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The Journal of Neuroscience, November 1, 1998, 18(21):8614-8624
Continuous and Transient Vesicle Cycling at a Ribbon Synapse
Ned C.
Rouze and
Eric A.
Schwartz
Department of Pharmacological and Physiological Sciences, The
University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
Optical methods were used to study the Ca2+
dependence of vesicle cycling in bipolar cells isolated from goldfish
retinas. Uniformly raising the Ca2+ concentration to
between 0.8 and 20 µM produced a continuous vesicle cycle
of balanced exocytosis and endocytosis with a maximum rate equivalent
to the turnover of the entire surface membrane of a terminal every 2 min (or ~900 vesicles sec
1). Increasing the
Ca2+ concentration above 20 µM
inhibited continuous vesicle cycling. In contrast, influx of
Ca2+ through voltage-gated channels produced a
transient burst of exocytosis that increased the surface area of a
terminal by a maximum of 12% (equivalent to the addition of 13,000 vesicles). Endocytosis was delayed until after Ca2+
influx stopped and the average Ca2+ concentration in
the terminal declined. Hence, a single terminal has mechanisms for both
continuous and transient vesicle cycling.
Key words:
exocytosis; endocytosis; vesicle cycling; synaptic
vesicle; bipolar cell; retina
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INTRODUCTION |
Ca2+ influx has
long been known to be an effective trigger for regulated exocytosis
(Katz and Miledi, 1967
). The coupling between Ca2+
and fusion has been studied extensively in neuroendocrine cells. Most
studies have demonstrated that Ca2+ concentrations
above a few micromolar are sufficient to trigger exocytosis (see, for
example, Jankowski et al., 1992). Less is known about vesicle cycling
in presynaptic nerve endings, which are usually small and consequently
have been experimentally inaccessible. Only recently have the
Ca2+ dependence and kinetics of vesicle cycling been
studied in large synaptic terminals containing ribbon synapses. A
change in membrane capacitance has been used to monitor vesicle fusion
in retinal bipolar and photoreceptor cells. Surprisingly, two different
views have emerged. Goldfish bipolar cells (Heidelberger et al., 1994; von Gersdorff and Matthews, 1994a) transiently released vesicles when
the Ca2+ concentration exceeded 20 µM.
In contrast, salamander rod photoreceptors (Rieke and Schwartz, 1996)
sustained a continuous vesicle cycle when the Ca2+
concentration rose above 1 µM. A similar conclusion was
reached after staining goldfish bipolar cells with a fluorescent dye
and following the fate of tagged vesicles (Lagnado et al., 1996)
(however, see the ). Thus, the experiments paint pictures of
synaptic vesicle cycling that differ in two important respects. First, the Ca2+ concentrations required for release differ
>20-fold. Second, the entire process of release and vesicle resupply
is either transient or continuous.
We have now reconciled these divergent views. Vesicle cycling was
observed after bipolar cell membranes were stained with FM1-43 or
FM4-64. Relatively low Ca2+ concentrations
(0.8-20 µM) stimulated exocytosis and sustained a
continuous vesicle cycle with maximal rates sufficient to replace the
entire surface of a terminal every 2 min. Higher concentrations (>20
µM) inhibited continuous cycling. Large increases in the local concentration near voltage-gated channels produced a transient burst of exocytosis. A rapid wave of compensatory endocytosis was
initiated after Ca2+ influx stopped and the average
Ca2+ concentration declined.
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MATERIALS AND METHODS |
Cell isolation. Goldfish (Carassius
auratus) were killed by severing the cervical spinal cord and
pithing the brain, following guidelines of the University of Chicago
Institutional Animal Care and Use Committee. The procedure for
dissociating a retina and maintaining solitary cells was similar to
that described by Bader et al. (1982). Solitary bipolar cells
were identified by their characteristic morphology (Fig.
1).
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Figure 1.
Hoffman modulation micrograph of a solitary
bipolar cell. The squares indicate regions of interest
that were saved during an experiment. The circles
indicate areas in which the total fluorescence was measured (see text).
Scale bar, 10 µm.
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In our initial experiments, dissociated cells were plated into dishes
whose bottom was a glass coverslip. A styryl dye stained cells and
adsorbed to the glass coverslip. Unbinding of the dye from the
coverslip was very slow. Quantitative measurement of vesicle cycling
was difficult when images were contaminated by a large background
signal. Fortunately, unbinding of the dye was much faster when cells
were plated onto a coverslip that had been coated with a thin layer of
silicone plastic (Sylgard 184; Dow Corning, Midland, MI). Cells adhered
to the silicone plastic if it was first covered with 0.25 mg
ml1 concanavalin A in 1 M NaCl for 20 min,
rinsed with distilled water, and air dried.
The extracellular saline contained (in mM): NaCl, 120; KCl,
2.5; CaCl2, 0.2; MgCl2, 1;
Na-pyruvate, 2; Na-lactate, 2; glucose, 10; and HEPES, 10; the pH was
adjusted to 7.4 with NaOH, and the saline was supplemented with
vitamins (1:100) and amino acids (1:100) formulated for Eagle's
minimum essential medium (Life Technologies, Gaithersburg, MD).
The osmolality was ~260 mOsm kg1. Cells were
loaded with fura-2 by incubating in normal extracellular saline
supplemented with 2 µM fura-2 AM plus 16 µM pluronic acid for 1 hr. For some experiments (see Fig.
4), cells were loaded with mag-fura-5 and fluo-3 by incubating
in 1 µM mag-fura-5 AM, 1 µM fluo-3 AM, plus
16 µM pluronic acid for 1 hr. Experiments were performed
at 20-23°C.
Optical system and image analysis. Light from a 75 W Xe
short arc lamp was used to excite dye fluorescence. Excitation
wavelength was selected with a filter changer (Lambda 10-2; Sutter
Instruments, Novato, CA) containing narrow-band (15 nm) interference
filters with transmission maxima at 331, 380, and 485 nm. The filtered light was reflected into a microscope with a 510 nm dichroic mirror and
was imaged by an objective (Fluar 40x/1.3; Carl Zeiss) to a
150-µm-diameter spot centered in the field of view. The maximum intensity within this spot was 1.4 W m2 at
331 nm, 5.6 W m2 at 380 nm, and 180 W
m2 at 485 nm. Fluorescent light collected by the
objective passed through a second filter changer containing a 520-580
nm bandpass filter or an RG630 Schott glass long-pass filter before
being imaged onto a CCD camera (model AT200; Photometrics, Tucson, AZ). Each pixel imaged a 0.16 µm × 0.16 µm region in the plane of
focus. The operation of shutters and filter wheels was controlled by a
computer. Because of overlap in both their excitation and emission spectra, fluo-3 and FM1-43 were not used together in the same experiment. Control experiments demonstrated that the combinations of
wavelengths, light intensities, and dye concentrations actually used in
the experiments allowed the interleaved measurement of Ca2+ concentration and either FM1-43 or FM4-64
fluorescence without crossover between the signals. In some experiments
we used both FM1-43 and FM4-64. In these experiments, we corrected
for the crossover of light emitted by FM1-43 in the FM4-64 detection
band (see Fig. 3).
At the beginning of each experiment, a Hoffman interference image of
the entire cell was recorded (Fig. 1), and two regions of interest
~20 µm square were selected (Fig. 1, outlined by
squares). One region included the synaptic terminal; the
other region included a portion of the soma. Fluorescent images of the
two regions produced by 100 msec exposures were collected for the
required filter combinations and saved for later analysis.
Normally, several movies were made after an experiment was completed.
For example, the measurement of Ca2+ concentration
and FM1-43 fluorescence required six movies. One movie (produced by
485 nm exciting light) imaged FM1-43 fluorescence in the terminal.
Second (produced by 331 nm light) and third (produced by 380 nm light)
movies imaged fura-2 fluorescence in the terminal and were used to
calculate the average, cytoplasmic Ca2+
concentration. A similar set of three movies imaged FM1-43 and fura-2
fluorescence in the soma. The ability to identify potential problems
was greatly facilitated by recording a movie of fluorescent images
instead of integrating all of the light in a microscope field with a
photomultiplier tube. Preliminary experiments demonstrated that bright
331 and 380 nm lights adversely affected the morphology of stained
cells. Consequently, light intensity and repetition rate for fura-2 and
mag-fura-5 measurements were minimized. We rejected the rare
experiments in which a cell significantly changed shape.
The total fluorescence in the terminal was measured by summing the
intensities in pixels within a circular area slightly larger than the
terminal (Fig. 1, circle designated t). The
fluorescence in the soma was measured by summing the intensities of
pixels within a circular subregion ~8 µm in diameter over a portion
of the soma (Fig. 1, circle designated s).
Background fluorescence of FM1-43 and FM4-64, measured in a region
~5 µm distant from a cell, was subtracted from each pixel. For the
calcium indicator dyes, background fluorescence measured away from the
cell was negligible.
We assumed that the fluorescence of dye molecules was unchanged during
endocytosis. Results (see Figs. 5, 10) indicate that vesicles quickly
diffused throughout a terminal. Because vesicles appeared to be
uniformly distributed, we were able to use the fluorescence in an
optical section to calculate the total amount of endocytosed membrane
distributed throughout a terminal. A microscope objective collects
light from an image depth of 4
/(NA)2, where is the
wavelength of light and NA is the numerical aperture of the objective.
Hence the thickness of an optical section was 1.4-1.6 µm for
experiments that imaged FM1-43 or FM4-64. Because these values are
much less than the 10 µm diameter of a terminal, the geometry can be
described as a plane intersecting a sphere. If
p is the ratio of the area of a circular disk
to the length of its perimeter and s is the
ratio of the volume of a sphere to its surface area, then we can
measure p and estimate
s after multiplying by a geometrical
correction factor, 
= s/p = 2/3. Because
bipolar cell somata are not spherical, a similar procedure is not
appropriate. Instead, results from somata are in arbitrary units and
have been scaled to facilitate a qualitative comparison.
Calcium measurement. The fluorescence of fura-2 or
mag-fura-5 was excited by light of 331 and 380 nm. Emission was
detected between 520 and 580 nm. Intracellular Ca2+
concentration was calculated from the relation (Grynkiewicz et al.,
1985): [Ca2+] = k
(R 
Rmin)/(Rmax R), where R is the ratio of the fluorescence intensity excited at 331 nm divided by the intensity excited at 380 nm,
Rmax is the ratio produced by a saturating
Ca2+ concentration, Rmin is
the ratio produced by a minimum concentration, is the ratio of
emission intensity stimulated at 380 nm in the absence of
Ca2+ to that in the presence of an excess of
Ca2+, and k is an apparent affinity
constant. Affinity constants were determined by superfusing 20 cells
with either 10 µM ionomycin or 10 µM
A23187 and a series of Ca2+ concentrations
set with Ca2+ buffers (see below). Each
concentration was superfused until a steady state was achieved. The
affinity constant was 237 nM for fura-2 and 10 µM for mag-fura-5. Rmin,
Rmax, and were measured for each
cell. Rmin was estimated at the beginning of
each experiment while a cell was superfused with a low
Ca2+ concentration; Rmax and
were measured by superfusing at the end of each experiment with
saline containing 10 µM ionomycin, 4 mM
Ca2+, 40 mM K+, and 1 µM BayK8466.
The 485 nm light excited fluo-3. Emission was detected between 520 and
580 nm. The Ca2+ concentration was calculated from
the relation (Grynkiewicz et al., 1985): [Ca2+] = k(F Fmin)/(Fmax F), where F is the fluorescence intensity, Fmin is produced by a minimum
Ca2+ concentration, Fmax is
produced by a saturating Ca2+ concentration, and
k is the apparent affinity constant. Minimum and maximum
fluorescence were measured as described above for the ratiometric dyes.
The affinity constant for fluo-3, also measured as described above, was
0.9 µM (see also Rieke and Schwartz, 1996).
Solutions and control of Ca2+
concentration. During an experiment, a cell was continuously
superfused with solutions ejected from a four-barrelled pipette. Each
barrel ended in a ~50 µM square opening. Each barrel
was connected to a reservoir and pressure head through a solenoid
valve. Solution changes were made by simultaneously opening and closing
valves. Control experiments with fluorescent dyes demonstrated that
solution changes were 95% complete in <100 msec. When an experiment
required five different solutions, an additional single-barrelled
pipette was used.
The superfused solutions were similar to the extracellular saline
described above. Ca2+ concentrations were set with
Ca2+ buffers; 1 mM
bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid
(BAPTA) was used for free Ca2+ concentrations
between 0.1 and 2 µM, and 1 mM
N-(2-acetamido)-2-iminodiacetic acid (ADA) was used
for concentrations between 10 and 200 µM. Concentrations
above 200 µM were unbuffered. Salines containing ionomycin and ADA lacked Mg2+. A high
Ca2+ and K+ saline contained (in
mM): NaCl, 82.5; KCl, 40; CaCl2, 4; and MgCl2, 1; pyruvate, lactate, glucose, and HEPES
levels were as in the normal saline. Experiments were conducted on
only one cell in a dish.
The concentration of BAPTA or ADA in stock solutions used to make
Ca2+ buffers was determined by titrating with 1.0N
CaCl2 (BDH Laboratory Supplies) and measuring the free
Ca2+ concentration with a Ca2+
electrode. After the concentration of each stock solution was known,
salines with a specific free Ca2+ concentration were
made according to recipes calculated with the computer program MAXC
v1.70 (Bers et al., 1994).
Kinetics of membrane staining. The kinetics of membrane
staining depended on the dye concentration. Our procedure for measuring the kinetics is explained in Figure 2. An
image of a stained terminal is shown in Figure 2A.
The intensity of pixels along the radial solid line is
plotted in Figure 2B. The maximum intensity occurs at
the surface membrane (Fig. 2B, arrow). To
increase the accuracy with which the intensity of membrane fluorescence
is measured, we rotated the inclination of the radial line and
integrated the membrane signal around the terminal circumference
excluding the segment containing the axon (as indicated by the
dotted line in Fig. 2A). The
intensity of membrane fluorescence was measured in a sequence of images
and plotted as a function of time (Fig. 2C). The cell was
exposed to two concentrations of FM1-43, first 5 and then 20 µM. When the dye was applied, the membrane stained with a
nonexponential time course. When the dye was removed, the membrane
destained with an approximately exponential time course.
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Figure 2.
Kinetics of membrane staining depended on dye
concentration. A, Image of a terminal stained with 20 µM FM1-43. B, The intensity of pixels
along the solid line in A plotted as a
function of radial distance. The arrow indicates the
location of the surface membrane. C, The intensity in
the surface membrane plotted as a function of time. The cell was
superfused with two concentrations of FM1-43 as indicated by the
horizontal bars. The peak intensity along a radial spoke
(see B) was integrated around the circumference, except
for the region from which the axon arose (as indicated by the
dotted line in A). D,
Intensity of membrane staining as a function of dye concentration.
E, The t1/2 for the onset of
staining as a function of dye concentration. F, Apparent
exponential time constant for destaining as a function of dye
concentration.
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The procedure illustrated in Figure 2, A-C, was used to
obtain the data in Figure 2, D-F. In each experiment, a
cell was exposed to two dye concentrations. To compare experiments, we
normalized the steady state fluorescence to the intensity produced by
20 µM FM1-43. The intensity of membrane staining was
proportional to dye concentration (Fig. 2D). The time
to the half-maximum amplitude t1/2 decreased
approximately in inverse proportion to the concentration (Fig.
2E). Destaining was approximately described by a
single exponential with a time constant that also depended on
concentration (Fig. 2F).
A linear relation between dye concentration and fluorescence intensity
indicates that the number of dye molecules in the membrane is not
limited by a small and fixed number of binding sites. A simple model
would describe the movement of dye into the membrane as the
partitioning between an aqueous and lipid phase accomplished in a
single kinetic step. However, in this case, staining and destaining
would have exponential time courses that are independent of
concentration. This was not the case. The nonexponential time course
for staining and a change in kinetics with dye concentration indicate
that dye molecules equilibrate in a series of states.
The time course of membrane staining and destaining will obviously
limit the time resolution for measurements of vesicle cycling. A
concentration of 20 µM provided a reasonable resolution
for membrane staining. However, to observe membrane destaining with a
resolution finer than several tens of seconds, we needed an additional
technique.
Resonance energy transfer. We have discovered an easy method
to increase the time resolution of membrane destaining. FM4-64 rapidly
quenched the fluorescence of FM1-43. An example is shown in Figure
3A. A bipolar cell was
superfused first with FM4-64, next with FM1-43, and finally with both
dyes together. The emission intensities in the two detection channels
are superimposed in Figure 3A (upper traces).
Crossover from FM1-43 into the FM4-64 detection channel (Fig.
3A, thin upper trace) was measured, and the
corrected trace is plotted below. The essential observation came when
the two dyes were applied together. Now, the fluorescence of FM1-43
was strongly quenched (Fig. 3A, asterisk). Dye
intensity was reduced 94%. In addition, there was an apparent 14%
increase in FM4-64 fluorescence. This behavior suggested the procedure in Figure 3B. First, a cell was superfused with a pulse of
FM1-43. When the dye was removed, fluorescence in the FM1-43 channel
(Fig. 3B, thick trace) declined with a time
constant of 6.7 sec. Next, a pulse of FM1-43 was immediately followed
by a pulse of FM4-64. Now, FM1-43 fluorescence was quickly quenched.
The fluorescence in the surface membrane disappeared in <1.4 sec.
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Figure 3.
FM4-64 quenched FM1-43 fluorescence. Bipolar
cells were superfused with 20 µM FM1-43, 20 µM FM4-64, or both dyes together as indicated
(superfusions in this and the following figures indicated by
horizontal bars). A, The
upper pair of superimposed traces is the
fluorescence measured in the two detection channels. The thick
trace is the fluorescence detected between 520 and 580 nm; the
thin trace is the fluorescence detected with a 630 nm
long-pass filter. When the two dyes are given together, the
fluorescence of FM1-43 (asterisk) was less than that
when FM1-43 was delivered alone. FM4-64 fluorescence, estimated by
subtracting the crossover of FM1-43 fluorescence into the 630 nm
channel, is plotted in the lower trace. The small
glitches in the corrected trace (arrowhead) are produced
because two spectral components of FM1-43 fluorescence leave the
membrane at different rates. The time constants for FM1-43 destaining
are 6.7 sec (thick trace) and 3.5 sec (thin
trace). B, FM4-64 can be used to improve the
resolution of membrane destaining. The thick trace is
the intensity for emission between 520 and 580 nm. When FM1-43 was
delivered alone, the time constant of membrane destaining was 6.7 sec.
When FM1-43 was followed by a pulse of FM4-64, fluorescence of
FM1-43 in the surface membrane disappeared in <1.4 sec.
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Sources and abbreviations. Ionomycin, FM1-43, FM4-64,
fluo-3 AM, fura-2 AM, and mag-fura-5 AM were obtained from Molecular Probes (Eugene, OR). Papain was purchased from Worthington (Freehold, NJ). All other chemicals were obtained from Sigma (St. Louis, MO)..
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RESULTS |
FM1-43 is a lipophilic dye that partitions into cell membranes.
The dye has little fluorescence in aqueous solution and is much more
fluorescent in a membrane environment. Positive charges are believed to
prevent dye molecules from flipping between the outer and the inner
membrane leaflet. Consequently, extracellular FM1-43 has been used to
tag the outer leaflet of the surface membrane and then to visualize the
bits that are pulled into the cytoplasm by endocytosis (Betz et al.,
1992, 1996). However, as described in the , the ability of dye
to cross the cell membrane can complicate its use as a membrane tag. We
designed experiments to minimize this problem.
Ca2+ dependence of continuous
vesicle cycling
To explore the relation between Ca2+
concentration and vesicle cycling, we adopted two strategies to produce
a nearly uniform change in intracellular Ca2+
concentration. The first procedure relied on a Ca2+
ionophore, ionomycin, and the entry of Ca2+ from the
extracellular saline. The second procedure relied on a proton
ionophore, monensin, and the release of Ca2+ from
intracellular, acidic organelles. Both procedures were able to raise
the cytoplasmic Ca2+ concentration.
For the first procedure, cells were superfused with ionomycin (10 µM) and a Ca2+ buffer. Changing the
free Ca2+ concentration in the extracellular saline
was followed by a change in the intracellular concentration. During a
typical 1-3 min application, the intracellular concentration reached
3-25% of the extracellular concentration. An example is illustrated
in Figure 4. The cell was first
superfused with ionomycin and 100 nM
Ca2+. The addition of FM4-64 (Vida and Emr, 1995)
stained the surface membrane and produced a fluorescence change
(labeled s). Next, when the extracellular
Ca2+ concentration was increased to 50 µM, the intracellular concentration followed with a rise
to ~1.7 µM. At the same time, the fluorescence of
FM4-64 increased (labeled vx). We believe
that the increase in fluorescence is produced as exocytosis adds
vesicle membrane to the surface membrane. The rate of continuous
exocytosis Cx was calculated as
Cx = vx/s 
t, where
is a geometrical constant (see Materials and Methods) and
t is the time interval in which vx was measured.
Cx has units of membrane equivalents
(abbreviated MEq) per unit time. Raising the intracellular
concentration from 0.09 to 1.7 µM increased
Cx from 0.01 to 0.33 MEq
min1. At the same time, there was little change in
the fluorescence imaged from the soma.
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Figure 4.
Continuous exocytosis is stimulated by a low
Ca2+ concentration and inhibited by a high
Ca2+ concentration. Cells were continuously
superfused with salines containing 10 µM ionomycin. The
fluorescence intensity produced by 20 µM FM4-64 is
plotted by the upper set of traces; the
intracellular Ca2+ concentration is plotted by the
lower set of traces. Thick
traces in this and the following figures were measured in the
terminal; thin traces were measured in the soma.
Continuous exocytosis was measured as Cx = vx/s t.
Salines containing 100 nM, 50 µM, and 600 µM extracellular Ca2+ were applied as
indicated by the dashed vertical lines.
Ca2+ concentrations below 10 µM were
reported by fluo-3; concentrations above 10 µM were
reported by mag-fura-5. Increasing the intracellular
Ca2+ from 0.09 to 1.7 µM increased
Cx to 0.33 MEq min1.
A further rise in the intracellular Ca2+ to between
50 and 140 µM reduced Cx to
0.06 MEq min1.
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We expected the rate of vesicle cycling to increase with the
Ca2+ concentration. Surprisingly, when the
experiment was continued by increasing the extracellular
Ca2+ concentration to 600 µM, the
intracellular concentration rose to between 50 and 140 µM, and the rate of FM4-64 fluorescence change actually
declined. In this experiment Cx was
reduced from 0.33 to 0.06 MEq min1. A similar
result was observed in 21 experiments.
Images acquired during an experiment provide information about the fate
of membrane added to the surface by exocytosis. The following
experiment used the ability of FM4-64 to quench the fluorescence of
FM1-43 in the surface membrane (see Materials and Methods; Fig. 3).
Afterward, we quickly observed the distribution of FM1-43-labeled
vesicles in the cytoplasm (Fig. 5). Image
a was recorded 24 sec after the terminal was stained with
FM1-43. At this moment the Ca2+ concentration was
~1 µM. The profile of the terminal is nearly round and
smooth. Image b was taken 68 sec later, when the
intracellular Ca2+ concentration had increased to 4 µM. Small patches of fluorescence are located immediately
beneath the surface membrane, the surface contour itself is slightly
irregular with sites of local expansion or dilation, and the interior
of the terminal has a uniform weak fluorescence. Finally, image
c was taken 15 sec after the fluorescence in the surface
membrane was quenched so that only the fluorescence of FM1-43 in
endocytosed vesicles was detected. The fluorescence of FM1-43 was
almost uniformly distributed, indicating that endocytosed vesicles
quickly diffused throughout the cytoplasm.
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Figure 5.
Endocytosed vesicles quickly diffuse throughout
the terminal. Cells were superfused with 10 µM ionomycin
and Ca2+ buffers. a-c, Images
recorded at the times marked by the arrows are at the
right. Intracellular Ca2+
concentration in this and the following figures was reported by fura-2
fluorescence. The cell was superfused first with 100 nM
Ca2+, then with 20 µM FM1-43 in a
saline containing 100 µM Ca2+, and
finally with 20 µM FM4-64 in a saline containing 100 nM Ca2+. Scale bar, 10 µm.
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The rate of steady endocytosis was estimated by first raising the
intracellular Ca2+ concentration and then measuring
the amount of FM1-43 accumulated during a 12 sec test pulse (Fig.
6). The rate of continuous endocytosis Cn was calculated as
Cn = vn/s t, where
vn is defined in the figure. For the two
measurements in Figure 6, the Ca2+ concentration was
1.3 µM, and Cn was 0.33 and
0.22 MEq min1.
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Figure 6.
Continuous endocytosis is stimulated by a low
Ca2+ concentration. A bipolar cell was continuously
superfused with salines containing 10 µM ionomycin. The
timing bars indicate 12 sec applications of 20 µM FM1-43. The cell was superfused first with a saline
containing 100 nM Ca2+ and then with 30 µM Ca2+ as indicated. s
is the fluorescence intensity of the surface membrane;
vn is the fluorescence that remains
trapped in the cytoplasm and is attributed to endocytosis. The rate of
continuous endocytosis Cn was measured as
Cn = vn/s t. For
the two measurements, Cn was 0.33 and 0.22 MEq min1.
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Figures 4 and 6 provide two methods for measuring rates of continuous
vesicle cycling. A summary of 102 experiments is presented in Figure
7. Crosses were measured by
the procedure in Figure 4; closed symbols were
measured by the procedure in Figure 6. Vesicle cycling in the terminal
was not observed at Ca2+ concentrations below 0.8 µM. A high rate of cycling was sustained at
Ca2+ concentrations between 0.8 and 20 µM. Increasing the Ca2+ concentration
above 20 µM decreased the rate of cycling. The maximum
rate of vesicle cycling in Figure 7 was ~0.5 MEq
min1. Because the membrane surface area of a
terminal (with a diameter of 10 µm) is 110,000 times the surface area
of a 30 nm synaptic vesicle, this corresponds to a rate of 900 vesicles
sec1.
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Figure 7.
Rate of continuous vesicle cycling in bipolar cell
terminals plotted as a function of the intracellular
Ca2+ concentration. Circles are for
measurements of endocytosis from 60 cells permeabilized with ionomycin
as described in Figure 6. Triangles are for 18 unpermeabilized cells measured during the resting state by the same
procedure. Crosses are for measurements of exocytosis
from 24 cells measured by the procedure illustrated in Figure 4.
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Ionomycin allowed Ca2+ to enter the cytoplasm from
the extracellular saline. An alternative procedure was to raise
intracellular Ca2+ without a change in extracellular
Ca2+ concentration. For this purpose we used
monensin, an ionophore that disrupts the pH-dependent accumulation of
Ca2+ in intracellular organelles (Fasolato et al.,
1991). The subsequent release of Ca2+ raises
cytoplasmic Ca2+ concentration. The procedure is
illustrated in Figure 8. The cell was
continuously superfused with an extracellular saline containing 100 nM Ca2+ (and no ionomycin). When 2 µM monensin was added, the Ca2+
concentration in the terminal cytoplasm increased transiently to nearly
6 µM. This increase in Ca2+
concentration was not produced by an influx from the extracellular saline (where the Ca2+ concentration was only 100 nM) but was instead produced by the release of
Ca2+ from intracellular organelles. The rise in
Ca2+ concentration was accompanied by an increase in
FM4-64 fluorescence. A similar result was observed in eight
experiments.
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Figure 8.
The release of Ca2+ from
intracellular organelles raises the cytoplasmic Ca2+
concentration and stimulates exocytosis. The cell was continuously
superfused with an extracellular saline buffered to contain 100 nM free Ca2+. The lower timing
trace indicates the application of 20 µM FM4-64.
The upper timing trace indicates the addition of 2 µM monensin.
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Both the experiments with ionomycin and monensin demonstrate that a
rise in intraterminal Ca2+ concentration to between
0.8 and 20 µM produced a continuous cycle of vesicle
exocytosis and endocytosis. The maximum rate was sufficient to turn
over the surface membrane of a terminal every 2 min.
Transient vesicle cycling initiated by Ca2+
entry through voltage-gated channels
Normally, fusion is triggered when Ca2+ enters
through voltage-gated channels. Ca2+ first
accumulates at the cytoplasmic end of each open channel and then
diffuses into the cytoplasm. The result is many small domains of high
Ca2+ concentration that quickly dissolve. The
following experiments investigated vesicle cycling triggered by
depolarization and Ca2+ influx. In each experiment,
cells were initially superfused with a saline containing 100 nM Ca2+ (to minimize premature
Ca2+ entry during a control period). Exocytosis was
triggered by superfusion with a saline containing 4 mM
Ca2+ and 40 mM
K+.
Exocytosis induced by depolarization and Ca2+ influx
is illustrated in Figure 9. First,
FM4-64 stained the surface membrane (labeled s). Next, a 4 sec exposure to the high Ca2+ and
K+ saline depolarized, increased the average
Ca2+ concentration in the terminal, and produced a
step increase in FM4-64 fluorescence (labeled
vx). We assume that the abrupt increase in
the fluorescence of the terminal was proportional to the membrane added
to the surface by exocytosis. The amount of exocytosis
Tx was calculated as
Tx = vx/s. In Figure 9,
Tx was 0.13 and 0.08 MEq. In contrast,
depolarization did not increase fluorescence in the soma (Fig. 9,
thin trace). In 25 experiments
Tx varied from being barely detectable to
~0.13 MEq.
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Figure 9.
Exocytosis was induced by depolarization and
Ca2+ influx. A bipolar cell was superfused with an
extracellular saline containing 100 nM
Ca2+. The lower timing trace
indicates the application of 20 µM FM4-64. The
upper timing trace indicates 4 sec applications of a
high Ca2+ and K+ saline (4 mM Ca2+ and 40 mM
K+). vx is the
intensity increase produced by vesicle exocytosis. Transient exocytosis
Tx was measured as
Tx = vx/s. For the two
applications of high Ca2+ and K+
saline, Tx was 0.13 and 0.08 MEq.
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The previous measurements were confirmed by observing endocytosis.
Cells were first superfused with a saline containing 100 nM
Ca2+ to minimize Ca2+ entry and
to prevent Ca2+-triggered vesicle cycling. An 18 sec
exposure to FM1-43 stained the surface membrane of the terminal (Fig.
10, thick upper trace). As
expected, a 6 sec exposure to high Ca2+ and
K+ saline produced a Ca2+ influx
and raised the average cytoplasmic Ca2+
concentration (thick lower trace). After FM1-43 was removed
from the extracellular saline, the surface membrane destained, and fluorescence remained trapped in the cytoplasm of the terminal (labeled
vn). The amount of endocytosis
Tn was measured as
Tn = vn/s. In this case,
Tn was 0.08 MEq. In contrast, little or no
dye was trapped in the soma (Fig. 10, thin upper trace). In addition, no endocytosis occurred when FM4-64 was applied as a control
at the end of the experiment (Fig. 10, middle traces). In 22 experiments Tn was 0.049 ± 0.030 MEq
(equal to 5500 vesicles). The largest values were ~0.12 MEq
(equal to 13,000 vesicles).
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Figure 10.
Endocytosis was induced by depolarization and
Ca2+ influx. A bipolar cell was superfused with an
extracellular saline containing 100 nM
Ca2+. Timing traces indicate an 18 sec application of 20 µM FM1-43, an 18 sec application
of 20 µM FM4-64, and 6 sec applications of a high
Ca2+ and K+ saline.
s is the fluorescence intensity of the surface membrane.
vn is the fluorescence of endocytosed
vesicles that remain trapped in the cytoplasm. Transient endocytosis
was measured as Tn = vn/s. After superfusion with
the high Ca2+ and K+ saline,
Tn was 0.08 MEq. Insets,
Images of the terminal taken at the times marked a and
b are shown. The gain in b is eight times
the gain in a. Scale bar, 10 µm.
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Images of the terminal are shown in two insets in Figure 10.
Image a was taken during the time the membrane was stained
with FM1-43. Image b was taken after the dye was removed
and the membrane destained. There is no evidence of localization of
newly endocytosed vesicles beneath the surface membrane. Instead,
stained vesicles appear to be uniformly distributed throughout the
interior of the terminal.
A second application of high Ca2+ and
K+ saline without dye did not release a significant
fraction of the labeled vesicles. We were never able to label vesicles,
destain the surface membrane, and then release the accumulated dye in a
second bout of vesicle cycling. Of course, this is not surprising if
newly endocytosed vesicles diffuse rapidly and mix with the entire
intraterminal vesicle population. A single burst of exocytosis
typically labeled ~10,000 vesicles. For comparison, a terminal
contains 5-10 × 105 vesicles (see von
Gersdorff et al., 1996). Thus, only 1-2% would be labeled, and a
decrease in fluorescence by a second bout of exocytosis would be
difficult to measure.
Vesicle cycling induced by depolarization depended on
Ca2+ entry through voltage-gated
Ca2+ channels and stopped when
Ca2+ channels were blocked. An example is shown in
Figure 11. The cell was exposed to high
Ca2+ and K+ saline three times.
The first exposure produced a bout of endocytosis with
Tn = 0.08 MEq. Afterward, 50 µM nisoldipine was added to the solutions containing
FM1-43 to block voltage-gated channels (see Heidelberger and Matthews,
1992). The subsequent application of high Ca2+ and
K+ saline plus nisoldipine produced a much smaller
change in the average intraterminal Ca2+
concentration. Evidently, most Ca2+ channels were
blocked. At the same time, there was little endocytosis, and
Tn = 0.006 MEq (or a reduction of 92%). A
repeated application of high Ca2+ and
K+ saline plus nisoldipine produced a similar,
barely detectable increase in the average Ca2+
concentration and Tn = 0.003 MEq.
Increasing the extracellular concentrations of K+
and Ca2+ while pharmacologically blocking
Ca2+ influx did not produce vesicle cycling. The
same result was observed in 11 experiments. Depolarization stimulated
vesicle cycling only when Ca2+ was able to influx
through voltage-gated channels.
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Figure 11.
Pharmacologically blocking voltage-gated
Ca2+ channels blocked Ca2+ influx
and endocytosis. A bipolar cell was superfused first with an
extracellular saline containing 100 nM
Ca2+ and then three times with FM1-43 and high
Ca2+ and K+ saline. Each
application was a sequence of 20 µM FM1-43 in control
saline for 4 sec, FM1-43 in high Ca2+ and
K+ saline for 4 sec, and finally FM1-43 in control
saline for 12 sec. Nisoldipine (50 µM) was added to the
extracellular salines during the second and third applications.
Tn (measured as illustrated in Fig. 10)
for the three sequential applications was 0.08, 0.006, and 0.003 MEq.
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We were next interested to know whether Ca2+ influx
through voltage-gated channels produced a relatively steady rate of
vesicle cycling or a burst of cycling that developed quickly and soon subsided. Long and short episodes of Ca2+ entry were
promoted by superfusing with a saline containing high Ca2+ and K+. An example is shown
in Figure 12. After a 3 sec exposure to
high Ca2+ and K+ saline,
Tn(3) was 0.11 MEq. Lengthening the
depolarization to 15 sec produced a very similar value;
Tn(15) = 0.12 MEq. Finally, the 15 sec
exposure was repeated with 1 µM BayK8466 added to the high Ca2+ and K+ saline to
prolong the open time of Ca2+ channels (see
Heidelberger and Matthews, 1992). As expected, the average cytoplasmic
Ca2+ concentration rose to a higher level, but
Tn was still 0.11 MEq. In 15 experiments
the ratio
Tn(3)/Tn(15)
was 1.06 ± 0.49. Each pulse of high Ca2+ and
K+ produced an approximately equal increment in dye
accumulation. An experiment described above (see Fig. 10) demonstrates
that labeled vesicles quickly mixed with a 100× larger vesicle pool.
Consequently, only ~1% of the vesicles labeled during one bout of
cycling were released in a subsequent bout of cycling, and each
increment represents labeled vesicles added in a new round of
exocytosis with little loss of previously accumulated vesicles. Thus,
equal steps of accumulation indicate that the majority of exocytosis
initiated by superfusion with a high Ca2+ and
K+ saline is completed in a single, transient
burst.
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Figure 12.
Vesicle cycling triggered by
Ca2+ influx was transient. The cell was superfused
three times with a 30 sec pulse of 20 µM FM1-43.
Transient endocytosis Tn was measured as
shown in Figure 10. The first application included a 3 sec pulse of
high Ca2+ and K+;
Tn = 0.11 MEq. The second application
included a 15 sec pulse of high Ca2+ and
K+; Tn = 0.12 MEq. The
third application was similar but added 1 µM BayK8466 to
the high Ca2+ and K+ saline to
prolong the open time of Ca2+ channels;
Tn = 0.11 MEq. Increasing the duration or
amount of Ca2+ entry did not alter transient
exocytosis. The control saline contained 100 nM
Ca2+. Each pulse of FM1-43 extended 12 sec beyond
the end of the high Ca2+ and K+
application.
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Capacitance measurements by von Gersdorff and Matthews (1994b) indicate
that endocytosis does not occur while the Ca2+
concentration is elevated. The following experiment (Fig.
13) provides additional
insight. A cell was exposed to two pulses of high
Ca2+ and K+ saline. The first
application was used to identify the amounts of endocytosis that
occurred first during and then after Ca2+ entry. At
the end of the first application, the exposure to FM1-43 and the high
Ca2+ and K+ saline ended
together, and FM1-43 fluorescence in the surface membrane was quenched
with FM4-64. Vesicles endocytosed during exposure to high
Ca2+ and K+ saline should be
labeled with FM1-43, but vesicles endocytosed after the return to the
low Ca2+ saline should be labeled with FM4-64.
Tn measured by the FM1-43 pulse was only
0.004 MEq. Tn measured by the FM4-64
pulse was 0.05 MEq. The second application of FM1-43 was used to
measure the total amount of endocytosis. Exposure to FM1-43 continued 12 sec beyond the 6 sec pulse of high Ca2+ and
K+ saline. Consequently, vesicles endocytosed both
during and after the pulse of Ca2+ and
K+ saline were labeled with FM1-43. In this case
Tn was 0.07 MEq. The entire experiment
indicates that little endocytosis occurred during superfusion with high
Ca2+ and K+ saline but that a
significant amount occurred after return to the control saline.
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Figure 13.
Endocytosis was delayed until after
Ca2+ influx stopped. The timing of endocytosis was
measured with 18 sec applications of 20 µM FM1-43
combined with 6 sec pulses of high Ca2+ and
K+ saline. The first applications of FM1-43 and
high Ca2+ and K+ saline ended
together and were immediately followed by a pulse of 20 µM FM4-64 in a saline containing 100 nM
Ca2+. The FM4-64 quenched the fluorescence of
FM1-43 in the surface membrane (see Fig. 3) but not in vesicles
trapped in the cytoplasm. Tn measured (as
shown in Fig. 10) with FM1-43 was 0.004 MEq and indicated that little
endocytosis occurred during superfusion with the high
Ca2+ and K+ saline.
Tn measured with FM4-64 was 0.05 MEq and
indicated that significant endocytosis occurred after return to the
control saline. The second application of FM1-43 measured the total
amount of endocytosis; Tn = 0.07 MEq.
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DISCUSSION |
Electrical and optical experiments now provide different
perspectives of synaptic vesicle cycling. Both methods have been used
to study ribbon synapses in the large synaptic endings of goldfish
bipolar cells. Capacitance measurements are best suited to detect rapid
events. Heidelberger et al. (1994) have measured the change in
electrical capacitance produced when a jump in Ca2+
concentration promotes the rapid fusion of a limited number of vesicles. In contrast, fluorescence from styryl dyes cannot resolve rapid events but is best suited to detect slow, continuous, or maintained changes. As we explain below, the two methods provide complementary views of synaptic vesicle dynamics.
The electrical measurements reveal that a rapid, saturating increase in
the intracellular Ca2+ concentration triggers the
fusion of ~3000 vesicles with a time constant of 0.35 msec
(Heidelberger et al., 1994), too short for an individual fusion site to
be used more than once. Consequently, all 3000 vesicles must be
predocked and ready for release. The rate constant for the last step in
vesicle fusion accelerates from 1 sec1 when the
Ca2+ concentration is ~10 µM to 1000 sec1 when the concentration reaches 200 µM [see Heidelberger et al. (1994), their Fig.
3a]. The optical measurements reported here demonstrate
that a Ca2+ concentration between 0.8 and 20 µM can maintain a continuous cycle of balanced exocytosis
and endocytosis with a maximal rate of 900 vesicles
sec1 (Fig. 7) or ~0.3 vesicle
sec1 at each release site. The continuous cycle
slows when the Ca2+ concentration rises above 20 µM. Both the electrophysiological and optical
measurements demonstrate that Ca2+ influx through
voltage-gated channels produces a burst of exocytosis but little or no
endocytosis until the average Ca2+ concentration
falls. Moreover, the maximum number of vesicles released appears to be
greater than the number of fusion sites (i.e., 10,000 vesicles vs 3,000 fusion sites), implying that several vesicles fuse at each site before
cycling stops.
Our results differ from those of Lagnado et al. (1996). We have
observed that (1) depolarization by high K+
produces a transient burst of vesicle cycling, (2) newly endocytosed vesicles rapidly diffuse and mix with the total intraterminal vesicle
population, and (3) a high rate of continuous cycling is sustained at
concentrations between 0.8 and 20 µM intracellular Ca2+ and stops at higher concentrations. Lagnado et
al. (1996) claim that (1) depolarization by high K+
produces continuous vesicle cycling, (2) newly endocytosed vesicles accumulate beneath the surface membrane, and (3) continuous cycling is
sustained at all Ca2+ concentrations above 0.3 µM Ca2+. We cannot explain these
differences.
We expect the function of fusion sites to depend critically on the
location of Ca2+ channels and illustrate our view
with two examples. One extreme behavior would occur in a "synapse"
with only one Ca2+ channel and an immediately
adjacent fusion site. In this case, the Ca2+
concentration in a microdomain at the cytoplasmic end of the channel
will tightly control vesicle cycling. In contrast, another extreme
behavior would occur in a "synapse" with a Ca2+
channel far from a fusion site. In this case, release would be controlled by the average Ca2+ concentration in the
cytoplasm. Many channel openings would raise the average cytoplasmic
Ca2+ concentration and sustain a continuous cycle of
repeated exocytosis and endocytosis. Of course, these idealized
synapses illustrate extreme behaviors. But the examples indicate how
dynamic behavior may depend on the number and relative spatial
distribution of channels and fusion sites. Many central synapses have
only one, or a few, fusion sites (see Redman, 1990; Korn and Faber,
1991; Ryan et al., 1997) and consequently may approach either of the two extreme behaviors described above. However, cells with ribbon synapses have many fusion sites (Raviola and Gilula, 1975; von Gersdorff et al., 1996) and, combining the behavior of both models, should mediate both transient and continuous release.
Bipolar cells, like rod photoreceptors (Rieke and Schwartz, 1996),
sustain high rates of vesicle cycling at relatively low (0.8-20
µM) Ca2+ concentrations. This is the
result of having many release sites that operate in parallel. Although
the rate of turnover at an individual release site is estimated as only
0.3 vesicle sec1, the net effect of simultaneously
maintaining release at a large number of sites is an enormous membrane
flux, equal in a bipolar cell to the replacement of the entire surface
area of a terminal every 2 min. Neurons with ribbon
synapses
photoreceptors, hair cells, and retinal bipolar
cellsappear to be specialized for the massive, parallel release of
many vesicles.
An endocytosed vesicle passes through an uncertain number of
intermediate steps before being ready for a new round of fusion. If
newly endocytosed vesicles had to be immediately reused, then continuous release would be limited by the time required to produce a
mature, fusion-competent vesicle. Significantly, endocytosed vesicles
are not immobilized near release sites but quickly diffuse throughout
the terminal (Figs. 5, 10). A total of 5-10 × 105 vesicles (von Gersdorff et al., 1996) is equal
to ~500-1000 vesicles per release site. Hence, vesicles may
continuously move to docking sites from an abundant reserve. A large
number of release sites and a surfeit of vesicles are required to
maintain a high rate of continuous release.
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FOOTNOTES |
Received May 14, 1998; revised Aug. 7, 1998; accepted Aug. 13, 1998.
This work was supported by a grant from the National Institutes of
Health.
Correspondence should be addressed to Dr. Eric Schwartz, Department of
Pharmacological and Physiological Sciences, The University of Chicago,
947 East 58th Street, Chicago, IL 60637.
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APPENDIX: : FM DYES CROSS THE SURFACE MEMBRANE |
When a bipolar cell was superfused with FM1-43 for several
minutes, dye sometimes accumulated in the cytoplasm even without stimulation or a change in experimental condition. For example, in
Figure 14A, a cell
was permeabilized with a Ca2+ ionophore (10 µM ionomycin) and continuously superfused with 100 nM Ca2+. The intracellular
Ca2+ concentration remained constant at ~100
nM throughout the experiment. Nonetheless, FM1-43
initially stained the surface membrane and then, after a significant
delay, entered the cytoplasm.
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Figure 14.
Spontaneous entry of FM1-43. A,
The cell was continuously superfused with 10 µM ionomycin
and 100 nM Ca2+. Intracellular
Ca2+ concentration was measured by fura-2
fluorescence (closed circles). The application of 20 µM FM1-43 is indicated by the horizontal timing
bar. a-c, The three images at the
right were taken at the times indicated by
arrows. B, Dye crosses the membrane of a
fixed terminal. The cell was fixed with 2% formaldehyde for 1 hr. The
application of 15 µM FM1-43 plus 15 µM
FM4-64 is indicated by the horizontal timing bar.
d, e, The two images were taken at the
time indicated by the arrows. Data were corrected for
crossover of FM1-43 fluorescence into the FM4-64 detection band.
FM1-43 and FM4-64 accumulated in the cytoplasm with different time
courses. Scale bars in c and e, 10 µm.
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The spontaneous entry of dye did not depend on vesicle cycling.
Instead, the following experiment demonstrates that it was produced by
dye molecules crossing the surface membrane. Cells were first fixed
with 2% formaldehyde for 1 hr and afterward superfused with a saline
containing both FM1-43 and FM4-64. The result is shown in Figure
14B. There are two essential observations. First, FM1-43 accumulated in the cytoplasm of a fixed cell. Thus, spontaneous entry did not require vesicle cycling. This conclusion is reinforced by
the second observation; FM1-43 accumulated in the cytoplasm with a
different time course than did FM4-64. Of course, the two dyes would
be accumulated with the same time course if the two dyes tagged the
surface membrane and were internalized together in the membrane of
endocytosed vesicles. Instead, the accumulation of FM4-64 was much
slower. The results are easily explained if FM1-43 molecules cross the
lipid bilayer and the other dye, FM4-64, crosses more slowly.
Images recorded during spontaneous dye entry (Fig. 14) differed in two
ways from images recorded during vesicle cycling (Fig. 5). First,
fluorescent foci did not develop beneath the surface membrane, and the
surface contour did not become irregular. Second, dye preferentially
accumulated in the center of a terminal and formed a fluorescent spot
occupying perhaps half the internal cross-sectional area. A similar
central spot developed when FM1-43 was applied to cells that had been
permeabilized by a brief exposure to 20 µM digitonin
(data not shown). Thus, the central spot was a region that avidly
stained when free dye was available in the cytoplasm.
Several observations indicate that styryl dyes form a series of states
when they partition into membranes. The complex kinetics of membrane
staining and destaining observed in Figure 2 indicates that the dye has
more than one state in a membrane. In addition, the glitches seen in
Figure 3 (at the arrowhead) are produced because spectral
components of FM1-43 fluorescence leave the membrane at different
rates. The correlation of spectral and kinetic components indicates
distinct states. Finally, the ability of FM4-64 to quench FM1-43
indicates that dye molecules can be closely packed (see Stryer, 1978).
Hence, dyes may aggregate. Thus, we were not surprised to find that
FM1-43 facilitated its own entry. For example, Figure 15 shows the result from a cell
superfused with two concentrations of FM1-43. When the cell was first
superfused for several minutes with 5 µM FM1-43, there
was a steady, slow rate of cytoplasmic accumulation (Fig. 15,
lower trace). Next, when the concentration was
increased to 40 µM, the rate of entry (normalized by the
dye concentration) increased fivefold (viz., from 0.074 to 0.38 MEq min1).
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Figure 15.
Increasing the FM1-43 concentration facilitates
spontaneous entry. A cell was permeabilized with ionomycin and
superfused with a saline buffered to contain 100 nM
Ca2+. After a control period, 5 and then 40 µM FM1-43 were added. Upper trace, Total
fluorescence measured in sequential images of the terminal plotted as a
function of time. Lower trace, Normalized fluorescence
plotted as a function of time. The fluorescence in the surface membrane
and that in the cytoplasm (multiplied by the geometric factor ) were
summed, normalized by the dye concentration, and then scaled so that
the normalized fluorescence in the surface membrane had a value of
1.
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We believe that dye molecules equilibrate between a series of states.
Some of these transitions may be fast and others quite slow. Moreover,
some states may depend on or be nucleated by specific membrane
molecules. Eventually, dye molecules might cross the surface membrane
either by flipping from the outer to the inner leaflet or by
interacting with the lipids that surround channels, opening channels,
and then passing through the open pore. The experiments in Figure
16, A and B,
provide a hint for the mechanism of dye entry. Cells were loaded with
fura-2 AM and superfused alternately with salines that contained either
2 mM Mg2+ or 2 mM
Cd2+. Fura-2 was used to detect the entry of
Cd2+. Normally, the permeability of
Cd2+ is low. Thus, in each of seven control
experiments, there was only a small change in the ratio of fura-2
fluorescence elicited by 331 and 380 nm light (Fig.
16A). In contrast, a different picture was observed
in eight experiments when a cell was also superfused with FM1-43 (Fig.
16B). Now, fura-2 fluorescence reported a significant increase in intracellular divalent ion concentration each time the cell
was exposed to Cd2+. Because the extracellular
saline lacked Ca2+, the change in fura-2
fluorescence is attributed to the entry of Cd2+.
Thus, continuous exposure to FM1-43 increased the membrane
permeability of Cd2+. In addition, each exposure to
Cd2+ decreased the simultaneous entry of FM1-43
and, after the removal of Cd2+, increased the
subsequent entry of FM1-43. Although Cd2+ and
FM1-43 interacted in a complex manner, the results imply that both
cooperate to increase membrane permeability, most likely by opening a
channel.
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Figure 16.
The combination of Cd2+ and
FM1-43 increased membrane permeability to both Cd2+
and FM1-43. Cells were loaded with fura-2. A,
Cd2+ does not normally enter a terminal. The cell
was superfused alternately with an extracellular saline containing 2 mM Mg2+ (no added
Ca2+) and a saline containing 2 mM
Cd2+. There was little change in the ratio of fura-2
fluorescence produced by 331 and 380 nm light, indicating that
Cd2+ did not enter. B,
Cd2+ enters a terminal stained with FM1-43. The
cell was superfused with an extracellular saline containing 2 mM Mg2+ (no added
Ca2+). FM1-43 (20 µM) was added to
the saline. When Mg2+ was replaced by
Cd2+, fura-2 fluorescence indicted an increase in
intracellular, divalent ions. After returning to the
Mg2+-rich saline, the rate of FM1-43 entry
increased.
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Experiments on vesicle cycling can be contaminated by spontaneous dye
entry. The characteristic delay before rapid intracellular staining was
variable. Rapid entry could be triggered by a brief exposure to
Cd2+ (Fig. 16), and we have the impression that the
transition may sometimes be initiated in the absence of
Cd2+ by bright light or exposure to a hydrophobic
drug. Once dye entry started, it was not reversible. Therefore, our
strategy was (1) to use FM1-43 only for short exposures (<120 sec in
Fig. 5 and <30 sec in all other experiments) and FM4-64, which
appeared to permeate more slowly (Fig. 14B), for
longer exposures, (2) to record images of the terminal in every
experiment and to reject an experiment if a bright fluorescent spot
formed in the center of the terminal (as in Fig. 14), and (3) to elicit
multiple periods of vesicle cycling and to include controls to be
certain that all measurements were repeatable.
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D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim
Lysosomes Are the Major Vesicular Compartment Undergoing Ca2+-Regulated Exocytosis from Cortical Astrocytes
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[Abstract]
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G. Matthews and P. Sterling
Evidence That Vesicles Undergo Compound Fusion on the Synaptic Ribbon
J. Neurosci.,
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[Abstract]
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D. Zenisek
Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals
PNAS,
March 25, 2008;
105(12):
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[Abstract]
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B. Innocenti and R. Heidelberger
Mechanisms Contributing to Tonic Release at the Cone Photoreceptor Ribbon Synapse
J Neurophysiol,
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[Abstract]
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M. R. Coggins, C. P. Grabner, W. Almers, and D. Zenisek
Stimulated exocytosis of endosomes in goldfish retinal bipolar neurons
J. Physiol.,
November 1, 2007;
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[Abstract]
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Y. Serulle, M. Sugimori, and R. R. Llinas
Imaging synaptosomal calcium concentration microdomains and vesicle fusion by using total internal reflection fluorescent microscopy
PNAS,
January 30, 2007;
104(5):
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[Abstract]
[Full Text]
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M. J. Palmer
Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals
J. Physiol.,
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[Abstract]
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P. E. MacDonald, L. Eliasson, and P. Rorsman
Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells
J. Cell Sci.,
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[Abstract]
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H. Teng and R. S Wilkinson
'Delayed' endocytosis is regulated by extracellular Ca2+ in snake motor boutons
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[Abstract]
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M. Kreft, D. Krizaj, S. Grilc, and R. Zorec
Properties of Exocytotic Response in Vertebrate Photoreceptors
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[Abstract]
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C. Paillart, J. Li, G. Matthews, and P. Sterling
Endocytosis and Vesicle Recycling at a Ribbon Synapse
J. Neurosci.,
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[Abstract]
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C. E. Armstrong-Gold and F. Rieke
Bandpass Filtering at the Rod to Second-Order Cell Synapse in Salamander (Ambystoma tigrinum) Retina
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[Abstract]
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D. Zenisek, V. Davila, L. Wan, and W. Almers
Imaging Calcium Entry Sites and Ribbon Structures in Two Presynaptic Cells
J. Neurosci.,
April 1, 2003;
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M. Holt, A. Cooke, M. M. Wu, and L. Lagnado
Bulk Membrane Retrieval in the Synaptic Terminal of Retinal Bipolar Cells
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February 15, 2003;
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R. Heidelberger, Z.-Y. Zhou, and G. Matthews
Multiple Components of Membrane Retrieval in Synaptic Terminals Revealed by Changes in Hydrostatic Pressure
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J. Hurtado, S. Borges, and M. Wilson
Na+-Ca2+ Exchanger Controls the Gain of the Ca2+ Amplifier in the Dendrites of Amacrine Cells
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P. Congar and L.-E. Trudeau
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C. B. Griesinger, C. D. Richards, and J. F. Ashmore
FM1-43 Reveals Membrane Recycling in Adult Inner Hair Cells of the Mammalian Cochlea
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May 15, 2002;
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B. Volgyi, D. Xin, and S. A Bloomfield
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G. Neves, A. Gomis, and L. Lagnado
Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells
PNAS,
November 29, 2001;
(2001)
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[Abstract]
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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
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September 15, 2001;
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[Abstract]
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R. Heidelberger
ATP Is Required at an Early Step in Compensatory Endocytosis in Synaptic Terminals
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P. Chen, T.-C. Hwang, and K. D. Gillis
The Relationship between Camp, Ca2+, and Transport of Cftr to the Plasma Membrane
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[Abstract]
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G. Kilic, J. K Angleson, A. J Cochilla, I. Nussinovitch, and W. J Betz
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May 1, 2001;
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M. A. Freed
Parallel Cone Bipolar Pathways to a Ganglion Cell Use Different Rates and Amplitudes of Quantal Excitation
J. Neurosci.,
June 1, 2000;
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T. Euler and R. H. Masland
Light-Evoked Responses of Bipolar Cells in a Mammalian Retina
J Neurophysiol,
April 1, 2000;
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[Abstract]
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S. A Bloomfield and D. Xin
Surround inhibition of mammalian AII amacrine cells is generated in the proximal retina
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March 15, 2000;
523(3):
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[Abstract]
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J. Burrone and L. Lagnado
Synaptic Depression and the Kinetics of Exocytosis in Retinal Bipolar Cells
J. Neurosci.,
January 15, 2000;
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[Abstract]
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G Stenbeck and M. Horton
A new specialized cell-matrix interaction in actively resorbing osteoclasts
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[Abstract]
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A. Gomis, J. Burrone, and L. Lagnado
Two Actions of Calcium Regulate the Supply of Releasable Vesicles at the Ribbon Synapse of Retinal Bipolar Cells
J. Neurosci.,
August 1, 1999;
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[Abstract]
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O. Prange and T. H. Murphy
Correlation of Miniature Synaptic Activity and Evoked Release Probability in Cultures of Cortical Neurons
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August 1, 1999;
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[Abstract]
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G. Neves, A. Gomis, and L. Lagnado
Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells
PNAS,
December 18, 2001;
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[Abstract]
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Top
Abstract
Introduction
