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The Journal of Neuroscience, August 1, 1999, 19(15):6309-6317
Two Actions of Calcium Regulate the Supply of Releasable Vesicles
at the Ribbon Synapse of Retinal Bipolar Cells
Ana
Gomis,
Juan
Burrone, and
Leon
Lagnado
Medical Research Council Laboratory of Molecular Biology, Cambridge
CB2 2QH, United Kingdom
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ABSTRACT |
Ribbon synapses of sensory neurons are able to sustain high rates
of exocytosis in response to maintained depolarization, but it is not
known how this is achieved. Using the capacitance technique, we have
found that Ca2+ regulates the supply of releasable
vesicles at the ribbon synapse of depolarizing bipolar cells from the
retina of goldfish. Ca2+ had two actions that could
be differentiated by introduction of the Ca2+
chelator EGTA; one action stimulated refilling of the rapidly releasable pool of vesicles from a reserve pool, and a second action stimulated recruitment of vesicles to the reserve pool. The
capacity of the reserve pool was ~3500 vesicles, which is similar to
the number that can attach to the ribbons. These results suggest that
continuous exocytosis at ribbon synapses is maintained by the
Ca2+-dependent translocation of vesicles from the
cytoplasm, through the ribbon, to release sites on the plasma membrane.
Key words:
synapse; vesicle; calcium; exocytosis; refilling; synaptic depression; retina; depolarizing bipolar cell; ribbon synapse; EGTA
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INTRODUCTION |
The processes that make synaptic
vesicles available for exocytosis play an important role in determining
the efficiency of synaptic transmission during ongoing activity. For
instance, many synapses exhibit short-term depression after a period of
activity, and this is at least partly because of a decrease in
the number of rapidly releasable vesicles docked at the plasma membrane
(Del Castillo and Katz, 1954 ; Betz, 1970; Zucker, 1989; Rosenmund and Stevens, 1996; Dittman and Regehr, 1998). The question of how vesicles
become available for exocytosis is of particular importance at ribbon
synapses, which can support continuous high rates of exocytosis in
response to maintained depolarization (Dowling and Ripps, 1973; Parsons
et al., 1994; Lagnado et al., 1996; Rieke and Schwartz, 1996). Ribbon
synapses are found in sensory neurons that generate graded voltage
signals rather than action potentials, including photoreceptors and
bipolar cells in the retina (Gray and Pease, 1971; Rao-Mirotznik et
al., 1995; von Gersdorff et al., 1996) and auditory and vestibular hair
cells in the ear (Jacobs and Hudspeth, 1990; Lenzi et al., 1999).
Electron micrographs of these neurons show that the regions in which
synaptic vesicles dock to the plasma membrane are associated with an
electron-dense structure, called a ribbon or synaptic body, to which
vesicles attach by short filaments. This arrangement suggests that
vesicles attached to the ribbon form a reserve pool that supplies the
active zone on the plasma membrane (Gray and Pease, 1971), although it has been difficult to test this idea experimentally (Burns and Augustine, 1995; Lenzi et al., 1999). Recently, our knowledge of the
structure of ribbon synapses has been complemented by functional studies of exocytosis using capacitance and optical techniques. In
particular, we have a relatively large amount of information about
vesicle cycling at the ribbon synapse of depolarizing bipolar cells
from the goldfish retina (Tachibana et al., 1993; Heidelberger et al.,
1994; von Gersdorff and Matthews, 1994; Lagnado et al., 1996). An
important feature of this synapse is that the size of the rapidly
releasable pool of vesicles (RRP) can be directly measured using the
capacitance technique (Mennerick and Matthews, 1996; Neves and Lagnado,
1999).
We have investigated the refilling of the RRP in goldfish bipolar cells
during different patterns of stimulation and after introduction of the
Ca2+ chelator EGTA. Our results indicate that
Ca2+ entering through Ca2+
channels has two effects that stimulate the supply of vesicles to the
RRP. Over the short-term, Ca2+ stimulated refilling
of the RRP from a reserve pool, and over the long-term,
Ca2+ stimulated the recruitment of vesicles to the
reserve pool. The ability of residual Ca2+ to
stimulate refilling of the RRP for periods of 5-10 sec indicates that
this action occurs at free Ca2+ concentrations in
the range of 1 µM or below. The capacity of the reserve
pool was estimated to be ~3500 vesicles, which is similar to the
total number attached to the ribbons, calculated from serial EM
reconstructions (von Gersdorff et al., 1996). These results therefore
provide functional evidence for the idea that the ribbon acts to supply
vesicles to the active zone on the plasma membrane.
Ca2+-dependent trafficking of vesicles through the
ribbon to the RRP may act to maintain high rates of exocytosis that
occur during maintained depolarizing responses (Lagnado et al., 1996;
Rouze and Schwartz, 1998; Neves and Lagnado, 1999).
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MATERIALS AND METHODS |
Isolated synaptic terminals of depolarizing bipolar cells from
the goldfish retina were obtained by enzymatic dissociation (Burrone
and Lagnado, 1997). Terminals were voltage-clamped using the perforated
patch technique, and capacitance measurements were made using the
piecewise linear method (Neher and Marty, 1982). Our implementation of
this method is described 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 two-phase lock-in amplifier, the phase of
which was set using the "capacitance dither" technique, allowing
independent measurement of changes in membrane capacitance and
conductance (Gillis, 1995). The capacitance signal was calibrated by
dithering the capacitance by 100 fF at the beginning of the 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 50-300 msec, ending 20 msec before depolarization. The "after"
measurement was averaged over a period of 50-300 msec, beginning 20 msec after repolarization.
The capacity of the RRP was measured after various stimulation patterns
by measuring the capacitance increase elicited by a 20 msec
depolarization to 0 mV. At this potential, the Ca2+
current is maximally activated. A 20 msec stimulus was sufficient to
ensure that the whole of the RRP was exhausted, because it is normally
released with a time constant of 2-4 msec (see Results). This duration
stimulus also provided a safety margin that ensured depletion of the
RRP when the Ca2+ current was partially inactivated,
as sometimes occurred immediately after the introduction of a large
Ca2+ load by prolonged depolarization.
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. The
solution in the patch pipette contained (in mM): 110 caesium gluconate, 4 MgCl2, 3 Na2ATP, 1 Na2GTP, 10 TEACl, 0.4 BAPTA, and 20 HEPES, with 250 µg/ml nystatin (260 mOsm/l, pH 7.2).
In some experiments terminals were loaded with the
Ca2+ chelator EGTA by incubation in 0.1 or 0.2 mM EGTA-AM (0.1% DMSO; Molecular Probes, Eugene,
OR) for 15-20 min.
All measurements are given as mean ± SEM.
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RESULTS |
Measuring the rapidly releasable pool of vesicles
In depolarizing bipolar cells, there is a clear kinetic
distinction between the first and second phases of exocytosis elicited by strongly activating the Ca2+ current (Mennerick
and Matthews, 1996; Neves and Lagnado, 1999). Here, we define the pool
of vesicles involved in the first phase as the RRP. The approach we
used to measure the size of the RRP is derived from the behavior shown
in Figure 1. Figure 1A
shows the capacitance response to two pairs of depolarizing stimuli to
10 mV, a potential at which the Ca2+ current is
maximally activated. The interval between the stimuli was 100 msec. The
duration of the first stimulus was varied between 0.5 and 20 msec so as
to release a varying proportion of the RRP. These responses are plotted
as the filled circles in Figure 1B. In this example, the maximum amplitude of the first phase of exocytosis was 45 fF, equivalent to ~1700 vesicles, assuming the capacitance of
a single vesicle is 26 aF (Neves and Lagnado, 1999). The RRP was
exhausted with a time constant of 4 msec, so a 20 msec depolarization was more than sufficient to release all of it. Here, we term a 20 msec
depolarization to a potential that maximally activates the
Ca2+ current (10 or 0 mV) the "emptying"
stimulus, and we used it to probe the size of the RRP after various
patterns of stimulation.
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Figure 1.
Measuring the rapidly releasable pool of vesicles.
A, Capacitance increases elicited by a double-pulse
protocol. The first depolarization from 70 to 10 mV lasted 1 msec
in the example on the left and 10 msec in the example on
the right. In each case, the second stimulus was a 20 msec depolarization, delivered after a delay of 100 msec, to release
the remainder of the RRP (the emptying stimulus). The rest period
between stimulation episodes was 1-3 min. The size of the RRP at the
beginning of each stimulus episode was equivalent to a total response
of ~45 fF. The Ca2+ currents are shown below.
B, The capacitance increase elicited by the first
stimulus plotted as a function of its duration (filled
circles). The bold line through the
points is a saturating exponential with a time constant
of 3.6 msec and a maximum amplitude of 45 fF. The open
circles plot the capacitance increase elicited by the emptying
stimulus. The dashed line is a mirror image of the
bold line (see Results).
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Evidence that this approach does indeed measure changes in the size of
the RRP is shown in Figure 1B in which the open
circles plot the response to the emptying stimulus applied
after variable amounts of the RRP were released. The decline in the
measured size of the RRP mirrored the amount of rapid exocytosis
elicited by the first stimulus. The dashed line shows
the time course over which the RRP was expected to decline, assuming
that the RRP was equivalent to 45 fF at the start of each trial and
that there was negligible refilling in the 100 msec period between
stimuli. The decline in the measured size of the RRP mirrored the
amount of rapid exocytosis elicited by the first stimulus in similar experiments on eight other cells.
Refilling of the RRP after a brief stimulus
To assess how the RRP was refilled after a brief stimulus, two
emptying stimuli were applied with varying delays, as shown in Figure
2A. After a delay of
100 msec, there was only a small response to the second stimulus (Fig.
2A, top left), averaging 6% of the first.
The Ca2+ currents flowing during each stimulus were
very similar (Fig. 2A, inset), so
depression of the second response was not a result of inactivation of
the Ca2+ current. As the interval between stimuli
was increased, the second response recovered, reflecting refilling of
the RRP (Fig. 2A, middle,
right). Results from this type of experiment are collected in Figure 2B, which plots the refilling of the RRP as
a function of the interval between stimuli. There were two phases of
refilling: 30% of the RRP was refilled with a time constant of 0.64 sec, and the remainder with a time constant of 31 sec.
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Figure 2.
Refilling of the RRP after complete depletion by a
20 msec stimulus. A, The capacitance response to two 20 msec depolarizations is shown for intervals of 100 msec
(left), 3 sec (middle), and 40 sec
(right). In each case, the stimulus was a depolarization
from 70 to 0 mV. The inset in the left
panel is a superimposition of the Ca2+
currents elicited by two stimuli (calibration: 20 msec, 200 pA).
Currents were leak subtracted using a single leak pulse (a 20 mV
depolarization from 70 mV). B, The time course of
refilling of the RRP measured from the type of experiment shown in
A. The amplitude of the second response is expressed as
a percentage of the first for various intervals. The
line fitted through the points is a
double-exponential, with 30% of sites refilled with a time constant of
0.64 sec and the remainder with a time constant of 31 sec. The number
of observations for each interval are as follows: 100 msec, 7; 250 msec, 1; 500 msec, 24; 1 sec, 14; 2 sec, 13; 5 sec, 20; 10 sec, 10; 20 sec, 4; 30 sec, 3; 40 sec, 3; 60 sec, 3.
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Behavior qualitatively similar to that shown in Figure 2 has also been
observed at synapses from the CNS of mammals in which there are fast
and slow phases of recovery from synaptic depression. The fast phase
can be inhibited by the Ca2+ chelator EGTA,
indicating that it reflects the transient acceleration of refilling by
residual Ca2+ (Dittman and Regehr, 1998; Stevens and
Wesseling, 1998; Wang and Kaczmarek, 1998). Below, we present evidence
that residual Ca2+ also stimulates refilling of the
RRP in the synaptic terminal of bipolar cells. However, at this ribbon
synapse, Ca2+ appears to stimulate refilling of the
RRP by two actions, one of which is more sensitive to EGTA.
Refilling of the RRP was stimulated by calcium
When the duration of the depolarizing stimulus was increased,
refilling of the RRP was accelerated above the basal rate for a longer
period. The protocol used to measure refilling after a longer stimulus
is shown in Figure 3A,
Test. First, the emptying stimulus was applied to measure
the initial size of the RRP. Next, a 500 msec depolarization to 0 mV
was delivered after a delay of 200 msec. Finally, the emptying stimulus
was applied again to measure the RRP after a variable delay. In the
example shown in Figure 3A, 91% of the RRP was refilled 10 sec after a 500 msec depolarization (Test), but only ~44%
was refilled 10 sec after the emptying stimulus delivered alone
(Control). The time course of refilling after
depolarizations lasting 20 (filled circles) and
500 (open circles) msec is compared in Figure
3C. The basal rate constant of refilling, measured from the
slow phase after a brief stimulus, was 0.03/sec (Fig.
2B). Figure 3C shows that after a 500 msec
depolarization, refilling was accelerated above the basal rate for
5-10 sec until the RRP was almost completely replenished. In
comparison, it took ~60 sec to refill 90% of the RRP after a 20 msec
depolarization (Fig. 2B).
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Figure 3.
Refilling of the RRP after a long stimulus.
A, Recovery of the capacitance response 10 sec after a
20 msec depolarization (Control) is compared with
that measured 10 sec after a 500 msec depolarization
(Test). Note that in the Test protocol,
the 500 msec stimulus was preceded by a 20 msec depolarization to
measure the initial size of the RRP. In this terminal, the RRP
recovered 91% of its initial size 10 sec after a 500 msec
depolarization but only 44% in the controls immediately before and
after. B, Recovery of the capacitance response 1 sec
after a 20 msec depolarization is compared with that measured 1 sec
after a 500 msec depolarization. In this terminal, the RRP did not
exhibit any measurable recovery 1 sec after the 500 msec stimulus but
recovered 17% in the control immediately before and 21% in the one
after. The inset in the middle panel is a
superimposition of the Ca2+ currents elicited by the
two emptying stimuli (calibration: 20 msec, 200 pA). C,
The time course of refilling of the RRP measured from the type of
experiment shown in A and B. The
open circles show refilling measured after a 500 msec
depolarization; the amplitude of the response to the second depleting
stimulus is expressed as a percentage of the first for various
intervals. The open squares show results obtained in
terminals loaded with EGTA (see Results and Fig. 5). The closed
circles provide a comparison with the results in Figure
2B, showing refilling measured after a 20 msec
depolarization.
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Although the net effect of a longer stimulus was to potentiate
refilling of the RRP, at short delays the RRP was significantly smaller
(Fig. 3C). For instance, in the example shown in Figure 3B (same terminal as Fig. 3A), there did not
appear to be any refilling of the RRP 1 sec after a 500 msec
depolarization, whereas ~19% was refilled 1 sec after a 20 msec
depolarization. The delayed growth in the size of the RRP after a 500 msec stimulus is probably because residual Ca2+
continued to stimulate exocytosis after closure of
Ca2+ channels. Capacitance and optical measurements
in bipolar cell terminals have demonstrated that residual
Ca2+ can continue to drive exocytosis for periods of
1 sec or more, at rates of 400-800 vesicles/sec (Neves and Lagnado,
1999). We would expect that vesicles accumulate in the RRP only after
their removal by exocytosis has ceased, which can take ~1 sec after a
500 msec depolarization to 0 mV. Consistent with the results in Figure
3C, exocytosis does not continue after a 20 msec stimulus that introduces a smaller amount of Ca2+ (Neves and
Lagnado, 1999).
An idea of the changes in free cytoplasmic
[Ca2+] that occurred after stimulation
could be obtained using Ca2+-activated conductances
in the plasma membrane that are activated by residual
Ca2+ and generate a slowly decaying tail current
(Okada et al., 1995). Measurements of cytoplasmic
[Ca2+] have demonstrated that the amplitude of
this current is correlated with the spatially averaged
[Ca2+], and it declines with a time constant that
is similar to the fall in [Ca2+] (Neves and
Lagnado, 1999). Figure
4A shows that the
Ca2+-activated tail current was much larger and
longer after a 500 msec depolarization than after a 20 msec
depolarization.
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Figure 4.
The rise in cytoplasmic
[Ca2+] was reduced in terminals loaded with EGTA.
A, A comparison of the currents elicited by a 20 and 500 msec depolarization from 70 to 0 mV in a terminal with endogenous
Ca2+ buffers (bold line) and a
terminal additionally loaded with EGTA (thin line).
These terminals were used for this comparison because the
Ca2+ currents were of similar amplitude. EGTA
blocked the Ca2+-activated tail current that decayed
slowly after the 500 msec stimulus. The latter part of the decay in the
tail current occurred with a time constant of 0.51 sec in the control.
B, The tail currents after the 20 msec stimuli shown on
expanded scales. The bold line fitted to the control
trace has a time constant of 0.11 sec. C,
EGTA did not completely block the Ca2+-activated
tail current after a 500 msec stimulus; expansion of
trace in A. The bold line
fitted to the trace has a time constant of 0.43 sec.
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We tested whether residual Ca2+ might be the
stimulus that accelerated refilling after a longer stimulus by loading
terminals with the Ca2+ chelator EGTA so as to
reduce the rise in cytoplasmic [Ca2+]. Terminals
were loaded by incubation in the AM ester (see Materials and Methods).
Buffering the rise in [Ca2+] greatly reduced the
Ca2+-activated tail current (Okada et al., 1995).
This was most clearly observed after a 500 msec depolarization (Fig.
4A,C), but it was also apparent
after a 20 msec depolarization that caused a small activation of the
tail current (Fig. 4B). The tail current averaged 50 ± 6 pA (n = 20) immediately after a 500 msec
depolarization in a control terminal but only 10 ± 2 pA
(n = 12) after loading with EGTA. Experiments in
which we have altered the internal concentration of
Ca2+ buffers through a patch pipette indicate that
at least 2 mM EGTA is required to cause a similar
inhibition of the tail current (J. Burrone and L. Lagnado,
unpublished observations; Okada et al., 1995).
EGTA shortened the accelerated phase of refilling that followed a long
stimulus. Examples of these measurements are shown in Figure
5A, and averaged results for
delays of 10 and 5 sec are plotted as the open squares
in Figure 3C to allow a comparison with cells that did not
contain any exogenous Ca2+ buffers. After a 500 msec
depolarization, the acceleration of refilling above its basal rate
normally continued for ~10 sec (open circles), but
buffering the rise in cytoplasmic [Ca2+] blocked
refilling at delays longer than 5 sec (open squares). EGTA did not, however, block the initial accelerated phase of refilling. Figure 3C shows that 43 ± 4% of the RRP
was refilled over the first 5 sec, although there was no further
refilling over the next 5 sec. The refilling of 43% of the RRP in the
first 5 sec indicates an average rate constant of 0.09/sec over this period. In comparison, the basal rate constant of refilling was 0.03/sec in control terminals, as measured from the second phase of the
double-exponential fit in Figure 2B. The initial rate
constant of refilling in EGTA-loaded terminals was therefore at least
three times that measured in control terminals at resting levels of Ca2+.
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Figure 5.
EGTA inhibited the second phase of exocytosis and
the acceleration of refilling caused by a prolonged stimulus.
A, Recovery of the capacitance response after a 500 msec
depolarization is compared in a terminal with endogenous
Ca2+ buffers (left) and a terminal
additionally loaded with EGTA (right). In each case, the
size of the RRP was measured after a delay of 10 sec. EGTA had two
effects: a 500 msec depolarization caused less exocytosis, and recovery
of the RRP was reduced. B, EGTA caused a small reduction
in the capacitance response to the emptying stimulus. Only the first
response in each terminal was used for this comparison because EGTA
inhibited refilling of the RRP over the long term (see Results).
C, EGTA inhibited exocytosis from the second pool of
vesicles. The capacitance response to a 500 msec depolarization is
shown relative to the response to the emptying stimulus delivered 200 msec before. All measurements were made from terminals in which these
were the first stimuli.
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The effects of EGTA on the first and second phases
of exocytosis
We compared the effects of EGTA on refilling of the RRP
with its effects on the different kinetic components of exocytosis. EGTA had a small effect on exocytosis from the RRP. The first response
to a 20 msec depolarization averaged 55.5 ± 4.7 fF
(n = 58) in control terminals and 40.6 ± 3.6 fF
(n = 38) in terminals loaded with EGTA (Fig.
5B). The somewhat smaller response in the presence of EGTA
might reflect a slowing of exocytosis from the RRP and/or a decrease in
the initial size of the pool. The latter possibility seems more likely
because the kinetics of fast exocytosis are little affected by EGTA
(Mennerick and Matthews, 1996), whereas EGTA inhibited refilling of the
RRP over long time scales (see below). Our estimate of the initial size
of the RRP in EGTA-loaded terminals is therefore likely to be a lower
limit, especially in view of the fact that isolated bipolar cell
terminals can spontaneously depolarize, triggering exocytosis (Burrone
and Lagnado, 1997). In an effort to ensure that estimates of the size
of the RRP were made before exocytosis had been stimulated to any
appreciable extent, cells were loaded with EGTA in the absence of
external Ca2+, and Ca2+ was
replaced only after Ca2+ channels were held closed
by voltage-clamping the terminal.
EGTA had a stronger inhibitory effect on the second, slower, phase of
exocytosis. Figure 5A compares responses to a 20 and 500 msec stimulus, first under control conditions and then in a terminal
loaded with EGTA. Considering only the first stimulus episode in a
given terminal, a 500 msec depolarization normally released 1.56 ± 0.24 (n = 22) times as many vesicles as the
preceding emptying stimulus. However, after loading with EGTA, this
ratio was reduced to 0.73 ± 0.17 (n = 10) (Fig.
5C). We propose that these two actions of EGTA, inhibition
of the second phase of exocytosis and reduced refilling after a long
stimulus, reflects the involvement of the same set of vesicles, which
undergo Ca2+-dependent transfer to the RRP (see Discussion).
Recruitment to a reserve pool was stimulated by calcium
To investigate refilling over longer time scales, the RRP was
repeatedly exhausted by delivering trains of emptying stimuli at a
frequency of 0.1 Hz for durations of 3 min or more. The second response
in a train normally released 40-50% as many vesicles as the first
(Fig.
6A,B),
as would be expected from the results obtained with a double-pulse
protocol (Fig. 2B). The amplitude of the capacitance
response elicited by the emptying stimulus then remained constant,
indicating that the same fraction of the RRP was refilled after each
emptying stimulus and that the average rate of refilling was fixed
(Fig. 6A). The cumulative response during these
stimulus trains, relative to the initial size of the RRP, is plotted as
the filled circles in Figure 6C. The first response averaged 50 ± 15 fF (n = 18). The RRP
could be refilled at least ten times over, equivalent to the transfer
of at least 20,000 vesicles, without any apparent depletion of the
reservoir from which it was supplied.
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Figure 6.
EGTA inhibited refilling of a reserve pool of
vesicles. A, Capacitance responses during a train of
emptying stimuli (20 msec depolarizations to 0 mV) delivered at a
frequency of 0.1 Hz. The top is from a control terminal,
and the bottom is from a terminal loaded with EGTA. The
figures below the responses indicate the stimulus number in the train.
B, Averaged results from the type of experiment shown in
A. The capacitance responses during the stimulus train
are plotted relative to the first response, which averaged 50 fF in
control terminals (n = 18; closed
symbols) and 33 fF in terminals loaded with EGTA
(n = 16; open symbols). In control
terminals, the second response was 45% of the first, and subsequent
responses were not significantly different. In terminals loaded with
EGTA, the response to the emptying stimulus gradually declined to zero.
C, The accumulated amount of exocytosis averaged from
the type of experiment shown in A. In control terminals,
refilling of the RRP occurred at a constant average rate, as indicated
by the solid line. In terminals loaded with EGTA, the
maximum number of vesicles that could be transferred to the RRP was 2.7 times its initial size, as indicated by the dashed
line.
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When terminals were loaded with EGTA, the rate of refilling was not
affected at first, because the second response was still ~50% of the
first (Fig. 6A,B). Thus, EGTA did
not directly affect the transfer of vesicles to the RRP after a
brief stimulus. Over the long term, however, the rate of refilling
gradually declined during a train, until no more vesicles could be
transferred to the RRP and the capacitance response was abolished (Fig.
6A,B). The cumulative response
during these stimulus trains is plotted as the open
circles in Figure 6C. In the presence of EGTA,
the maximum number of vesicles transferred to the RRP averaged 2.7 times its initial size, or ~3500 ± 400 vesicles (the amplitude of the first capacitance response in this set of EGTA-loaded terminals was 33.4 ± 3.5 fF; n = 16). In other words, only
~3500 vesicles could be rapidly transferred from a reserve pool to
the RRP in a terminal loaded with EGTA. A simple explanation for this
result is that refilling of the RRP occurs from a reserve pool, which has an initial capacity of ~3500 vesicles, and EGTA blocks the transfer of vesicles to this pool, causing it to gradually empty (see Discussion).
If EGTA inhibits refilling of the reserve pool by buffering
Ca2+ transients, then it should be possible to
overcome this effect by introducing a larger Ca2+
load into the terminal. A demonstration that this is the case is shown
in Figure 7. In this experiment, both the
RRP and reserve pool were completely exhausted by applying a train of
emptying stimuli, as in Figure 6. After a 150 sec period of rest,
application of the emptying stimulus elicited a response of only 2 fF,
confirming that the RRP was refilled very slowly at resting levels of
Ca2+. A larger number of vesicles were released by a
500 msec depolarization, and refilling of the RRP was stimulated, as
demonstrated by the much larger response to the emptying stimulus
applied after a delay of 10 sec. Similar behavior was observed in four
other terminals. These results provide further support for the idea
that Ca2+ stimulates the transfer of vesicles to the
reserve pool and then to the RRP.
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Figure 7.
The effects of EGTA were partially overcome by
introduction of a large Ca2+ load. Capacitance
response from a terminal loaded with EGTA, after complete depletion of
both the RRP and reserve pool by a train of stimuli. Before this
stimulation episode, the terminal was at rest for 150 sec. The first
emptying stimulus elicited a negligible response. A 500 msec
depolarization stimulated exocytosis and caused the RRP to refill, as
demonstrated by the much larger response to the second emptying
stimulus.
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DISCUSSION |
Two basic observations indicate that Ca2+
stimulates refilling of the RRP in the synaptic terminal of
depolarizing bipolar cells. First, accelerated refilling of the RRP was
prolonged by introducing a larger Ca2+ load into the
terminal (Fig. 3C), and this effect could be inhibited by
buffering the rise in cytoplasmic [Ca2+] with EGTA
(Fig. 5). Second, EGTA limited the number of vesicles that could be
released by repetitive brief stimuli (Fig. 6), and this effect could be
counteracted by introducing a larger Ca2+ load into
the terminal (Fig. 7).
Vesicle pools in the bipolar cell terminal
After brief depolarizations, EGTA blocked the
Ca2+-dependent transfer of all but a small fraction
of available vesicles, although it had no immediate effect on refilling
of the RRP (Fig. 6). The differential action of EGTA allows us to
provide a functional definition for two pools of vesicles involved in
refilling of the RRP: a reserve pool of ~3500 vesicles that refills
the RRP over the short term, and a much larger reservoir that maintains the supply of vesicles over the long term. Figure
8 shows a simple model of vesicle
transfer in the bipolar cell terminal that seeks to integrate these
results with previous anatomical and physiological work. Features of
this model are discussed below.
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Figure 8.
Vesicle pools in the synaptic terminal of
depolarizing bipolar cells. Evidence for this model is summarized in
Discussion. The numbers in
parentheses indicate the rates of the vesicle
transport when the Ca2+ current is maximally
activated.
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It is possible to define at least three pools of vesicles in the
bipolar cell terminal. When the Ca2+ current is
activated maximally, the most rapid phase of exocytosis involves the
release of 1200-1800 vesicles within ~10 msec (Heidelberger et al.,
1994; Mennerick and Matthews, 1996; Neves and Lagnado, 1999). A similar
number of vesicles are docked to the plasma membrane beneath the
ribbon, suggesting that these correspond to the RRP (von Gersdorff et
al., 1996). The second phase of exocytosis involves the release of
~4400 vesicles over 1 sec (Neves and Lagnado, 1999). Two pieces of
evidence indicate that these vesicles constitute the reserve pool that
is used to refill the RRP after closure of Ca2+
channels. First, the reserve pool contained a similar number of
vesicles, ~3500 (Fig. 6). Second, EGTA inhibited the second phase of
exocytosis and also reduced refilling of the RRP stimulated by residual
Ca2+ (Fig. 5). The anatomy of the bipolar cell
terminal immediately suggests that the reserve pool of vesicles is
attached to the ribbon behind the plasma membrane, and this idea is
supported by EM measurements indicating that there are ~4800 vesicles
attached to the ribbons (von Gersdorff et al., 1996). The third
component of exocytosis observed during maintained depolarization
occurs at a rate of ~1000 vesicles/sec but does not fatigue for
periods of many tens of seconds (Lagnado et al., 1996; Rouze and
Schwartz, 1998; Neves and Lagnado, 1999). Measurements made with
FM1-43 indicate that this continuous process of exocytosis is
supported by a reservoir of ~750,000 vesicles that is efficiently
replenished by endocytosis (Lagnado et al., 1996). EM measurements
indicate that there are a similar number of vesicles in the cytoplasm, free of the ribbons (von Gersdorff et al., 1996).
In the model shown in Figure 8, the rate constants
K1 and K2 are both
increased by Ca2+, but during brief periods of
Ca2+ influx, EGTA is more effective at preventing
the increase in K2. When the
Ca2+ current is activated maximally, the second
phase of exocytosis occurs at a rate of ~4500 vesicles/sec (Neves and
Lagnado, 1999), so the maximum rate constant for the transfer of
vesicles from the reserve pool to the RRP,
K1, is ~1.3/sec (assuming the capacity of the reserve pool is 3500 vesicles). As might be expected, the rate
of refilling that we measured after closure of Ca2+
channels was somewhat slower, occurring with a rate constant of
~0.5/sec in the few hundred milliseconds after a 20 msec
depolarization (Fig. 2B). It appears that
Ca2+ could modulate the rate of refilling over a
range of ~40-fold, because the basal value of
K1 at resting levels of Ca2+
was ~0.03/sec (Fig. 2B). After the first two pools
of vesicles have been exhausted, continuous exocytosis occurs at a rate
of ~1000 vesicles/sec (Neves and Lagnado, 1999), indicating that the
maximum rate constant for the transfer of vesicles from the reservoir
to the reserve pool, K2, is ~1.3 × 10 3/sec.
Two actions of Ca2+
The differential effect of EGTA on vesicle transfer to the RRP and
reserve pool (Figs. 6, 7) might be explained if the
Ca2+-binding molecule regulating
K2 was further from Ca2+
channels than the Ca2+-binding molecule regulating
K1. During brief periods of
Ca2+ influx, the molecule regulating
K2 would be subject to smaller rises in
[Ca2+] that are more effectively buffered by EGTA
(Neher, 1998).
The EGTA concentrations we introduced into the terminals were
sufficient to strongly buffer Ca2+ at the
Ca2+-dependent conductances in the plasma membrane
(Fig. 4). Two factors might explain why they were not sufficient to
inhibit refilling of the RRP after a brief stimulus (Fig. 6). First,
the Ca2+-sensor for refilling may be closer to
Ca2+ channels than the
Ca2+-dependent conductances, so that it senses a
larger rise in [Ca2+] while
Ca2+ channels are open. Second, the
Ca2+-sensor for refilling may have a higher affinity
for Ca2+ than the Ca2+-dependent
conductances. The latter idea is supported by the observation that
residual Ca2+ stimulated refilling for ~10 sec
after a 500 msec depolarization (Fig. 3C), whereas the
Ca2+-activated tail current had decayed completely
after 2 sec (Fig. 4). Neves and Lagnado (1999) used furaptra, a
Ca2+-indicator of relatively low affinity, to
measure the cytoplasmic [Ca2+] in the bipolar cell
terminal. Strongly activating the Ca2+ current for 1 sec caused the spatially averaged [Ca2+] to reach
~25 µM. After repolarization, the
[Ca2+] fell to undetectably low levels with a time
constant of ~0.7 sec, causing the Ca2+-activated
tail current to decay similarly (Neves and Lagnado, 1999, their
Fig. 10). Together, these measurements indicate that residual
Ca2+ stimulated the transfer of vesicles at
concentrations that did not activate Ca2+-dependent
conductances at the plasma membrane and that were close to the limit of
detection with furaptra. In the experiments of Neves and Lagnado
(1999), it was quite possible to detect Ca2+ levels
of 1.5 µM with furaptra, so this figure provides an upper limit for the [Ca2+] sufficient to stimulate the
transfer of vesicles to the RRP.
What is the relationship between transient and
continuous exocytosis?
Lagnado et al. (1996) and Rouze and Schwartz (1998) have
demonstrated that continuous exocytosis at the bipolar cell synapse can
be driven by Ca2+ concentrations of the order of 1 µM. Refilling of the RRP probably occurs at similar low
levels of Ca2+, suggesting that continuous
exocytosis might occur by the trafficking of vesicles from the bulk
cytoplasm, through the reserve pool, to the RRP (Fig. 8). The initial
rapid phase of exocytosis requires much higher levels of
Ca2+; it is half-maximal at ~200 µM
Ca2+ (Heidelberger et al., 1994). So, if the model
presented in Figure 8 is correct, the final
Ca2+-dependent step during the first phase of
release is probably different from that controlling the later phases.
This might occur if vesicles docked at release sites could undergo
exocytosis from one of two states: state 1 in which fusion requires
Ca2+ to bind to a sensor of high affinity (or does
not require Ca2+ at all), and state 2 in which
fusion requires Ca2+ to bind a sensor of low
affinity. If all docked vesicles were in state 2 after a period of
rest, the first phase of exocytosis measured experimentally would have
a requirement for high levels of Ca2+. But if
vesicles arrived at release sites in state 1, the continuous component
of exocytosis would occur at lower Ca2+
concentrations. The idea of exocytosis from two different states is not
new. In other neurons in which the kinetics of exocytosis have been
characterized, the fast component of exocytosis synchronous with the
action potential is followed by a slower asynchronous component that
appears to be driven by residual Ca2+ (Zucker, 1989;
Delaney and Tank, 1994; Goda and Stevens, 1994). Geppert et al. (1994)
found that the synchronous component of exocytosis was blocked in
hippocampal neurons cultured from mice in which synaptotagmin I had
been knocked out, although the asynchronous component appeared intact.
At the moment, we do not have any information about the proteins that
are involved in these vesicle movements, although Heidelberger (1998)
has demonstrated that the hydrolysis of ATP is required for the
refilling of the RRP in the terminal of bipolar cells.
A comparison with other synapses
The ribbon synapses of hair cells and photoreceptors also support
continuous exocytosis at rates that indicate that release sites are
efficiently replenished (Parsons et al., 1994; Rieke and Schwartz,
1996). Recently, Lenzi et al. (1999) used electron tomography to
investigate the structure of ribbon synapses in saccular hair cells and
found that it was not possible to reduce the number of
ribbon-associated vesicles by continuous depolarization, leading to the
conclusion that the transfer of vesicles to the ribbon was not
rate-limiting during continuous exocytosis. It may therefore be that
the Ca2+-dependent transport of vesicles through
ribbons also occurs in hair cells.
In neurons that fire action potentials, residual
Ca2+ accelerates recovery from synaptic depression
after repetitive high-frequency stimulation (Dittman and Regehr, 1998;
Stevens and Wesseling, 1998; Wang and Kaczmarek, 1998). This effect can
be completely blocked by EGTA, which, as pointed out by Wang and
Kaczmarek (1998), suggests that the reserve pool of vesicles is some
way from the RRP. In contrast, EGTA did not inhibit the transfer of
vesicles to the RRP at the ribbon synapse of bipolar cells (Figs. 6,
7). This functional difference might reflect the introduction of lower concentrations of EGTA into the bipolar cell terminal, or it might be
caused by the different anatomical arrangement of the reserve pool of
vesicles in "classical" synapses and ribbon synapses. At a typical
synapse in the CNS, there may be 100-200 vesicles per active zone, of
which 10-20 may be docked, while the remainder form a cluster
extending ~500 nm from the presynaptic membrane in which they are
linked to each other and the cytoskeleton (Burns and Augustine, 1995).
At ribbon synapses, the reserve pool of vesicles appears to be more
organized (Gray and Pease, 1971; Jacobs and Hudspeth, 1990;
Rao-Mirotznik et al., 1995; Lenzi et al., 1999). In goldfish
bipolar cells, vesicles dock to the plasma membrane in two rows of 11, on either side of the ribbon, and each face of the ribbon has
approximately five rows of 11 vesicles attached by short filaments (von
Gersdorff et al., 1996). The ribbon is only one vesicle diameter from
the presynaptic membrane, and extends ~150 nm. This arrangement may
hold vesicles of the reserve pool closer to the RRP and
Ca2+ channels than is the case at other synapses.
| |
FOOTNOTES |
Received March 19, 1999; revised May 13, 1999; accepted May 14, 1999.
A.G. was supported by the European Community. We thank Guilherme Neves
for helpful discussions.
Correspondence should be addressed to Leon Lagnado, Medical Research
Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
| |
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TOP
ABSTRACT
INTRODUCTION
