 |
 Previous Article | Next Article 
The Journal of Neuroscience, January 15, 2000, 20(2):568-578
Synaptic Depression and the Kinetics of Exocytosis in Retinal
Bipolar Cells
Juan
Burrone and
Leon
Lagnado
Medical Research Council Laboratory of Molecular Biology, Cambridge
CB2 2QH, United Kingdom
 |
ABSTRACT |
The capacitance technique was used to investigate exocytosis at the
ribbon synapse of depolarizing bipolar cells from the goldfish retina.
When the Ca2+ current was activated strongly, the
rapidly releasable pool of vesicles (RRP) was released with a single
rate-constant of ~300-500 sec 1. However, when
the Ca2+ current was activated weakly by
depolarization in the physiological range ( 45 to 25 mV), exocytosis
from the RRP occurred in two phases. After the release of 20% or more
of the RRP, the rate-constant of exocytosis fell by a factor of 4-10.
Thus, synaptic depression was caused by a reduced sensitivity to
Ca2+ influx, as well as simple depletion of the RRP.
In the resting state, the rate of exocytosis varied with the amplitude
of the Ca2+ current raised to the power of 2. In the
depressed state, the sensitivity to Ca2+ influx was
reduced approximately fourfold. The initial phase of exocytosis
accelerated e-fold for every 2.1 mV depolarization over the
physiological range and averaged 120 sec1 at 25 mV.
The synapse of depolarizing bipolar cells therefore responds to a step
depolarization in a manner similar to a high-pass filter. This
transformation appears to be determined by the presence of rapidly
releasable vesicles with differing sensitivities to
Ca2+ influx. This might occur if vesicles were
docked to the plasma membrane at different distances from
Ca2+ channels. These results suggest that the ribbon
synapse of depolarizing bipolar cells may be a site of adaptation in
the retina.
Key words:
synapse; vesicle; exocytosis; depression; retina; depolarizing bipolar cell
| |
INTRODUCTION |
Many synapses exhibit
activity-dependent changes in the efficiency of synaptic transmission.
A particularly rapid form of synaptic plasticity is depression, which
is at least partly caused by a decrease in the number of
vesicles that can be rapidly released when
Ca2+ channels open (Liley and North, 1953 ;
del Castillo and Katz, 1954; Betz, 1970; Dittman and Regehr, 1998).
This "rapidly releasable pool" (RRP) is thought to correspond to
the vesicles that are docked to the plasma membrane at the active zone
(Burns and Augustine, 1995). A few studies have suggested a second
mechanism of synaptic depression: a decrease in the efficiency of
exocytosis in response to Ca2+ influx
(Furukawa and Matsura, 1978; Korn et al., 1984; Hsu et al., 1996;
Bellingham and Walmsley, 1999; Wu and Borst, 1999).
We have investigated exocytosis at the ribbon synapse of depolarizing
bipolar cells from the goldfish retina. We find that synaptic
depression is caused by a decrease in the efficiency of exocytosis, as
well as depletion of vesicles. These two causes of synaptic depression
were separated by measuring both the amount of exocytosis and the size
of the RRP. The RRP was defined on the basis of capacitance
measurements (Mennerick and Matthews, 1996; Sakaba et al., 1997; Gomis
et al., 1999; Neves and Lagnado, 1999) and fluorescence measurements
(Neves and Lagnado, 1999), which demonstrate that depolarizing bipolar
cells contain a finite pool of vesicles that can be completely released
within 10-20 msec when the Ca2+ current
is activated strongly. An alternative term that has been used to
describe this group of vesicles is the "ultrafast pool" (Mennerick
and Matthews, 1996). Drawing on electron microscopy performed by von
Gersdorff et al. (1996), it has been suggested that these
vesicles are docked to the plasma membrane under the ribbons (Mennerick
and Matthews, 1996; Gomis et al., 1999). An important advantage of
depolarizing bipolar cells for the study of synaptic depression is that
changes in the size of the RRP that occur after stimulation can be
measured relatively directly (Gomis et al., 1999).
We paid particular attention to exocytosis from the RRP when the
Ca2+ current was activated weakly, because
this is the situation physiologically. The L-type
Ca2+ current that controls exocytosis at
the synapse of bipolar cells begins to activate at approximately 45
mV (Tachibana et al., 1993; Burrone and Lagnado, 1997), whereas the
depolarizing response to the strongest light does not exceed 30 to
25 mV (Werblin and Dowling, 1969; Ashmore and Falk, 1980a). The
voltage-dependence of exocytosis was steepest at membrane potentials
below 30 mV, when the initial rate of release accelerated e-fold for
every 2.1 mV depolarization. Surprisingly, exocytosis from the RRP
occurred in two phases at these physiological potentials. After the
rapid release of 20% or more of the RRP, the rate-constant of
exocytosis from the RRP fell by a factor of 4-10. The major cause of
depression at this ribbon synapse was therefore a decreased sensitivity
of the exocytic machinery to Ca2+ influx.
The time course of exocytosis at physiological potentials appeared to
be determined by the presence of rapidly releasable vesicles with
differing sensitivities to the activation of the Ca2+ current. This might arise if vesicles
comprising the RRP docked to the plasma membrane at different distances
from Ca2+ channels. The results suggest
that the active zone of depolarizing bipolar cells is a site of
adaptation in the retina.
| |
MATERIALS AND METHODS |
Isolated synaptic terminals of depolarizing bipolar cells from
the goldfish retina were obtained by enzymatic dissociation using
methods described by Burrone and Lagnado (1997). The standard Ringer's
solution contained (in mM): 120 NaCl, 2.5 CaCl2, 2.5 KCl, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.3.
Terminals were voltage-clamped using the perforated patch technique
(Neves and Lagnado, 1999). The solution in the patch pipette contained
(in mM): 110 Cs gluconate, 4 MgCl2, 3 Na2ATP, 1 Na2GTP, 10 TEACl,
0.4 BAPTA, and 20 HEPES, with 250 µg/ml nystatin (260 mOsm), pH 7.2. Capacitance measurements were not made until the access resistance had
fallen below 30 M . The current at the holding potential of 70 mV
was usually 15 to 40 pA. A description of our implementation of the
piecewise linear technique (Neher and Marty, 1982) is provided by Neves
and Lagnado (1999). Briefly, a sinusoidal command voltage (50 mV
peak-to-peak, 2 kHz) was superimposed onto a holding potential of 70
mV. The output of the patch-clamp amplifier was analyzed by a hardware
lock-in amplifier. The phase of the two orthogonal outputs was set
using the "capacitance dither" method, allowing independent
measurements of changes in membrane capacitance and conductance
(Gillis, 1995). The capacitance signal was calibrated by dithering the
whole-cell capacitance compensation by 100 fF at the beginning of each
recording episode. The change in capacitance elicited by the stimulus
( Cm) was measured by subtracting
the capacitance signal before the stimulus from that measured after.
The "before" measurement was averaged over a period of 20-50 msec,
ending 20 msec before depolarization. The "after" measurement was
averaged over a period of 20-50 msec beginning 20 msec after
repolarization. Neves and Lagnado (1999) found that, after the
introduction of a large Ca2+ load, there
was a Ca2+-activated tail current, but
this conductance change was not large enough to "reflect" onto the
capacitance measurement. In this study, the
Ca2+ loads and associated conductance
changes were considerably smaller. Where capacitance measurements are
converted to vesicle numbers, it is assumed that the capacitance of a
single vesicle is 26 aF (von Gersdorff et al., 1996; Neves and Lagnado,
1999).
To measure the current-voltage relationship for the L-type
Ca2+ current, voltage ramps were applied
(from 60 to +40 mV at 100 mV/sec1).
Series resistance was compensated 70-80% with a lag of 10 µsec, and
the records were leak subtracted.
Measurements are given as mean ± SEM.
| |
RESULTS |
Fast and slow phases of exocytosis at physiological voltages
We have investigated exocytosis at the synaptic terminal of
depolarizing bipolar cells at membrane potentials at which the Ca2+ current is activated weakly. This is
the normal operating condition of this synapse because the L-type
Ca2+ current that controls exocytosis
begins to activate at approximately 45 mV, whereas the depolarizing
response to the strongest light does not exceed 30 to 25 mV
(Werblin and Dowling, 1969; Ashmore and Falk, 1980a). Figure
1, A and B, shows
an experiment designed to measure the time course of exocytosis at a
membrane potential of 33 mV. The left side of Figure
1A shows capacitance responses to two depolarizing
stimuli delivered 200 msec apart from a holding potential of 70 mV.
The first stimulus, a 20 msec depolarization to 33 mV, activated the
Ca2+ current weakly and elicited a
response of 16 fF. The second stimulus, a 20 msec depolarization to
10 mV, activated the Ca2+ current
strongly and elicited a response of 52 fF. This strong stimulus was
termed the "emptying stimulus," because it was sufficient to empty
the remainder of the RRP (Mennerick and Matthews, 1996; Gomis et al.,
1999). The right side of Figure 1A shows a
repetition of this protocol in which the duration of the depolarization
to 33 mV was increased to 500 msec. The time course of the
capacitance increase at 33 mV is shown by the filled
circles in Figure 1B in which two phases of
exocytosis are evident. An important aspect of this experiment is that
the first phase of release only involved ~20% of the RRP.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 1.
Exocytosis evoked by Ca2+
currents. A, Capacitance responses evoked by pairs of
depolarizing stimuli. The first stimulus, to 33 mV, had a duration of
20 msec in the left trace and 500 msec in the
right trace. In each case, the second stimulus was a 20 msec depolarization to 10 mV, delivered after a delay of 200 msec
(the emptying stimulus; see Results). The leak-subtracted
Ca2+ currents evoked by each stimulus are shown
below on an expanded time scale. B, The filled
circles plot the size of the capacitance increase as a function
of the duration of the depolarization to 33 mV (same cell as in
A). The bold line through the
points is an empirical fit of a double-exponential
function, with 14 fF of the capacitance increase occurring with a
rate-constant of 77 sec1 and 70 fF with a
rate-constant of 2 sec1. The total size of the RRP
at the beginning of each stimulus episode, calculated from the summed
responses to the shorter stimuli, averaged 68 fF (heavy dashed
line). The light dashed line shows the time
course of exocytosis that would be predicted if the RRP were exocytosed
with a fixed rate-constant of 12 sec1. The
open circles plot the capacitance response elicited by
the emptying stimulus. The thin line is a mirror image
of the bold line. C, Capacitance
responses evoked by depolarizations to 28 mV lasting 5 msec
(left trace) and 500 msec (right trace).
In each case, an emptying stimulus was delivered 200 msec later.
D, The time course of exocytosis (filled
circles) and decline in the size of the RRP (open
circles) at a membrane potential of 28 mV (same cell as in
C). The size of the RRP at the beginning of each
stimulus episode averaged 76 fF (heavy dashed line),
which is equivalent to 2900 vesicles. The light dashed
line shows the time course of exocytosis that would be
predicted if the RRP were exocytosed with a fixed rate-constant of 27 sec1. The fit to the filled circles
(bold trace) is a double-exponential function with 24 fF
occurring with a rate-constant of 140 sec1 and 91 fF with a rate-constant of 1.4 sec1. The
thin line describing the decline in the size of the RRP
is a mirror image of the bold line.
|
|
Depletion of the RRP at a membrane potential of 33 mV is shown by the
open circles in Figure 1B, which plot the
responses to the emptying stimuli. The decline in the size of the RRP
mirrored the time course of exocytosis, at least over the first 300 msec, indicating that there was negligible refilling of the RRP on this time scale. A second example of this behavior is shown in Figure 1,
C and D, which shows the time course of the
capacitance increase at a potential of 28 mV. In this terminal, the
fast phase of exocytosis involved the release of ~32% of the RRP.
Again, the decline in the measured size of the RRP mirrored the time
course of exocytosis over the first 300 msec. However, some refilling of the RRP probably occurred at later times. For instance, Figure 1B indicates that the size of the RRP actually
increased between 300 and 500 msec, although exocytosis continued.
The rate of exocytosis is the time derivative of the capacitance
change, so the results in Figure 1, B and D,
indicate that a step depolarization caused a rapid initial burst of
exocytosis, followed by a slower phase of release. The synapse of
depolarizing bipolar cells therefore has some characteristics of a
high-pass filter, signaling most strongly the onset of the stimulus.
This initial transient involved a fraction of vesicles in the RRP, and
it was followed by exocytosis that was maintained for at least 0.5 sec.
The rate-constant of exocytosis from the RRP
We sought to quantify our measurements in terms that could be
related to the properties of exocytosis from the RRP. When the Ca2+ current in bipolar cells is activated
strongly, the whole of the RRP is released with a time course that can
be described as a single exponential (Mennerick and Matthews, 1996;
Gomis et al., 1999). That is, the increase in membrane capacitance
Cm is:
|
(1)
|
An example of the exponential depletion of the RRP is shown by
filled circles in Figure 6D. Such a time
course indicates a fixed rate-constant of release, k, so
that at any time the rate of increase in membrane capacitance
Cm is related to the remaining size of
the RRP (in femtofarads) by:
|
(2)
|
In the example shown in Figure 1B, the rate of
capacitance increase at 33 mV averaged 750 fF/sec over the first 20 msec of stimulation. Over this period, the size of the RRP averaged ~61 fF (measured using the emptying stimuli). So, applying
Equation 2, the rate-constant of release at the start of the
stimulus was 12 sec1. The dashed
curve in Figure 1B shows the time course of
exocytosis that would be expected if the whole of the RRP were released
with this rate-constant fixed, calculated using Equation 1. The actual measurements show that the rate-constant of exocytosis at 33 mV was
not fixed but fell significantly. Applying Equation 2 over the period
from 50 to 300 msec, the rate-constant of exocytosis averaged 3.4 sec1. In the example shown in Figure
1D, the rate-constant of exocytosis declined from 27 to 2.5 sec1 at a membrane potential of
28 mV. Similar behavior was observed in all 15 cells in which the
time course of exocytosis was measured at potentials below 25 mV.
The initial rate-constant of exocytosis from the RRP was ~40 times
slower at 33 mV (Fig. 1B) than at 10 mV
(Mennerick and Matthews 1996; Sakaba et al., 1997; Gomis et al., 1999;
Neves and Lagnado, 1999). The slowing of exocytosis was correlated with the smaller steady-state Ca2+ current at
more hyperpolarized potentials (Fig. 7), but it could not be accounted
for by the slower activation of the Ca2+
current. At 33 mV, the initial rate-constant of exocytosis was 12 sec1, whereas the
Ca2+ current was fully activated in
~3-4 msec. At 25 mV, the initial rate-constant of exocytosis was
120 sec1 (see Fig. 7), but the
Ca2+ current was almost fully activated in
2 msec (Fig. 2). At 10 mV, however, the
initial rate-constant of exocytosis can become limited by the
activation kinetics of the Ca2+ current
(Mennerick and Matthews, 1996).
View larger version (15K):
[in this window]
[in a new window]
|
Figure 2.
Measuring changes in the efficiency of exocytosis.
Increases in membrane capacitance in response to a pair of stimuli that
activated the Ca2+ current submaximally. The stimuli
were 2 msec depolarizations to 25 mV, with an interval of 100 msec.
The Ca2+ currents are shown superimposed. In each
case, Ca2+ influx carried 0.25 pC of charge. The
first response, C1, was 37 fF, and
the second response, C2, was 4 fF.
The remainder of the rapidly releasable pool of vesicles was exhausted
with a third stimulus, a 20 msec depolarization to 10 mV. This
emptying stimulus passed 4.14 pC of charge and elicited a capacitance
increase, C3, of 32 fF. After
accounting for the reduced size of the RRP, the second response was
21% of the first, i.e., the efficiency of exocytosis was reduced by
79% (see Results).
|
|
When the Ca2+ current was activated
submaximally, a double-exponential function provided an empirical
description of the capacitance increase measured after various delays,
as shown by the bold lines in Figure 1B,D.
Such a description might be expected if the RRP, which has been defined
by strongly activating the Ca2+ current,
was actually composed of two populations of vesicles, released with
different rate-constants when the Ca2+
current was activated weakly. However, a second possibility is that all
vesicles within the RRP were equivalent at the beginning of the
stimulus, but the properties of the release process "adapted" with
time, causing a fall in the rate-constant of release (Hsu et al.,
1996). Our measurements of rate-constants have been made in terms of
the whole of the RRP, which can be precisely defined and directly
measured using our stimulation protocol, rather than a putative
subpopulation of this pool. When the whole of the RRP appeared to be
released at a fixed rate (see Fig. 6D), the
rate-constant was obtained by fitting Equation 1. When two phases of
release were apparent (as in Fig.
1B,D), we measured rate-constants
over defined periods by applying Equation 2. The rate-constant at the start of the stimulus, calculated as in the examples above, was termed
the "initial rate-constant." Note that the protocol we used in
Figure 1 allowed independent measurement of the amount of exocytosis
and the size of the RRP at different delays after the beginning of the stimulus.
Changes in the efficiency of exocytosis
A decrease in the rate of exocytosis in response to a fixed
stimulus is a common form of synaptic plasticity termed "synaptic depression," and at some synapses it is thought to be attributable to
a decrease in the number of vesicles available for release (Zucker,
1989; Dittman and Regehr, 1998; Wang and Kaczmarek, 1998). The results
in Figure 1 indicate that synaptic depression in depolarizing bipolar
cells also has a second cause: a decrease in the probability with which
vesicles are released from the RRP. To investigate this contribution to
synaptic depression, we used a stimulus protocol designed to measure
both paired-pulse depression and depletion of the RRP. In the example
shown in Figure 2, the two submaximal stimuli (2 msec depolarizations
to 25 mV) were delivered 100 msec apart. The first response,
C1, was 37 fF, and the second response, C2, was 4 fF. The
Ca2+ currents elicited by the stimuli were
identical (as shown by the overlay), so the reduction in the second
response could not be explained by inactivation of the
Ca2+ current. To estimate how much
depression was caused by depletion of the vesicles available for rapid
release, an emptying stimulus was applied 200 msec after the second
pulse. The remainder of the RRP elicited a capacitance increase of 32 fF, indicating that profound synaptic depression had occurred, even
though there were a large number of vesicles still available for rapid release.
The total size of the RRP at the start of the experiment in Figure 2
was calculated as the sum of the three responses,
C1 + C2 + C3. The percentage of the RRP
released by the first stimulus (R1)
was then estimated as:
which in this case was 51%. The size of the RRP when the second
stimulus was applied was calculated as
C2 + C3, and the percentage of the RRP
released by the second stimulus (R2)
as:
which in this case was 11%. The efficiency E of the
second stimulus compared with the first was calculated as:
and the percentage decrease in efficiency as 100 E. Thus, after taking into account changes in the size of
the RRP, the second response was 21% of the first, and the efficiency
of exocytosis was reduced by 79%. This method seeks to quantify
synaptic depression over and above changes in the size of the RRP, but
it assumes that there is a negligible supply of new vesicles to the RRP
over the period that the measurements are made. The longest interval between the end of the first stimulus and beginning of the third was
350 msec, and the results in Figure 1 indicate that there is little
refilling over this time scale because the decline in the size of the
RRP mirrored the amount of exocytosis (see also Gomis et al.,
1999).
The decreased efficiency of exocytosis measured using the double-pulse
protocol varied with the amount of the RRP released by the first pulse.
Figure 3A shows examples of
responses to 2 msec depolarizations to 28 mV (left trace)
and 15 mV (right trace). The capacitance response to the
first pulse to 15 mV was 27 fF, the response to the second was 7 fF,
and the efficiency was reduced by 64%. The first pulse to 28 mV, on
the other hand, elicited a much smaller response of 7 fF, and the
second pulse elicited a similar response of 6 fF, indicating that
the efficiency of exocytosis was unchanged. Results from this type
of experiment performed on 16 terminals are collected together in
Figure 3B, which plots the relationship between the decrease
in the efficiency of exocytosis and the percentage of the RRP released
by the first pulse. The efficiency was reduced by 70-80% after the
release of ~40% or more of the RRP. These results therefore predict
that the rate-constant of exocytosis would, on average, decrease by a
factor of 4-5, which is in agreement with measurements of the kinetics
of exocytosis made in Figure 1.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 3.
The decrease in the efficiency of exocytosis was
dependent on the amount of the RRP released. A,
Capacitance responses to paired-pulse stimuli lasting 2 msec. The
left trace shows responses to depolarizations to 28
mV, and the right trace shows responses to
depolarizations to 15 mV from the same terminal. The interval between
pulses was 100 msec, and each pair was followed by an emptying stimulus
delivered after 200 msec. B, The relationship
between the decrease in the efficiency of exocytosis and depletion of
the rapidly releasable pool of vesicles. Results collected from 16 terminals. The paired stimuli were 2-20 msec in duration, and
interpulse intervals were 50-200 msec. The decreased efficiency of
exocytosis reached a maximal value of ~76% (dashed
line) when >40% of the RRP was released.
|
|
Sensitivity of the exocytic machinery to calcium influx
Decreases in the efficiency of exocytosis could be characterized
as a decrease in the sensitivity of the exocytic apparatus to calcium
influx. Figure 4 shows the relationship
between the percentage of the RRP released by a 20 msec depolarization
and QCa, the integral of the
Ca2+ current flowing during the stimulus.
The filled circles show this relationship measured from rest
(that is, from the first response in a paired-pulse protocol of the
type shown in Fig. 2). The results could be described by a Hill
function of the form, R = Rmax QCah/(QCah + Q1/2h),
where R is the percentage of the RRP released,
Rmax is the maximum released,
h is the Hill coefficient,
QCa is the charged carried by
Ca2+ influx, and
Q1/2 is the charge carried for
half-maximal release. The curve fitted to the points in Figure 4 has
h = 2, indicating that
Ca2+ acted cooperatively to stimulate
exocytosis, as has been found at other synapses (Dodge and Rahamimoff,
1967; Augustine and Charlton, 1986; Mintz et al., 1995). Release was
half-maximal at Q1/2 = 1.3 pC. The
open circles in Figure 4 show measurements made when the
synapse was depressed and the efficiency of exocytosis reduced to its
minimum. These measurements were made from the second response in a
paired-pulse protocol in which the first pulse released at least 30%
of the RRP. Q1/2 increased to ~2.7
pC, but there was no obvious change in the degree of cooperativity, as
shown by the limiting slopes in a double-logarithmic plot (Fig.
4B). So, for small amounts of calcium influx, the
rate of release in the depressed state was reduced by a factor of
approximately (2.7/1.3)2, or
~4.3-fold.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 4.
Synaptic depression was associated with a decrease
in the Ca2+ sensitivity of exocytosis.
A, The percentage of the RRP released by a 20 msec
stimulus as a function of the integral of the Ca2+
current. The measurements were collected from the type of experiments
shown in Figure 3B. Each point is the
average of four measurements. The filled circles are
measurements made from rest. The open circles are
measurements made within 200 msec of a conditioning stimulus, which
released >30% of the RRP. The points were fit with a
Hill function R = RmaxQCah/(Q1/2h + QCah), with h = 2, Q1/2 = 1.3 pC for terminals at rest
(bold trace), and 2.7 pC for terminals in the depressed
state (thin line). Rmax was
90% because ~10% of the RRP was refilled in the 100-200 msec
interval between the test stimulus and emptying stimulus.
B, The results from A are plotted on a
double-logarithmic scale. The limiting slope was similar at rest and in
the depressed state, indicating that there was no significant change in
the cooperativity with which Ca2+ activated
exocytosis.
|
|
The results presented above indicate that the RRP is not homogenous.
Although it is released with a single rate-constant when the
Ca2+ current is activated strongly, weak
activation of the Ca2+ current reveals the
presence of vesicles with differing sensitivities to
Ca2+ influx. One possibility is that
vesicles comprising the RRP experience different rises in
[Ca2+] because they are docked at
different distances from Ca2+ channels
(see Discussion).
Recovery from synaptic depression
It has usually been proposed that recovery from synaptic
depression reflects refilling of the RRP with new fusion-competent vesicles (Kusano and Landau, 1975; Dittman and Regehr, 1998; Stevens and Wesseling, 1998; Wang and Kaczmarek, 1998), but the interpretation will be more complicated if synaptic depression is also caused by an
activity-dependent decrease in the efficiency of exocytosis (Hsu et
al., 1996; Bellingham and Walmsley, 1999; Wu and Borst, 1999). Gomis et
al. (1999) measured refilling of the RRP in bipolar cells independently
of any possible changes in the efficiency of exocytosis by using
super-saturating stimuli that always caused complete depletion of the
RRP. They found that, after a brief stimulus, ~30% of the RRP was
refilled with a time constant of ~0.6 sec and the remainder with a
time constant of ~30 sec.
We sought to measure how the efficiency of exocytosis recovered after a
stimulus independently of refilling of the RRP. The protocol we used is
shown in Figure 5A. The
left side shows that 10% of the RRP was released by the
second of two 20 msec depolarizations to 26 mV, delivered with an
interval of 200 msec. The reduction in the efficiency of exocytosis
after this short interval was 82%, quantified using the method
described above (Fig. 2). When the interval to the second stimulus was
increased to 5 sec, it released 38% of the RRP, reflecting an
increased efficiency (Fig. 5A, right). To
quantify the change in the efficiency of exocytosis after this long
delay, it was necessary to assume that the first stimulus of the pair
released a fixed fraction of the RRP, and this fraction was measured
from a trial in which there was a short delay between this first
stimulus and the emptying stimulus (Fig. 5A,
left). Collected results are shown in Figure 5B,
which plots the decrease in the efficiency of exocytosis for the second
stimulus of a pair as a function of the interval between them. Recovery occurred with a time constant of ~6 sec (Fig. 5B). These
measurements can be compared with those made by Wu and Borst (1999),
who found that depression at a giant calyx-type synapse in the auditory brainstem was associated with a reduction in the probability of release
from the RRP. Recovery of release probability occurred with a half-time
of ~20 sec.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 5.
Recovery of synaptic efficiency. A,
Capacitance responses to a pair of weak stimuli (20 msec
depolarizations to 26 mV) with an interval between pulses of 200 msec
(left trace) and 5 sec (right trace).
Each pair was followed by an emptying stimulus. B, The
decrease in efficiency at various intervals after a conditioning
stimulus. The submaximal stimulus pairs were all 20 msec in duration,
and the membrane potential was selected to cause the first stimulus of
the pair to release 40-60% of the RRP. Points are the
average of three to seven measurements. The curve
describing the recovery of synaptic efficiency is an exponential with a
time constant of 5.8 sec.
|
|
The voltage-dependence of rapid exocytosis
The initial phase of exocytosis elicited by submaximal activation
of the Ca2+ current was strongly dependent
on the potential to which the membrane was stepped. Figure
6, A and B,
compares the time course of the capacitance increase at 30 and 26
mV in the same cell. The initial rate of exocytosis (in femtofarads per
second) was measured by drawing a straight line through the
points over short intervals (Fig. 6B). This quantity
was divided by the resting size of the RRP (in femtofarads), measured
from the response to the emptying stimulus delivered from rest, to
obtain the initial rate-constant of release (Eq. 2). At 30 mV, the
rate-constant was ~20 sec1, whereas at
26 mV it was approximately six times faster.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 6.
Measuring the initial rate of
exocytosis. A, Capacitance responses evoked by
depolarizations to 30 mV, lasting 10 msec (left), 20 msec (middle), and 100 msec (right). Each
stimulus was followed by an emptying stimulus delivered with a delay of
100 msec. B, The relationship between the size of the
capacitance increase and the duration of the depolarization to 30 mV
(filled circles). The open circles
show this relationship at 26 mV for the same cell. The dashed
line shows the average size of the RRP at the beginning of
these trials. The initial rates of exocytosis were estimated by drawing
a straight line (by eye) over the initial phase of the response. At
30 mV, the rate-constant was ~20 sec1, whereas
at 26 mV, the rate-constant was ~120 sec1.
C, Capacitance responses evoked by depolarizations to
35 mV, lasting 5 msec (left), 20 msec
(middle), and 50 msec (right).
D, The time course of exocytosis for depolarizations to
10 mV (filled circles), 35 mV (open
circles), and 38 mV (filled squares).
The results are expressed as a percentage of the RRP measured from the
summed responses to the test stimulus and emptying stimulus. All
measurements from the same cell. The time course of exocytosis at 10
mV could be described as a single exponential. The rate-constants were
385, 37, and 6.6 sec1 for depolarizations to 10,
35 and 38 mV, respectively.
|
|
In the experiments shown in Figure 1, the size of the RRP was
relatively constant from trial to trial, as indicated by the fixed
amplitude of the summed responses when the test stimulus and emptying
stimulus were delivered within 300 msec of each other. More usually,
the size of the RRP varied, even with a recovery time of 2 min or more
between trials, and there was often a tendency for the size of the RRP
to decline during the course of an experiment. To measure the
rate-constant of exocytosis when this occurred, we normalized responses
to a test stimulus to the size of the RRP measured within the same
trial. An example of this approach is shown in Figure 6, C
and D. The three trials in Figure 6C use the
double-pulse protocol to measure the time course of exocytosis at 35
mV. The open circles in Figure 6D plot the
time course of exocytosis at 35 mV, with each response expressed as a
percentage of the RRP measured from the summed responses to the test
stimulus and emptying stimulus. The initial rate-constant of exocytosis was 37 sec1 at 35 mV. Also shown in
Figure 6D are measurements made in the same cell at
potentials of 10 and 38 mV, when the initial rate-constants were
385 and 6.6 sec1.
The small error arising from refilling of the RRP in the interval
between the test stimulus and emptying stimulus can be seen in Figure
6D in which the filled circles plot
results from trials in which the test depolarizations were to 10 mV.
The amplitude of the test response saturated at ~92% of the summed
responses, indicating that ~8% of the RRP was refilled during the
100 msec delay between two emptying stimuli (see also Gomis et al.,
1999). Generally, the initial rate-constant of exocytosis was measured using stimuli releasing <50% of the RRP, so the errors arising from
the supply of new vesicles would be expected to be <5%.
The initial rate-constant of exocytosis as a function of membrane
potential is plotted linearly in Figure
7A and semilogarithmically in
Figure 7B (results collected from 16 cells). Between 37
and 30 mV, the rate-constant of exocytosis changed e-fold for every 2.1 mV change in voltage. We were not able to measure capacitance responses to brief stimuli at more hyperpolarized potentials, presumably because the rate of exocytosis was too low. At potentials above 30 mV, the rate of exocytosis was much less steeply
voltage-dependent, changing e-fold in ~10 mV. At 10 mV, the
rate-constant of exocytosis from the RRP was 400-500
sec1, which is similar to measurements
made by Mennerick and Matthews (1996), Sakaba et al. (1997), and Neves
and Lagnado (1999).
View larger version (16K):
[in this window]
[in a new window]
|
Figure 7.
The voltage-dependence of rapid exocytosis.
A, The initial rate-constant of exocytosis plotted as a
function of the membrane voltage (right axis). For
membrane potentials ranging from 37 to 33 mV, the initial rate of
exocytosis was calculated using a stimulus lasting 20 msec that
released <50% of the RRP. For membrane potentials from 30 to 25
mV, measurements were made using a stimulus lasting 2 msec that
released <50% of the RRP. For stronger depolarizations, the
rate-constant of exocytosis was calculated from single-exponential fits
to the time course of the capacitance increase, as in Figure
6D. Each point is the average of
measurements on four to eight terminals. The continuous
line shows the voltage-dependence of the
Ca2+ current, normalized to its maximum. The
measurement was made using a ramp change in membrane potential (see
Materials and Methods) and is an average from 10 detached terminals.
B, The results in A plotted
semilogarithmically. The rate-constant of exocytosis changed e-fold in
2.1 mV at membrane potentials below 30 mV (bold dotted
line), whereas the Ca2+ current changed
e-fold in 6.6 mV over the same range (bold line).
Between 30 and 10 mV, the rate-constant of exocytosis changed
e-fold in every 10 mV (thin dotted line).
|
|
Calcium acted cooperatively to trigger exocytosis
Changes in membrane potential modulate the exocytosis of synaptic
vesicles through the opening of voltage-dependent
Ca2+ channels (Katz and Miledi, 1967;
Llinas et al., 1981; Augustine et al., 1985). The
Ca2+ current and rate-constant of rapid
exocytosis in bipolar cell terminals are compared in Figure
7A. The continuous line (corresponding to the right
axis) is the voltage-dependence of the
Ca2+ current measured using ramped changes
in membrane potential (see Materials and Methods). When plotted
semilogarithmically (Fig. 7B), it can be seen that the
Ca2+ current was less strongly dependent
on voltage than the rate of exocytosis over the physiological range of
potentials. Whereas the rate-constant of exocytosis changed e-fold in
2.1 mV, the amplitude of the Ca2+ current
changed e-fold in 6.6 mV.
The steep voltage-dependence of exocytosis is probably related to the
supralinear relationship between the free
[Ca2+] and the rate of exocytosis
(Augustine and Charlton, 1986; Heidelberger et al., 1994). Assuming
that the free [Ca2+] at the
Ca2+ sensor is linearly related to the
amplitude of the Ca2+ current, the results
in Figure 6 suggest that Ca2+ activates
exocytosis with a cooperativity of (6.6/2.1 =) 3.3, which is similar to
the value of 2 measured more directly in Figure 4. A supralinear
relationship between exocytosis and Ca2+
influx has also been found at other synapses, with power-laws generally
varying between 2 and 4 (Dodge and Rahamimoff, 1967; Augustine and
Charlton, 1986; Llinas et al., 1982; Mintz et al., 1995). Perhaps the
most direct measurements of the
Ca2+-dependence of rapid exocytosis at a
synapse made to date are those of Heidelberger et al. (1994), who
controlled the free [Ca2+] inside the
bipolar cell terminal by photolysis of "caged"
Ca2+ and measured a cooperativity of 4.
The initial phase of exocytosis
A clear kinetic distinction between "fast" and "slow"
phases of capacitance increase could not be observed during very weak stimulation nor during particularly strong stimulation (when the whole
of the RRP appeared to be depleted with a single time constant). To
obtain a qualitative understanding of how the rapid output of this
synapse might vary, capacitance responses were measured after a 20 msec
depolarization to a variety of potentials (Fig. 8A). A duration of 20 msec was used because, when the rapid phase of exocytosis could be
distinguished, it was approximately complete within this time (Figs. 1,
6). The amount of exocytosis (expressed as a percentage of the RRP) is
plotted as a function of membrane potential in Figure
8B. An empirical description of these results was
obtained by a Hill function of the form R = RmaxVh/(V1/2h + Vh), where R is the % RRP
released and V is the amplitude of the voltage step above
the threshold for activation of the Ca2+
current (taken as 43 mV for the fit shown).
V1/2, when release was half-maximal,
corresponded to a membrane potential of 33 mV. At 33 mV, where the
voltage- dependence was at its steepest, an extra 6% of the RRP was
released for every 1 mV increase in the amplitude of the
depolarization. If the RRP contains 1800 vesicles, this represents the
release of an extra 90 vesicles per millivolt.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 8.
Voltage-dependence of exocytosis for 20 msec
pulses. A, Capacitance increases elicited by 20 msec
depolarizations to 37, 35, and 10 mV. Each test stimulus was
followed by the emptying stimulus. The Ca2+ currents
for each stimulus are shown below. B, The amount of
exocytosis elicited by a 20 msec depolarization to various membrane
potentials. Each point is the average of 4-16
measurements. Results collected from a total of 20 cells. The
capacitance increase to the test stimulus was expressed as a percentage
of the RRP. The line fitted through the
points is a Hill function (see Results).
|
|
A notable feature of the results in Figure 8B is that
the responses did not saturate until the membrane potential was stepped to approximately 25 mV. This suggests that only the largest voltage signals normally occurring in depolarizing bipolar cells will come
close to causing complete exhaustion of the RRP. Over most of the
physiological range of membrane potentials, the rapid phase of
exocytosis involves only a fraction of the RRP (Figs. 1, 6).
| |
DISCUSSION |
These results indicate that the ribbon synapse of depolarizing
bipolar cells adapts to a constant stimulus. A step depolarization elicited a rapid phase of exocytosis, followed by a slow phase that
reflected a decrease in the rate-constant of release. Synaptic depression therefore had two causes: depletion of the RRP and a
decrease in the efficiency of exocytosis from the RRP. The latter effect was the more important at membrane potentials that cover the
operating range in the retina.
The efficiency of exocytosis from the RRP
A number of studies have suggested that synaptic depression may
not be caused simply by depletion of the RRP. Furukawa and Matsura
(1978) recorded EPSPs at ribbon synapses between hair cells and
eighth nerve fibers in goldfish. A continuous sound caused "adaptive
rundown" of successive EPSPs, but even when the response had declined
to zero, an increase in sound intensity caused a new discharge. They
suggested that release sites in hair cells might have different
sensitivities to the amplitude of the depolarization, so that only a
few release sites were stimulated by a weak sound but a larger number
would respond to a loud sound. Support for this idea was provided by a
quantal analysis of changes in the amplitude of EPSPs after changes in
sound intensity. An increase in EPSP amplitude was associated with an
increase in n, the number of vesicles available for release
(Furukawa et al., 1978), whereas a decrease in EPSP amplitude was
associated with a decrease in n (Furukawa et al., 1982).
Qualitatively similar adaptation to a sensory stimulus has been
observed in the lateral line organ of fish that responds to vibration
(Flock and Russell, 1976). Blight and Llinas (1980) studied
transmission at the synapse between "T" fibers from stretch
receptors and motoneurons in the crab. They found that strong
presynaptic depolarization led to depression of transmission, causing
the postsynaptic response to decline after an initial transient. On
repolarization, there was a second strong postsynaptic response driven
by the calcium tail-current, indicating that the RRP still contained a
large number of vesicles.
Changes in the efficiency of exocytosis from the RRP may not be
confined to ribbon synapses. At the squid giant synapse, Kusano and
Landau (1975) found that increasing the amplitude of the presynaptic depolarization appeared to increase the number of vesicles available for rapid release. More recently, Wu and Borst (1999) studied a giant
calyx-type synapse in the auditory brainstem and found that depression
was associated with a reduction in the probability of release from the RRP.
It is interesting to relate the changes in the efficiency of exocytosis
that we observed to events at the active zone. Electron microscopy
performed by von Gersdorff et al. (1996) indicates that there are ~22
vesicles docked to the plasma membrane under each ribbon, and it has
been suggested that these vesicles comprise the RRP (Mennerick and
Matthews, 1996; Gomis et al., 1999). The decrease in the efficiency of
exocytosis was half-maximal after the release of 10-15% of the RRP
(Fig. 3B), which would correspond to only two to three
vesicles per active zone.
Factors that might affect the efficiency of exocytosis from
the RRP
We have considered three possible causes for synaptic depression
over and above depletion of the RRP. First, vesicles within the RRP
that are docked further away from Ca2+
channels might be released less efficiently than those docked more
closely. Second, some vesicles within the RRP might have a lower
intrinsic sensitivity to Ca2+ (independent
of their location). Third, there might be some form of negative
feedback that regulates the Ca2+
sensitivity of exocytosis and causes the rate-constant of exocytosis to
fall with a delay.
Full consideration of the first model would require an understanding of
the spatial organization of Ca2+ channels
and vesicles at the plasma membrane, as well as the properties of the
Ca2+ buffers and
Ca2+ sensor(s) for exocytosis. However,
some simple calculations indicate the feasibility of the idea that
vesicles docked at the active zone may not all experience the same
Ca2+ signal. In the presence of a mobile
buffer, the free concentration of which is relatively constant, the
steady-state free [Ca2+] near a single
Ca2+ channel is expected to fall
exponentially with the distance r (Neher, 1998):
where iCa is the single channel
current, F the Faraday constant, and
DCa is the diffusion coefficient for
free Ca2+. The spatial extent of free
Ca2+ will be determined by the space
constant  , which is a measure of the distance a
Ca2+ ion diffuses before it is intercepted
by a buffer molecule. is given by:
where kon is the rate-constant
with which Ca2+ binds the buffer, and
[B]o is the free concentration of
buffer. The value for [B]o is
obtained from [B]o = BtKd/(Kd/[Ca]b),
where Kd is the dissociation constant for the
buffer, [Ca]b is the basal free Ca2+ concentration, and
Bt is the total concentration of
mobile buffer. At the ribbon synapse of hair cells, there is a mobile
Ca2+ buffer, the concentration of which is
expected to be 1-2 mM if it binds
Ca2+ at a rate similar to BAPTA (Roberts,
1993; Tucker and Fettiplace, 1996). Similarly, the synaptic terminal of
depolarizing bipolar cells contains ~1.6 mM of
a BAPTA-like buffer (J. Burrone and L. Lagnado, unpublished
observations). The Kon for BAPTA is 6 × 108 M/sec, which yields a value of = 17 nm, assuming DCa = 220 µm2/sec, and
[Ca2+]b = 50 nM. If four Ca2+
ions act cooperatively to trigger exocytosis (Heidelberger et al.,
1994), a fivefold difference in the rate of release would reflect a
1.5-fold difference in the [Ca2+] at the
sensor. Such variations in the [Ca2+] at
the sensor might occur if the separation between the sensor and nearby
Ca2+ channels were subject to a
variability of only a few nanometers. A vesicle has a diameter of ~36
nm, so it seems possible that vesicles docked at the active zone may
have different probabilities of release because the
Ca2+ sensor that they are associated with
experiences slightly different rises in
[Ca2+].
The results of two studies suggest that vesicles docked near the
ribbons of hair cells may indeed have different sensitivities to
Ca2+ influx. First, Roberts (1993) made
numerical simulations of the [Ca2+] at
the ribbon synapse of hair cells, taking into consideration the
contribution of all the Ca2+ channels
localized to an active zone. The simulations predicted that the profile
of the time-averaged [Ca2+] near the
plasma membrane will vary greatly across the active zone, with peaks of
high [Ca2+] occurring under single
Ca2+channels (see also Zucker, 1989).
Second, electron tomography of these synapses indicates that probably
<50% of vesicles are docked within 50 nm of a
Ca2+ channel (Lenzi et al., 1999),
suggesting that vesicles toward the periphery of an active zone
experience a substantially smaller rise in
[Ca2+] than those toward the center. It
remains to be seen whether the arrangement of vesicles and
Ca2+ channels at the active zone of hair
cells might account for the variable sensitivity to depolarization
suggested by Furukawa et al. (1982).
A second possible cause of heterogeneity within the RRP is that
different vesicles require different Ca2+
concentrations to trigger their release. Recently, Blank et al. (1998) found that Ca2+
concentrations above threshold caused the fusion of only a
subpopulation of secretory vesicles in sea urchin eggs and proposed
that individual vesicles have different sensitivities to
Ca2+. However, it does not seem likely
that this mechanism operates in bipolar cells, given the results of
Heidelberger et al. (1994). They used caged
Ca2+ to impose spatially uniform rises in
free [Ca2+] in the synaptic terminal of
depolarizing bipolar cells and found that the resulting rise in
capacitance always followed a single exponential time course with a
fixed final amplitude, indicating that all vesicles within the RRP had
a fixed intrinsic sensitivity to Ca2+.
The possibility that the Ca2+ sensitivity
of the reaction triggering exocytosis might fall because of some form
of negative feedback has been raised by a few studies. Korn et al.
(1984) performed a quantal analysis at inhibitory synapses on the
Mauthner cell of goldfish and concluded that synaptic depression was
caused by a decrease in release probability rather than a decrease in quantal content. They suggested that vesicle fusion caused a transient refractoriness of the exocytic apparatus by disrupting the normal organization of the active zone (Triller and Korn, 1982). Hsu et al.
(1996), working on the squid giant synapse, found that the rate of
exocytosis elicited by a constant rise in
[Ca2+] fell with a time constant of 30 msec but could not account for this by depletion of the RRP. They
suggested that the exocytic machinery might adapt to the
Ca2+ signal and that the site of
adaptation might be the Ca2+-sensitive
molecule controlling the final step in release. A similar idea has been
proposed by Bellingham and Walmsley (1999), who found that synaptic
depression at the calyx of Held was modulated by
Ca2+ entry.
Our results do not rule out the possibility that the decreased
efficiency of exocytosis from the RRP reflects a time-dependent "adaptation" of the exocytic machinery of the type postulated by
Hsu et al. (1996). However, in our view, the simplest possibility with
the most evidence is that vesicles within the RRP are subject to
different rises in Ca2+ concentration
because they are different distances from
Ca2+ channels.
Operating characteristics of the bipolar cell synapse
A common feature of neurons possessing ribbon synapses is that
they do not fire sodium-dependent action potentials, but instead signal
with graded voltage changes that can be as small as a fraction of a
millivolt. In the dark-adapted retina, for instance, the absorption of
a single photon generates a signal of ~250 µV in a depolarizing
bipolar cell receiving synaptic inputs from rod photoreceptors (Ashmore
and Falk, 1980b). These small signals are reliably transmitted to
ganglion cells (Barlow et al., 1971), and this may depend on the steep
voltage-dependence of release from bipolar cells. The rate of
exocytosis at the terminal of depolarizing bipolar cells was at its
steepest at potentials below 30 mV, accelerating e-fold for every 2.1 mV depolarization (Fig. 7). Exocytosis at the ribbon synapse of rods
has a similar voltage sensitivity (Attwell et al., 1987; Belgum and
Copenhagen, 1988).
The voltage-dependence of exocytosis fell dramatically at potentials
above the physiological range (more than 30 mV). Matsui et al. (1998)
made paired recordings from depolarizing bipolar cells and synaptically
connected ganglion cells in the newt retina and also found that
synaptic gain decreased dramatically above 30 mV. It therefore seems
that the input-output properties of this synapse are matched to the
15-20 mV range in which it normally operates. Other ribbon synapses
respond to somewhat smaller voltage signals. For instance, the
oscillatory potentials that modulate exocytosis from hair cells have a
peak-to-peak amplitude of ~10 mV (Crawford and Fettiplace, 1981). The
operating range of the rod synapse is only ~5 mV because light causes
a hyperpolarization from a dark potential of 40 mV, whereas the
Ca2+ current turns off below 45 mV
(Attwell et al., 1987).
Ganglion cells postsynaptic to depolarizing bipolar cells show a
variety of behaviors in response to a step of light (Dowling, 1987).
Some are purely transient, responding only when the light is turned on.
Some are sustained, signaling with similar intensity throughout a step.
Others show a mixed transient-sustained response. It seems likely that
the transient component of exocytosis that we observe in depolarizing
bipolar cells contributes to the transient signals seen in postsynaptic
ganglion cells. Our results indicate that this differentiation is
performed by a subset of vesicles within the RRP that are particularly
sensitive to Ca2+ influx. In the retina,
this transformation plays an essential role in the processing of visual
signals (Lagnado, 1998). Feedback connections from amacrine cells onto
the synaptic terminal of depolarizing bipolar cells will also
contribute to the generation of transient output from this synapse
(Roska et al., 1998). It seems most likely that the sustained signals
that occur in postsynaptic ganglion cells are generated by the slower
continuous component of exocytosis that the synapse supports in
response to maintained depolarization (Lagnado et al., 1996;
Rouze and Schwartz, 1998; Neves and Lagnado, 1999).
Exocytosis from the synaptic terminal of depolarizing bipolar cells has
also been studied by detecting glutamate release using glutamate-activated channels in retinal horizontal cells (Sakaba et
al., 1997; von Gersdorff et al., 1998). As in the present study, von
Gersdorff et al. (1998) applied sustained depolarizations that
activated the Ca2+ current to varying
degrees. When the Ca2+ current was smaller
than ~140 pA, the glutamate-activated current in the "sniffer"
cell had two peaks separated by delays of 50-200 msec. This was
interpreted as indicating two discrete bouts of exocytosis. von
Gersdorff et al. (1998) suggested that the first bout corresponded to
the RRP (perhaps vesicles docked to the plasma membrane) and the second
bout to a reserve pool (perhaps vesicles on the ribbon). It was also
suggested that these two phases of release "fused" into one phase
that was complete within 100 msec when the
Ca2+ current was larger than ~140 pA.
Using the capacitance technique, we also find that exocytosis elicited
by Ca2+ currents smaller than ~100 pA
occurs in two phases. However, we have never observed two discrete
bouts of exocytosis. Furthermore, we found that the first phase of
exocytosis involved only a fraction of the RRP, whereas the second
phase primarily involved the remainder of the RRP. Only at delays
longer than 300 msec did we find evidence that vesicles from a reserve
pool might also be released (Fig. 1B,D). When the
Ca2+ current was larger than ~140 pA,
release of the RRP remained easily distinguished from release of the
reserve pool. Indeed, previous capacitance measurements (Mennerick and
Matthews, 1996; Sakaba et al., 1997; Gomis et al., 1999; Neves and
Lagnado, 1999) and FM1-43 measurements (Neves and Lagnado, 1999)
have demonstrated that release of the reserve pool occurs over a time
scale of several hundred milliseconds, even when the
Ca2+ current is large.
The use of the capacitance technique imposed an important limitation on
the stimuli that we were able to use: they always involved a transition
from a resting state at which all the Ca2+
channels were closed. Actually, the membrane potential of a
depolarizing bipolar cell will often vary over a range in which the
Ca2+ current is active. To understand the
operation of this synapse, it will therefore be necessary to understand
how ongoing exocytosis is modulated by changes in membrane potential.
Under these conditions, the size of the RRP will be dependent both on
the rate of exocytosis and the rate at which it is supplied with new
vesicles. Recent evidence indicates that refilling of the RRP is
stimulated by Ca2+. Mennerick and Matthews
(1996) found that EGTA inhibited the slow phase of exocytosis that
followed release of the RRP and suggested that the translocation of
vesicles might be rate-limiting during this process. However, they also
found that EGTA did not inhibit the refilling of the RRP that occurred
after stimulation and so concluded that a translocation process was not
the primary means of refilling the RRP once
Ca2+ channels had closed. In contrast,
Gomis et al. (1999) found that inhibition of the slow phase of
exocytosis by EGTA was correlated with reduced refilling of the RRP
after stimulation. They provided evidence that the supply of vesicles
for rapid exocytosis occurred through two distinct
Ca2+-dependent steps, one regulating the
movement of vesicles from a reserve pool to the RRP, and a second
regulating the transfer of vesicles to the reserve pool. We therefore
expect that the Ca2+-dependent
translocation of vesicles to the plasma membrane will accelerate the
supply of vesicles during stimulation, as well as recovery from
synaptic depression after stimulation. The same may be expected of
other ribbon synapses in which exocytosis is continuously modulated,
such as those in auditory and vestibular hair cells (Furukawa et al.,
1982; Parsons et al., 1994; Lenzi et al., 1999).
| |
FOOTNOTES |
Received July 9, 1999; revised Oct. 26, 1999; accepted Oct. 28, 1999.
We thank Ana Gomis and Guilherme Neves for discussions of this work.
Correspondence should be addressed to Leon Lagnado, Medical Research
Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH,
UK. E-mail: LL1{at}mrc-lmb.cam.ac.uk.
| |
REFERENCES |
-
Ashmore JF,
Falk G
(1980a)
Responses of rod bipolar cells in the dark-adapted the retina of the dogfish.
J Physiol (Lond)
300:115-150[Abstract/Free Full Text].
-
Ashmore JF,
Falk G
(1980b)
The single-photon signal in rod bipolar cells of the dogfish retina.
J Physiol (Lond)
300:151-166[Abstract/Free Full Text].
-
Attwell D,
Borges S,
Wu SM,
Wilson M
(1987)
Signal clipping by the rod output synapse.
Nature
328:522-524[Medline].
-
Augustine GJ,
Charlton MP
(1986)
Calcium dependence of presynaptic calcium current and post-synaptic response at the squid giant synapse.
J Physiol (Lond)
381:619-640[Abstract/Free Full Text].
-
Augustine GJ,
Charlton MP,
Smith SJ
(1985)
Calcium entry and transmitter release at voltage-clamped nerve terminals of squid.
J Physiol (Lond)
369:163-181.
-
Barlow HB,
Levick WR,
Yoon M
(1971)
Response to single quanta of light in retinal ganglion cells of the cat.
Vis Res Suppl
3:87-101.
-
Belgum JH,
Copenhagen DR
(1988)
Synaptic transfer of rod signals to horizontal and bipolar cells in the retina of the toad (Bufo marinus).
J Physiol (Lond)
396:225-245[Abstract/Free Full Text].
-
Bellingham MC,
Walmsley B
(1999)
A novel presynaptic inhibitory mechanism underlies paired pulse depression at a fast central synapse.
Neuron
23:159-170[Web of Science][Medline].
-
Betz WJ
(1970)
Depression of transmitter release at the neuromuscular junction of the frog.
J Physiol (Lond)
206:629-644[Abstract/Free Full Text].
-
Blank PS,
Cho M-S,
Vogel SS,
Kaplan D,
Kang A,
Malley J,
Zimmerberg J
(1998)
Submaximal responses in calcium-triggered exocytosis by differences in the calcium sensitivity of individual secretory vesicles.
J Gen Physiol
112:559-567[Abstract/Free Full Text].
-
Blight AR,
Llinas R
(1980)
The non-impulsive stretch-receptor complex of the crab: a study of depolarization-release coupling at a tonic sensorimotor synapse.
Philos Trans R Soc Lond B Biol Sci
290:219-276.
-
Burns ME,
Augustine GJ
(1995)
Synaptic structure and function: dynamic organization yields architectural precision.
Cell
83:187-194[Web of Science][Medline].
-
Burrone J,
Lagnado L
(1997)
Electrical resonance and Ca2+ influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina.
J Physiol (Lond)
505:571-584[Abstract/Free Full Text].
-
Crawford AC,
Fettiplace R
(1981)
An electrical tuning mechanism in turtle cochlear hair cells.
J Physiol (Lond)
312:377-412[Abstract/Free Full Text].
-
Del Castillo J,
Katz B
(1954)
Quantal components of the end-plate potential.
J Physiol (Lond)
124:560-573.
-
Dittman JS,
Regehr WG
(1998)
Calcium dependence and recovery kinetics of presynaptic depression at the climbing fiber to Purkinje cell synapse.
J Neurosci
18:6147-6162[Abstract/Free Full Text].
-
Dodge Jr FA,
Rahamimoff R
(1967)
Co-operative action of calcium ions in transmitter release at the neuromuscular junction.
J Physiol (Lond)
193:419-432[Abstract/Free Full Text].
-
Dowling JE
(1987)
In: The retina: an approachable part of the brain. Cambridge, MA: Harvard UP.
-
Flock A,
Russell I
(1976)
Inhibition by efferent nerve fibres: action on hair cells and afferent synaptic transmission in the lateral line canal organ of the burbot Lota lota.
J Physiol (Lond)
257:45-62.
-
Furukawa T,
Matsura S
(1978)
Adaptive rundown of excitatory post-synaptic potentials at synapses between hair-cells and afferent nerve fibres in goldfish.
J Physiol (Lond)
276:193-209[Abstract/Free Full Text].
-
Furukawa T,
Hayashida Y,
Matsura S
(1978)
Quantal analysis of the excitatory post-synaptic potentials at synapses between hair-cells and eighth nerve fibres synapses in goldfish.
J Physiol (Lond)
276:211-226[Abstract/Free Full Text].
-
Furukawa T,
Kuno M,
Matsura S
(1982)
Quantal analysis of a decremental response at hair-cell afferent fibre synapses in the goldfish sacculus.
J Physiol (Lond)
322:181-195[Abstract/Free Full Text].
-
Gillis KD
(1995)
Techniques for membrane capacitance measurements.
In: Single channel recording (Sakmann B,
Neher E,
eds), pp 155-198. New York: Plenum.
-
Gomis A,
Burrone J,
Lagnado L
(1999)
Two actions of calcium regulate the supply of releasable vesicles at the ribbon synapse of retinal bipolar cells.
J Neurosci
19:6309-6317[Abstract/Free Full Text].
-
Heidelberger R,
Heinemann C,
Neher E,
Matthews G
(1994)
Calcium dependence of the rate of exocytosis in a synaptic terminal.
Nature
371:513-515[Medline].
-
Hsu SF,
Augustine GJ,
Jackson MB
(1996)
Adaptation of Ca2+-triggered exocytosis in presynaptic terminals.
Neuron
17:501-512[Web of Science][Medline].
-
Katz B,
Miledi R
(1967)
A study of synaptic transmission in the absence of nerve impulses.
J Physiol (Lond)
167:23-38.
-
Korn H,
Faber DS,
Burnod Y,
Triller A
(1984)
Regulation of efficacy at a central synapse.
J Neurosci
4:125-130[Abstract].
-
Kusano K,
Landau EM
(1975)
Depression and recovery of transmission at the squid giant synapse.
J Physiol (Lond)
245:13-32[Abstract/Free Full Text].
-
Lagnado L
(1998)
Amacrine cells keep it short and sweet.
Curr Biol
8:R598-R600[Web of Science][Medline].
-
Lagnado L,
Gomis A,
Job C
(1996)
Continuous vesicle cycling in the synaptic terminal of retinal bipolar cells.
Neuron
17:957-967[Web of Science][Medline].
-
Lenzi D,
Runyeon JW,
Crum J,
Ellisman MH,
Roberts WM
(1999)
Synaptic vesicle populations in saccular hair cells reconstructed by electron tomography.
J Neurosci
19:119-132[Abstract/Free Full Text].
-
Liley AW,
North KAK
(1953)
An electrical investigation of effects of repetitive stimulation on mammalian neuromuscular junction.
J Neurophysiol
16:509-527[Free Full Text].
-
Llinas R,
Steinberg IZ,
Walton K
(1981)
Relationship between presynaptic calcium current and post synaptic potential in squid giant synapse.
Biophys J
33:323-352[Web of Science][Medline].
-
Llinas R,
Sugimori M,
Simon SM
(1982)
Transmission by presynaptic spike-like depolarization in the squid giant synapse.
Proc Natl Acad Sci USA
79:2415-2419[Abstract/Free Full Text].
-
Matsui K,
Hosoi N,
Tachibana M
(1998)
Excitatory synaptic transmission in the inner retina: paired recordings of bipolar cells and neurons of ganglion cell layer.
J Neurosci
18:4500-4510[Abstract/Free Full Text].
-
Mennerick S,
Matthews G
(1996)
Ultrafast exocytosis elicited by calcium current in synaptic terminals of retinal bipolar neurons.
Neuron
17:1241-1249[Web of Science][Medline].
-
Mennerick S,
Matthews G
(1998)
Rapid calcium-current kinetics in synaptic terminals of goldfish retinal bipolar neurons.
Vis Neurosci
15:1051-1056[Web of Science][Medline].
-
Mintz IM,
Sabatini BL,
Regehr WG
(1995)
Calcium control of transmitter release at a cerebellar synapse.
Neuron
15:675-688[Web of Science][Medline].
-
Neher E
(1998)
Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release.
Neuron
20:389-399[Web of Science][Medline].
-
Neher E,
Marty A
(1982)
Discrete changes of cell membrane capacity observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.
Proc Natl Acad Sci USA
79:6712-6716[Abstract/Free Full Text].
-
Neves G,
Lagnado L
(1999)
The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells.
J Physiol (Lond)
515:181-202[Abstract/Free Full Text].
-
Parsons TD,
Lenzi D,
Almers W,
Roberts WM
(1994)
Calcium-triggered exocytosis and endocytosis in an isolated presynaptic cell: capacitance measurements in saccular hair cells.
Neuron
13:875-883[Web of Science][Medline].
-
Roberts WM
(1993)
Spatial Ca2+ buffering in saccular hair cells.
Nature
363:74-76[Medline].
-
Roska B,
Nemeth E,
Werblin FS
(1998)
Response to change is facilitated by a three-neuron disinhibitory pathway in the tiger salamander retina.
J Neurosci
18:3451-3459[Abstract/Free Full Text].
-
Rouze NC,
Schwartz EA
(1998)
Continuous and transient vesicle cycling at a ribbon synapse.
J Neurosci
18:8614-8624[Abstract/Free Full Text].
-
Sakaba T,
Tachibana M,
Matsui K,
Minami N
(1997)
Two components of transmitter release in retinal bipolar cells: exocytosis and mobilization of synaptic vesicles.
Neurosci Res
27:357-370[Web of Science][Medline].
-
Stevens CF,
Wesseling JF
(1998)
Activity-dependent modulation of the rate at which synaptic vesicles become available to undergo exocytosis.
Neuron
21:415-424[Web of Science][Medline].
-
Tachibana M,
Okada T,
Arimura T,
Kobayashi K,
Piccolino M
(1993)
Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina.
J Neurosci
13:2898-2909[Abstract].
-
Triller A,
Korn H
(1982)
Transmission at a central inhibitory synapse. III. Ultrastructure of physiologically identified and stained terminals.
J Neurophysiol
48:708-736[Free Full Text].
-
Tucker TR,
Fettiplace R
(1996)
Monitoring calcium in turtle hair cells with a calcium-activated potassium channel.
J Physiol (Lond)
494:613-626[Abstract/Free Full Text].
-
von Gersdorff H,
Vardi E,
Matthews G,
Sterling P
(1996)
Evidence that vesicles on the synaptic ribbon of retinal bipolar neurons can be rapidly released.
Neuron
16:1221-1227[Web of Science][Medline].
-
von Gersdorff H,
Sakaba T,
Berglund K,
Tachibana M
(1998)
Submillisecond kinetics of glutamate release from a sensory synapse.
Neuron
21:1177-1188[Web of Science][Medline].
-
Wang LY,
Kaczmarek LK
(1998)
High-frequency firing helps replenish the readily releasable pool of synaptic vesicles.
Nature
394:384-388[Medline].
-
Werblin FS,
Dowling JE
(1969)
Organization of the inner retina of mudpuppy Necturus maculosus. II. Intracellular recording.
J Neurophysiol
32:335-339.
-
Wu L-G,
Borst JG
(1999)
The reduced release probability of releasable vesicles during recovery from short-term synaptic depression.
Neuron
23:821-832[Web of Science][Medline].
-
Zucker RS
(1989)
Short-term synaptic plasticity.
Annu Rev Neurosci
12:13-31[Web of Science][Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/202568-11$05.00/0
This article has been cited by other articles:
|
|
|
|
 
Q. Liu, G. Hollopeter, and E. M. Jorgensen
Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction
PNAS,
June 30, 2009;
106(26):
10823 - 10828.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Snellman, D. Zenisek, and S. Nawy
Switching between transient and sustained signalling at the rod bipolar-AII amacrine cell synapse of the mouse retina
J. Physiol.,
June 1, 2009;
587(11):
2443 - 2455.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Coggins and D. Zenisek
Evidence that Exocytosis Is Driven by Calcium Entry Through Multiple Calcium Channels in Goldfish Retinal Bipolar Cells
J Neurophysiol,
May 1, 2009;
101(5):
2601 - 2619.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L.-G. Wu, T. A. Ryan, and L. Lagnado
Modes of Vesicle Retrieval at Ribbon Synapses, Calyx-Type Synapses, and Small Central Synapses
J. Neurosci.,
October 31, 2007;
27(44):
11793 - 11802.
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Palmer
Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals
J. Physiol.,
May 1, 2006;
572(3):
747 - 762.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Rabl, L. Cadetti, and W. B. Thoreson
Paired-Pulse Depression at Photoreceptor Synapses
J. Neurosci.,
March 1, 2006;
26(9):
2555 - 2563.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Beaumont, A. Llobet, and L. Lagnado
Expansion of calcium microdomains regulates fast exocytosis at a ribbon synapse
PNAS,
July 26, 2005;
102(30):
10700 - 10705.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Palmer, C. Hull, J. Vigh, and H. von Gersdorff
Synaptic Cleft Acidification and Modulation of Short-Term Depression by Exocytosed Protons in Retinal Bipolar Cells
J. Neurosci.,
December 10, 2003;
23(36):
11332 - 11341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. H. Singer and J. S. Diamond
Sustained Ca2+ Entry Elicits Transient Postsynaptic Currents at a Retinal Ribbon Synapse
J. Neurosci.,
November 26, 2003;
23(34):
10923 - 10933.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. B. Sorensen, R. Fernandez-Chacon, T. C. Sudhof, and E. Neher
Examining Synaptotagmin 1 Function in Dense Core Vesicle Exocytosis under Direct Control of Ca2+
J. Gen. Physiol.,
August 25, 2003;
122(3):
265 - 276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Palmer, H. Taschenberger, C. Hull, L. Tremere, and H. von Gersdorff
Synaptic Activation of Presynaptic Glutamate Transporter Currents in Nerve Terminals
J. Neurosci.,
June 15, 2003;
23(12):
4831 - 4841.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Llobet, A. Cooke, and L. Lagnado
Exocytosis at the Ribbon Synapse of Retinal Bipolar Cells Studied in Patches of Presynaptic Membrane
J. Neurosci.,
April 1, 2003;
23(7):
2706 - 2714.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Holt, A. Cooke, M. M. Wu, and L. Lagnado
Bulk Membrane Retrieval in the Synaptic Terminal of Retinal Bipolar Cells
J. Neurosci.,
February 15, 2003;
23(4):
1329 - 1339.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X.-S. Wu and L.-G. Wu
Protein Kinase C Increases the Apparent Affinity of the Release Machinery to Ca2+ by Enhancing the Release Machinery Downstream of the Ca2+ Sensor
J. Neurosci.,
October 15, 2001;
21(20):
7928 - 7936.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Neves, A. Neef, and L. Lagnado
The actions of barium and strontium on exocytosis and endocytosis in the synaptic terminal of goldfish bipolar cells
J. Physiol.,
September 15, 2001;
535(3):
809 - 824.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Voets, T. Moser, P.-E. Lund, R. H. Chow, M. Geppert, T. C. Sudhof, and E. Neher
Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I
PNAS,
September 13, 2001;
(2001)
201398798.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Hunter and J. G. Milton
Synaptic Heterogeneity and Stimulus-Induced Modulation of Depression in Central Synapses
J. Neurosci.,
August 1, 2001;
21(15):
5781 - 5793.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Voets, T. Moser, P.-E. Lund, R. H. Chow, M. Geppert, T. C. Sudhof, and E. Neher
Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I
PNAS,
September 25, 2001;
98(20):
11680 - 11685.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
![[Ca<SUP>2+</SUP>]=<FR><NU>i<SUB><UP>Ca</UP></SUB></NU><DE>4&pgr;FD<SUB><UP>Ca</UP></SUB>r</DE></FR><UP>exp</UP>(<UP>−</UP>r/&lgr;),](/content/vol20/issue2/fulltext/568/img006.gif)
![&lgr;=<RAD><RCD>D<SUB><UP>Ca</UP></SUB>/k<SUB><UP>on</UP></SUB>[B]<SUB><UP>o</UP></SUB></RCD></RAD>,](/content/vol20/issue2/fulltext/568/img007.gif)
