The enhancement of the rapid recovery phase with increased
Cae suggests two main possibilities for its underlying
mechanism. (1) Recovery from depression is dependent on intracellular
calcium levels, and increased Cae leads to larger
stimulus-evoked calcium transients that accelerate recovery, and (2)
recovery from depression is somehow coupled to the probability of
release (a higher po leads to a more
pronounced rapid component of recovery). We distinguished between these
possibilities by introducing EGTA into presynaptic terminals using
EGTA-AM. Because EGTA binds calcium with slow kinetics, moderate
concentrations of the chelator can be introduced into the presynaptic
terminal without significantly affecting the large, brief, and
localized calcium transients that dictate release probability (Adler et
al., 1991). As we have shown previously, calcium levels in the tens to
hundreds of milliseconds after stimulation (residual calcium) are much
more effectively reduced by EGTA, and the slow calcium-binding
kinetics of EGTA allow it to speed the decay of calcium (Atluri and
Regehr, 1996). For example, exposure to 100 µM EGTA-AM
for 10 min shortens the half-decay time of presynaptic calcium
transients in parallel fibers evoked by a single stimulus from 40 to
2 msec.
Figure 6A shows an
example of a PPD time course determined before and after bath
application of 100 µM EGTA-AM in the presence of 4 mM Cae. The magnitude of depression at 10 msec
was unaltered, but recovery from depression was slowed. The lack of an
EGTA effect during the first few paired-pulse stimuli is consistent
with its slow on-rate, which prevents EGTA from affecting the early
phase of residual calcium decay. By monitoring PPD at an interpulse interval of 500 msec during a bath application of EGTA-AM, we observed
a nearly two-fold increase in the magnitude of depression accompanied
by a slight decrease in the EPSC amplitude (Fig. 6B). PPD at 500 msec increased by a factor of 1.9 ± 0.2 (mean ± SEM; n = 7). The PPD curves measured in 4 Cae with and without loading with EGTA-AM are compared in
Figure 6C. The EGTA PPD curve was fit to a sum of two
exponentials with Afast = 17%,
fast = 17 msec, Ainter = 65%,
and inter = 2.9 sec. In comparison with the 4 Cae PPD curve (Afast = 44%,
fast = 87 msec, Ainter = 38%,
and inter = 2.5 sec), the rapid recovery phase has been
greatly reduced in amplitude and accelerated. Figure
6D shows the same curves on a semilog plot to
emphasize the similar time courses of the intermediate recovery phase.
We conclude that this effect of EGTA on PPD was presynaptic in origin
because EGTA-AM would not alter EGTA levels in the postsynaptic cell,
which are set by the contents of the recording pipette. Furthermore, it
is highly unlikely that the minor reduction in the probability of
release by EGTA-AM could account for the changes in PPD time course;
EGTA-AM reduced the EPSC amplitude by just 11% (n = 5)
and reduced the magnitude of PPD at short interpulse intervals from 81 to 78% in 4 Cae. Hence, these experiments establish that
rapid recovery from depression depends on presynaptic residual calcium,
independent of the initial release probability.
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Figure 6.
The effect of intracellular EGTA on the rapid
recovery phase of depression. A, PPD recovery kinetics
in 4 Cae before (filled circles) and
after (open circles) exposure to 100 µM
EGTA-AM. Insets, Synaptic currents for brief interpulse
intervals before (top) and after (bottom)
loading with EGTA-AM. B, Left, Effects of
100 µM EGTA-AM on the control EPSC (filled
circles), the depressed EPSC (open circles), and
%PPD (bottom). Top right, Averages of 10 traces before (thin lines) and after
(thick lines) exposure to EGTA-AM. Interpulse interval
was 500 msec. Bottom right, Same traces
scaled to peak of first EPSC. C, Average time course of
PPD in 4 Cae before (closed circles) and
after (open circles; 12 experiments; mean ± SEM)
loading with EGTA-AM. Inset, Early interpulse intervals.
The PPD recovery curve after loading with EGTA-AM was fit to a double
exponential decay with amplitudes Afast = 17%, Ainter = 65%, fast = 17 msec, and inter = 2.9 sec. D, Semilog
plot of the data in C.
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Dynamics of use-dependent recovery during trains
The dynamics of recovery from depression and the predictions of
the calcium-dependent model were further explored using brief stimulus
trains in a variety of external calcium conditions. Figure 7, A and B, shows
the accumulation of depression after one or four pulses during a brief
20 Hz train while decreasing Cae from 4 to 1 mM. We found that
EPSC2/EPSC1 was affected to a greater degree than was EPSC5/EPSC1 when
external calcium was perturbed. This difference can be more clearly
seen when depression is plotted against stimulus pulse number for both
high and low Cae (Fig. 7C). The two curves
deviate substantially early in the train, whereas they begin to
converge as depression approaches a steady-state value. Thus, transient
depression is more sensitive to initial release probability than is
steady-state depression, as has been described for other synapses
(Markram and Tsodyks, 1996; Abbott et al., 1997; O'Donovan and Rinzel,
1997; Tsodyks and Markram, 1997).
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Figure 7.
Dynamics of presynaptic depression during stimulus
trains. A, Top, Peak EPSC for the first
pulse in a train of five stimuli given at 20 Hz while changing from 4 to 1 Cae. Bottom, Depression of the second
pulse relative to the first (open circles) and of the
fifth pulse relative to the first (filled
circles) during solution exchange. B,
Top, EPSC trains in 4 Cae (thin
lines) and 1 Cae (thick lines).
Bottom, Same traces normalized to the
first EPSC. Traces are averages of 5-10 trials each.
C, Depression magnitude plotted versus stimulus pulse
number for 4 Cae (open circles) and 1 Cae (closed circles). Dashed
lines are predictions from the recovery model (see
Results).
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Under conditions of increased calcium influx and high stimulus
frequencies, the calcium-dependent component of recovery was prominent,
as demonstrated in Figure 8. By comparing
the attenuation in EPSC amplitude during a brief 10 Hz train, we
observed a more pronounced depression in the presence of EGTA-AM,
consistent with the hypothesis that residual calcium participated in
the recovery from depression. The relationship between depression and
stimulus pulse number (Fig. 8B,D)
demonstrates that EGTA affects steady-state depression, in contrast to
manipulations that affected the probability of release (see above). In
some cases, we observed a partial recovery during the stimulus train
(Fig. 8A), but the magnitude of this effect was
highly variable from synapse to synapse.
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Figure 8.
Effects of perturbing presynaptic calcium dynamics
on depression during stimulus trains. A, EPSC train at
20 Hz in 4 Cae (thin lines) and with EGTA-AM
(thick lines). Traces are averages of
five trials and normalized to the peak of the first EPSC. The
arrow indicates depression in 4 Cae.
B, Depression magnitude plotted versus stimulus pulse
number for 20 Hz train in 4 Cae (open
circles) and 4 Cae and EGTA (filled
circles). Dashed lines are predictions from the
recovery model (see Results). C, Same as
A with a 10 Hz train. Note the scale bar change.
D, Same as B with a 10 Hz
train.
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Simple model for calcium-driven recovery from
presynaptic depression
Based on the experiments described in Figures 2-8, we propose a
simple model to account for depression at the climbing fiber synapse.
According to this model, a synaptic connection consists of
No independent release sites, each with
probability Pr of releasing neurotransmitter when an action potential arrives at the presynaptic terminal. Facilitation was not included because paired-pulse
enhancement of transmitter release was not observed under conditions of
low Pr (which would unmask facilitation by
reducing depression and enhancing facilitation). The distinction
between the availability of the site and the probability that
exocytosis occurs can be clarified by separating
Pr into two components; (1)
po is defined as the probability that a
site releases its transmitter given that it is release ready, and (2)
pn is the probability that a release site
is available to undergo vesicle fusion (Zucker, 1973; Korn and Faber,
1987). Thus, Pr = po·pn, and
quantal content m = No · Pr = No·pn·po.
If we let the number of available release sites N = No·pn, then
m = N·po. The
mean synaptic strength is given by EPSC1 = N·po·q, where
q is the magnitude of the average quantal response. If the
sites that undergo exocytosis require a nonzero recovery time, then
immediately after the first stimulus there are N·(1 po) sites capable of releasing
neurotransmitter. A second stimulus given just after the first will
elicit a response of size EPSC2 = N·(1 po)·po·q,
provided po at each site is unchanged and
assuming that pn = 0 at all
N·po sites that underwent
exocytosis. This part of the model is identical to a number of other
depletion schemes that have been proposed to account for the magnitude
of depression (Liley and North, 1952; Takeuchi, 1958; Elmqvist and Quastel, 1965; Betz, 1970). We separated release probability into the
two components defined above to clarify the origins of depression in
our model. Because both po and
pn are stochastic in nature, experimental
procedures based on trial-to-trial variation in quantal content, such
as quantal and nonstationary noise analysis, will provide estimates of
Pr but will not distinguish between
pn and po
(Kuno, 1964; Zucker, 1973; Bennett and Florin, 1974; Bennett et al.,
1976; Bennett and Fisher, 1977; Korn et al., 1982; Korn and Faber,
1987). By considering depletion as solely a reduction in
pn, depression could be interpreted as
either a reduction in the number of available release sites
(N = No·pn) or
as a reduction in release probability (Pr = po·pn).
Many models of recovery from depression use a first-order process, as
in Scheme I, that predicts an exponential (constant-rate) recovery,
PPD(t) = poe t/:
The time constant = (ko)1 is typically given a
value of several seconds, reflecting the time course of the return of
release sites to a release-ready state (Liley and North, 1952; Betz,
1970; Magleby, 1987). Scheme I fails to account for the properties of climbing fiber depression that we observe, such as multiexponential recovery and dependence on residual calcium.
By assuming that presynaptic residual calcium binds to a site on the
release apparatus causing an acceleration in the rate of recovery, we
were able to account for the properties of recovery from depression. We
propose that recovery from depression proceeds according to Scheme II.
On average, N·po release sites are
activated (and transiently refractory) and therefore must be removed
from the available pool. The refractory sites (R)
then recover through a calcium-bound intermediate state
(T) as represented below:
(See Materials and Methods for a mathematical treatment of Scheme
II). If the site is in the release-ready state, calcium influx causes
release of neurotransmitter with probability
po and drives the release site into a
refractory state in which release is not possible (i.e.,
pn = 0). The site then slowly returns to the release-ready state, and the rate of recovery is enhanced by
elevated residual calcium. Figure
9A shows the expected recovery from depression after an action potential. The rapid decay
(decay = 100 msec) of presynaptic residual calcium gives
rise to the fast component of recovery. After calcium has returned to
resting levels, recovery continues on a slow time scale
(recov = 3 sec). Figure 9B shows the model
predictions superimposed on the PPD data under four different
experimental conditions. On an expanded time scale (inset),
one can see that the model also successfully accounts for the loss of a
fast component when residual calcium transients are reduced by lowering
Cae and when residual calcium decay is shortened with EGTA.
In the four experimental conditions shown,
po was calculated by extrapolating the PPD
curves shown in Figure 4 to t = 0. The values used
were po = 0.42, 0.63, 0.81, and
0.78 in 1 Cae, 2 Cae, 4 Cae, and EGTA, respectively. The parameters KN, kmax,
and c were chosen to fit recovery in 2 Cae and then held fixed for the other conditions. However,
c was shortened in the presence of EGTA.
Relative calcium influx in either 4 or 1 Cae was adjusted
to give the best fit.
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Figure 9.
Model for calcium-dependent recovery from
presynaptic depression. A, Simulation of presynaptic
residual calcium (top), fraction of release sites
available (middle), and fraction of depressed sites
(bottom) after an action potential. B,
Summary PPD data fit with the calcium-dependent recovery model; 1 Cae (filled diamonds), 2 Cae (open circles), 4 Cae
(filled circles), and 4 Cae and EGTA
(open diamonds). Inset, Same data on an expanded time scale. Data points are mean ± SEM. Model parameters for fits are ko = 0.314 sec-1; kmax = 8 sec-1; KN = 1.05;
c = 120 msec (20 msec in EGTA); and
po = 0.38, 0.63, 0.81, and 0.78 in 1 Cae, 2 Cae, 4 Cae, and EGTA, respectively. Ca influx increased by
a factor of 2.5 going from 2 to 4 Cae and decreased by a
factor of 3.3 going from 2 to 1 Cae. C, Same
PPD data on an expanded time scale to compare rapid decay
components.
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The predictions of Scheme II were investigated during trains of action
potentials to explore further the contribution of residual calcium to
steady-state depression and recovery. The effect of changing from 4 to
1 Cae on synaptic depression produced by brief stimulus
trains is well described by Scheme II (Fig. 7C, dashed lines), as is the effect of speeding the decay of presynaptic residual calcium with EGTA-AM (Fig.
8B,D, dashed
lines). This model also captured many features of
frequency-dependent depression during brief stimulus over a large range
of stimulus frequencies, as shown in Figure
10. As the probability of release and
calcium influx were increased by elevating Cae,
steady-state depression increased more steeply with frequency. Scheme
II accounted for most of the depression using parameters derived from
PPD curves under similar experimental conditions. However, at high
frequencies in 4 Cae, EPSC amplitudes continued to
decrease below the predicted values, suggesting that some other process
may contribute to depression under these conditions.
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Figure 10.
Experimental summary of depression during
stimulus trains under various external calcium conditions.
A, Relative depression in 1 Cae during
trains of stimuli given at various frequencies for the second through
seventh pulse in a train. Data are mean ± SEM; 1 Hz
(filled circles; n = 6), 5 Hz
(open circles; n = 6), 10 Hz
(filled diamonds; n = 12), 20 Hz (open diamonds; n = 11), and 50 Hz (filled triangles; n = 7).
B, Relative depression in 2 Cae.
C, Relative depression in 4 Cae.
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Behavior of depression during more realistic spike trains
We then considered how presynaptic depression would contribute to
synaptic strength for realistic activity patterns. Inferior olivary
neurons normally fire at 1-4 Hz in vivo (Armstrong and Rawson, 1979), and climbing fibers are unlikely to be significantly depressed for such conditions. However, many other types of neurons fire irregularly at rates between 0.1 and 100 Hz (Kuffler et al., 1957;
Perkel et al., 1967; Softky and Koch, 1993). We used the climbing fiber
synapse as a model to investigate the dynamics of presynaptic
depression and the performance of our model during conditions of
activation that would be experienced by other types of synapses.
Although these experiments were conducted at 24°C and do not
quantitatively reflect the dynamics of depression that would be
experienced in vivo, they are instructive with respect to
the model and the qualitative dependence of synaptic strength on the
initial probability of release and recovery kinetics. Climbing fibers
were stimulated with Poisson spike trains at average rates between 1 and 20 Hz. Figure 11 illustrates
climbing fiber depression during a Poisson train for three different
experimental conditions. With low external calcium, little steady-state
depression was observed at 10 Hz (Fig. 11A).
Increasing external calcium enhanced synaptic depression as expected
from the PPD and regular stimulus train experiments (Fig.
11B). When calcium was increased to 4 mM, pulse-to-pulse fluctuations in EPSC amplitude were prominent at 5 Hz
(Fig. 11C). This striking variability resulted from the
interplay between release probability and recovery kinetics. As
po was increased with higher external
calcium, depression at high frequencies was augmented. In contrast,
recovery from depression was accelerated in high calcium so there was
relatively less depression at lower frequencies. The data shown in
Figure 11 are examples from single climbing fibers, and we observed a
large amount of variability in certain parameters from fiber to fiber.
For instance, po ranged between 0.45 and
0.8 in 2 Cae (with a mean of 0.63), whereas other parameters such as KN and
c varied to a lesser extent. Synapse-to-synapse variation is not surprising in young rats because climbing fiber synapses undergo elimination around this time (O'Leary et al., 1971; Crepel et al., 1976), and variations in presynaptic parameters may reflect these changes.
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Figure 11.
Examples of depression during Poisson train
stimuli under various external calcium conditions. A,
Climbing fiber stimulation in 1 Cae using a random spike
train with average rate of 10 Hz. Open circles
are predictions of the recovery model (Scheme II, see Materials and
Methods). Relative errors between the simulation and the data are shown
above. Model parameters were
ko = 0.31 sec-1,
kmax = 7.5 sec-1,
KN = 0.9, = 100 msec,
po = 0.16, kslow = 0.1, and  = 0.08. Calcium influx
was reduced by 3.7-fold relative to 2 Cae.
B, Same as A in 2 Cae. Model
parameters were ko = 0.31 sec-1, kmax = 7.5 sec-1, KN = 0.8, = 100 msec, po = 0.5, kslow = 0.1, and = 0.06. C, Same as A in 4 Cae. Model
parameters were ko = 0.31 sec-1, kmax = 8.5 sec-1, KN = 0.75, = 115 msec, po = 0.65, kslow = 0.1, and = 0.03. Calcium influx
was increased by 60% relative to 2 Cae. All data are
single trials.
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Although Scheme II could describe the first few EPSCs in a Poisson
train successfully under all conditions tested, a slower recovery
component was needed to simulate depression accurately over tens to
hundreds of stimuli. During prolonged stimulation of the climbing
fiber, we observed a gradual increase in depression that recovered in
<2 min. This long-lived depression has been described previously in
other climbing fiber preparations (Rawson and Tilokskulchai, 1981). For
simulations of Poisson stimulus trains (Fig. 11), it was necessary to
incorporate this slower component of depression. The simplest
modification of this scheme is the addition of a separate recovery
pathway with slower kinetics:
We did not explore the recovery kinetics of the slow pathway
(S) in this study, but trains repeated every 2 min
showed no signs of rundown. The recovery rate
kslow was therefore taken to be in the range of
10-60 sec. The probability that a refractory release site enters state
S is defined as . This parameter was adjusted to give the
best fit between simulations and Poisson train data. Typical values
were ~0.05, consistent with the observation that a slow depression
was prevalent after ~20 stimuli. Numerical simulations of Scheme II
were used in modeling responses to the Poisson stimuli shown in Figure
11 using a first-order Euler integration routine with time steps of 200 µsec. Simulations of Poisson stimuli captured most of the short-term
changes in synaptic efficacy over the time scale ranging from
milliseconds to seconds. Interestingly, the magnitude of the additional
slow component was inversely correlated with external calcium; lower
calcium accentuated the slow component analogous to its effect on the
intermediate recovery component.
Figure 12A shows the
behavior of the calcium-dependent recovery model during the first 10 pulses of a 10 Hz Poisson stimulus train. The filled
circles represent the predicted EPSC
magnitudes if no calcium-dependent recovery were to
occur but all other parameters were identical. A substantial deviation
between the model and the data can be seen within two or three stimuli,
indicating that calcium dependence was critical in maintaining synaptic
strength during the stimulation. In Figure 12B, we
plotted the steady-state attenuation of the climbing fiber EPSC during
a train of stimuli versus the frequency of stimulation; it is clear
that acceleration of recovery from depression substantially increases
synaptic strength relative to the calcium-independent recovery model.
The attenuation of climbing fiber EPSCs after seven pulses (see Fig.
10) is plotted against stimulus frequency on the same graph for
comparison (open circles). Even for the highly
simplified solution to Scheme II, calcium-dependent recovery provides a
better prediction of steady-state behavior than does a constant-rate
recovery model (Scheme I).
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Abstract
Introduction
