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The Journal of Neuroscience, May 1, 1998, 18(9):3233-3240
Attenuated Influx of Calcium Ions at Nerve Endings of csp
and shibire Mutant Drosophila
Joy A.
Umbach1,
Minoru
Saitoe2,
Yoshi
Kidokoro2, and
Cameron B.
Gundersen1
1 The Department of Molecular and Medical Pharmacology
and The Crump Institute for Biological Imaging, University of
California Los Angeles School of Medicine, Los Angeles, California
90095, and 2 Institute for Behavioral Sciences, Gunma
University School of Medicine, Maebashi 371, Japan
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ABSTRACT |
Previous work has shown that cysteine-string proteins (csps) are
synaptic vesicle proteins that are important for evoked
neurotransmitter release at Drosophila neuromuscular
junctions. Indirect evidence has implicated csps in a regulatory link
between synaptic vesicles and presynaptic calcium (Ca) channels. In
this report, we use Ca Crimson to monitor stimulus-dependent changes of
cytosolic Ca at motor nerve terminals of csp mutant
Drosophila. These mutants display temperature-sensitive
(TS) paralysis and a presynaptic failure of evoked synaptic
transmission. We show that this TS inhibition of neuromuscular
transmission is correlated with a block of Ca ion entry at nerve
endings of csp mutants. These data support the
hypothesis that csps mediate a regulatory interaction between synaptic
vesicles and presynaptic Ca channels. Moreover, these results predict
that if one depletes nerve endings of synaptic vesicles, one may see a
reduction of presynaptic Ca ion entry. Defects of the
dynamin gene in TS shibire mutant
Drosophila interfere with synaptic vesicle recycling and
lead to an activity-dependent depletion of these organelles. Our
results show that Ca influx is blocked at nerve terminals of
shibire mutant larvae at the same time that synaptic
transmission fails in these organisms. Thus, using two completely
independent Drosophila mutants, we demonstrate that
synaptic vesicles and csps are vital for the function of presynaptic Ca
channels.
Key words:
Ca imaging; cysteine string proteins; Ca channels; presynaptic function; shibire; Drosophila
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INTRODUCTION |
Cysteine-string proteins (csps) were
first described in Drosophila as synapse-specific antigens
(Zinsmaier et al., 1990 ). Independently, the cDNA encoding a
Torpedo csp was identified using a suppression cloning
strategy (Gundersen and Umbach, 1992). These latter investigations
revealed that csp antisense RNA inhibited the expression of N-type
calcium (Ca) channels in Xenopus oocytes that were
co-injected with Torpedo electric lobe mRNA. Concomitantly, csp sense RNA augmented the expression of these same Ca channels without significantly altering their kinetics or voltage dependence (Gundersen and Umbach, 1992). These results led to the hypothesis that
csps were either novel subunits or modulators of presynaptic Ca
channels (Gundersen and Umbach, 1992).
Two developments have refined considerably our understanding of csps
and their prospective function at synapses. First, studies of the
subcellular distribution of csps revealed that these proteins were
prominently associated with the P-face of synaptic vesicles from
unstimulated Torpedo electric organ (Mastrogiacomo et al., 1994), and csps have since been reported to be membrane-associated components of several other types of secretory organelles (for review,
see Buchner and Gundersen, 1997). These observations rendered it
unlikely that csps were integral subunits of presynaptic Ca channels,
and instead led us to propose that csps were participants in a
regulatory interaction between docked (or docking) synaptic vesicles
and presynaptic Ca channels (Mastrogiacomo et al., 1994; Umbach et al.,
1995). However, the nature and mechanism of this interaction remain to
be established.
The second important development is that Zinsmaier and colleagues
(1994) isolated csp mutant alleles of Drosophila.
These mutants developed slowly, showed sensory and motor deficits, and died prematurely (Zinsmaier et al., 1994). A particularly useful feature of these csp mutants was that they displayed
temperature-sensitive (TS) paralysis (Zinsmaier et al., 1994).
Subsequent investigations of the cellular basis of this TS paralytic
phenotype revealed that stimulus-evoked neurotransmitter release ceased
at temperatures above 30°C (Umbach et al., 1994). This block of
synaptic transmission was not caused by changes in axonal conduction or
in postsynaptic sensitivity to neurotransmitter. Instead, because
spontaneous quantal transmitter secretion persisted in these mutants
even when nerve impulses failed to evoke quantal transmitter release, we concluded that csp mutants were defective in
excitation-secretion coupling (Umbach et al., 1994). These
physiological results were consistent with the idea of a csp-Ca
channel link, but they did not exclude other explanations.
To investigate further the cause of the secretory failure in TS
csp mutants, we used ionic and pharmacological manipulations that either enhanced neurotransmitter secretion by augmenting Ca ion
entry via presynaptic Ca channels or raised presynaptic Ca ion activity
independently of Ca channels. Our results indicated that agents such as
 -latrotoxin and ionomycin, which bypass presynaptic Ca channels and
promote high frequency quantal discharges in wild-type Drosophila, were equally effective at promoting quantal
transmitter secretion in TS csp mutants (Umbach and
Gundersen, 1997). However, other manipulations (e.g., depolarization
using elevated KCl) that relied on the opening of presynaptic Ca
channels failed to enhance quantal transmitter release in
csp mutants (Umbach and Gundersen, 1997). These data
provided further indirect support for the hypothesis that presynaptic
Ca channels were blocked in csp mutants at the nonpermissive
temperature. The current investigations examine this issue more
directly. Here, we report the use of the fluorescent Ca-sensitive dye
Ca Crimson to monitor presynaptic Ca dynamics of wild-type and
csp mutant Drosophila at permissive and
nonpermissive temperatures. Moreover, we also monitored presynaptic Ca
dynamics of TS shibire mutant Drosophila. The
shibire mutation produces a conditional blockade of synaptic
vesicle recycling (Kosaka and Ikeda, 1983), and we hypothesized that
the absence of synaptic vesicles might interfere with the function of
presynaptic Ca channels. Our results argue that both synaptic vesicles
and csps are important for Ca channel function at nerve endings.
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MATERIALS AND METHODS |
Materials. Wild-type Drosophila were the
Canton S strain. csp mutants were the
cspu1null allele kindly supplied by Dr.
E. Buchner (University of Würzburg, Germany), and the
shibire mutants were the
shits1 allele (Grigliatti et al., 1973).
As before (Umbach et al., 1994), the csp mutant larvae were
preselected for TS paralytic behavior. Ca Crimson AM ester was obtained
from Molecular Probes (Eugene, OR). The de-esterified form of this dye
has a KD for Ca ions of 205 nM
(manufacturer's specifications). Fetal bovine serum,
dimethylsulfoxide, and cremophor were from Sigma (St. Louis, MO).
Schneider's medium was purchased from Life Technologies-BRL
(Gaithersburg, MD), and ionomycin was from Calbiochem (San Diego,
CA).
Ca imaging. The procedure we used to monitor presynaptic Ca
dynamics in type 1b boutons of Drosophila larvae was
modified from that described in Umbach et al. (1998). Ca Crimson AM
ester was dissolved in dimethylsulfoxide (at 5 mM) and
added to a final concentration of 10 µM in Schneider's
medium with 15% fetal bovine serum and 0.05% cremophor. Third instar
larvae were dissected and immobilized in a small, Sylgard-lined chamber
and incubated with dye-containing solution for 2-3 hr in the dark at
18-22°C. The preparation was rinsed with a conventional
Drosophila saline solution (Jan and Jan, 1976; Umbach et
al., 1994) with 2 mM CaCl2 and mounted on the
viewing stage of an upright Olympus (model BX50WI) fluorescence
microscope. A suction electrode was used to stimulate the motor nerves
innervating muscle fibers 6 and 7 of abdominal hemisegments 2, 3, or 4. Synaptic boutons were identified by their characteristic morphology,
along with the fact that Ca Crimson accumulates in these elements and
displays a detectable resting fluorescence (see Results) (Umbach et
al., 1998). Excitation of Ca Crimson fluorescence was achieved using a
single wavelength (580 nm) grating, and for individual images illumination was maintained for a duration of 270 msec. Photobleaching was minimized via a 25% neutral density filter (Olympus ND25). To
capture fluorescence emissions, we used a wide band yellow filter set
(Olympus WIY; dichroic mirror 600 nm; excitation filter 545-580 nm and
barrier filter 610 nm), and images were captured and stored using a
Hamamatsu cooled ccd camera (model C4880) and the Hamamatsu Argus 50 Ca
imaging system. Image files of 8 bits (512 × 483 pixels in area)
were later analyzed for fluorescence intensity on a 0-255 gray scale
using the Axon Imaging Workbench 2.1 software from Axon Instruments
(San Jose, CA). For each figure, the same pseudocolor scale (from blue
low, to red high) was added to the images. The color palette used the
central two-thirds of the 0-255 gray scale and was chosen to highlight
changes of Ca Crimson fluorescence.
Our standard protocol was to record fluorescent images of nerve
terminals immediately before and within 30 sec after a fixed stimulus
train (10 Hz for 2 min). The time course of the decay of Ca Crimson
fluorescence after this 10 Hz stimulus train is documented in Results.
This stimulation protocol was chosen because pilot experiments showed
that it yielded a reproducible and significant increase in the
intensity of fluorescence emission from Ca Crimson-loaded nerve endings
of wild-type preparations (see Fig. 1). Here, we need to emphasize two
procedural constraints that led us to select this stimulus paradigm.
First, for the experiments using csp and shibire
mutant preparations, we used visual observation to confirm when nerve
impulses were no longer eliciting muscle twitches at the nonpermissive
temperature. Because of this requirement that we verify by visual
observation the blockade of neuromuscular transmission, we could not
use neuromuscular blocking agents (and we also reasoned that it was
preferable to avoid the use of agents that might have indirect
presynaptic effects). However, because our preparations (with the
exception of those that became paralyzed) moved during the test
stimulus train, we needed several seconds (usually, 5-20 sec) to
refocus on the nerve terminal before capturing images for analysis of
Ca Crimson fluorescence. This secondary constraint forced us to select
a stimulation protocol that caused a prolonged and relatively stable
increase of nerve ending Ca Crimson fluorescence. This stable change in
turn allowed us to acquire suitable images for analysis. Moreover,
because the critical issue for these experiments was the question of
whether there was Ca entry at nerve endings of csp and
shibire mutants when synaptic transmission was blocked, we
also monitored Ca Crimson fluorescence in these paralyzed preparations
during the stimulus train (as well as immediately after).
To quantitate changes of Ca Crimson fluorescence for individual
synaptic boutons, we measured the intensity of Ca Crimson fluorescence
at each bouton before stimulation and after nerve stimulation. We
performed a background subtraction for each bouton by obtaining the
mean fluorescence intensity from at least 10 different background
regions (in the muscle), each the equivalent size of individual
boutons. This background value varied among preparations (ranging from
40 to 80 units on the 0-255 gray scale) and was obtained both at rest
and after nerve stimulation. Background was subtracted from the resting
or poststimulation fluorescence intensity associated with single
boutons to yield Frest and
Fstim, respectively. These parameters
were measured for a minimum of 10 boutons per neuromuscular junction
and are reported in Table 2. The ratio of
Fstim/Frest was
determined separately for each bouton to quantitate the change in
fluorescence of individual boutons, and the mean
Fstim/Frest value
was then determined for each neuromuscular junction by averaging the
individual bouton ratios. By computing this ratio one minimizes
differences among preparations that are caused by variable dye loading.
The stimulus-induced changes in Ca Crimson fluorescence were then
compared for the same boutons at permissive (22°C) and nonpermissive
(32°C) temperatures. These data comprise Table
1. To assess possible changes in the basal, bouton fluorescence of Ca Crimson between 22°C and 32°C, we
computed the ratio of Frest values for
individual boutons at these two temperatures and calculated the mean
Frest32/Frest22 for each neuromuscular junction. Frest at 32°C
was measured 15 min after heating to 32°C for wild-type and
csp mutants, and unless indicated otherwise for
shibire mutants, Frest at 32°C was
measured after a total 16-17 min incubation at 32°C, which included
the 5 min rest period after nerve stimulation that was used to deplete synaptic vesicles.
To assess the maximum detectable increase in Ca Crimson fluorescence
intensity in larval boutons, we used the calcium ionophore ionomycin
(10 µM). This reagent was applied in the presence of 5 mM CaCl2, and fluorescent images were
captured after 10 min in ionomycin and compared with resting
fluorescence before ionophore application.
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RESULTS |
The fluorescent, Ca-sensitive dye Ca Crimson was loaded into nerve
endings of wild-type Drosophila larvae. As reported (Umbach et al., 1998), this dye loads preferentially into presynaptic boutons.
Thus, our first goal was to establish conditions of nerve stimulation
that lead reproducibly to an increase of Ca Crimson fluorescence in
these nerve terminals. The protocol that we adopted (10 Hz stimulation
for 2 min) yields a significant increase (p < 0.01 by the paired Student's t test) in the mean intensity
of fluorescence emission from nerve terminal boutons immediately after
the stimulus train (Figs. 1,
2a,b). The ratio of
Fstim/Frest remains elevated for several tens of seconds before decaying
exponentially to the resting fluorescence level ~5 min after the
stimulus train (Fig. 1). We have not investigated systematically the
processes that contribute to this relatively sustained increase of Ca
Crimson fluorescence at motor endings of Drosophila larvae
under these conditions. However, four additional sets of observations
are important for interpreting these results. First, as shown in Figure 1, basal nerve terminal fluorescence (without stimulation of the nerve)
is very stable. In other words, the increase of Ca Crimson fluorescence
emission in the stimulated preparation of Figure 1 is a response to
nerve stimulation. Second, this stimulus-dependent change of Ca Crimson
fluorescence (Figs. 1, 2) is eliminated in solutions devoid of Ca
(containing 10 mM MgCl2 and 0.1 mM
EGTA with no added CaCl2) or when CdCl2
(1 mM) is added to the extracellular medium (data not
shown). These latter results indicate that the observed change of Ca
Crimson fluorescence is contingent on entry of extracellular Ca ions,
presumably via presynaptic Ca channels. Third, we have detected a
smaller
(Fstim/Frest < 1.1) and more rapid decay of fluorescence (a return to basal
fluorescence intensity occurs in <5 sec) in immobilized preparations
subjected to 10 Hz stimulation for 5 sec. However, the more robust
signal-to-noise ratio obtained with 2 min of 10 Hz stimulation led us
to select this paradigm for subsequent experiments. Fourth, we assessed the maximum increase of Ca Crimson fluorescence that could be detected
in these preparations using the calcium ionophore ionomycin (10 µM). Ionomycin treatment produces a nearly fourfold
increase of Ca Crimson fluorescence (3.7 ± 0.7 for
n = 5), which indicates that the 1.4- to 1.5-fold
changes (Fig. 1, Table 1) we detect with nerve stimulation are
subsaturating.
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Figure 1.
Ca Crimson fluorescence in larval neuromuscular
preparations with and without nerve stimulation. Separate nerve
terminals in a Ca Crimson-loaded preparation were analyzed for the
relative intensity of fluorescence emission in the absence of nerve
stimulation ( ) or immediately after (at the times indicated) 2 min
of 10 Hz stimulation of the motor nerve ( ). In the control
preparation () the motor nerve of the adjacent hemisegment was
stimulated (10 Hz, 2 min) to mimic the movement seen with stimulation
of the correct motor nerve (). Frest was
obtained from images captured 30 sec before the stimulus train.
Fstim is mean bouton fluorescence assessed
at the indicated times after stimulation. Data are mean values for at
least 10 boutons per terminal. The SD did not exceed 20% of the
mean.
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Figure 2.
Stimulus-dependent changes of Ca Crimson
fluorescence at a wild-type neuromuscular junction. Ca Crimson
fluorescence increases at presynaptic boutons as a function of nerve
stimulation at both 22 and 32°C (a, c: rest; b,
d: stimulated). Scale bar, 17 µm. The same magnification is
used in Figures 3 and 4.
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Images typical of those analyzed to produce Figure 1 are presented in
Figure 2a,b. Here, a wild-type larval preparation is shown before (Fig. 2a) and ~15 sec after (Fig.
2b) an episode of 10 Hz stimulation for 2 min. As
indicated by the pseudocolor scale (Fig. 2a,b), Ca
Crimson fluorescence emission increases appreciably in response to
nerve stimulation in this preparation at 22°C. Data summarized in
Table 1 show that the average value for
Fstim/Frest is
1.52 for four separate control preparations at 22°C. Using the paired
Student's t test, this mean increase of Ca Crimson
fluorescence (relative to rest) is significant at p < 0.01. Next, we warmed these preparations, and after 15 min at 32°C, a
stimulus train (10 Hz for 2 min) was delivered. This resulted in a
similar (to 22°C) stimulus-dependent increase of Ca Crimson
fluorescence (Fig. 2c,d, Table 1). Again, this
stimulus-dependent increase of Ca Crimson fluorescence at 32°C was
statistically significant (p < 0.01 by the
paired Student's t test). Mean bouton Frest and mean bouton
Fstim intensities are in Table
2. In addition to confirming the trend in
Table 1, the data in Table 2 and the ratios in Table
3 are important because they show that
there is no significant change in the resting fluorescence intensity of
Ca Crimson at 32°C versus 22°C. This stability of
Frest is necessary to maintain the fidelity of
detection of stimulus-dependent changes of presynaptic Ca.
Our next step was to investigate the effect of nerve stimulation on
presynaptic Ca ion activity in TS csp mutant larvae. As shown (Fig. 3a,b, Table
1), there was a significant increase (p < 0.05 by the paired Student's t test) in the intensity of Ca
Crimson fluorescence using the standard stimulation paradigm at 22°C
(again, significance was assessed by comparing
Fstim vs Frest at
individual boutons in these preparations, and as before the mean
intensity data are in Table 2). Interestingly, at 22°C the ratio
Fstim/Frest was
consistently lower in these mutants relative to wild-type controls
(Table 1), which indicated that presynaptic Ca ion activity was reduced
in these organisms even at room temperature (note that quantal content
was also lower in csp mutants relative to controls at
22°C) (Umbach et al., 1994).
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Figure 3.
Stimulus-dependence of Ca Crimson fluorescence at
a csp mutant neuromuscular junction. Nerve
impulse-dependent increases of Ca Crimson fluorescence are seen at
nerve endings at 22°C before (a, b) and after
(e, f) a 32°C challenge. At 32°C, nerve
impulses fail to alter Ca Crimson fluorescence (c,
d).
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Previous work had shown that when TS csp mutant larvae were
warmed to 30-32°C, there was a progressive decline of evoked
transmitter release that eventually gave way to a complete failure of
evoked responses and muscle twitch (Umbach et al., 1994; Umbach and
Gundersen, 1997). For the current experiments, we warmed the
preparations to 32°C and waited (10-15 min) until single nerve
impulses failed to elicit muscle contraction before recording the
impact of a stimulus train on Ca Crimson fluorescence. As illustrated
(Fig. 3c,d, Table 2), a standard episode of nerve
stimulation produced no increase in the intensity of Ca Crimson
fluorescence at motor nerve terminals of this csp mutant
preparation at 32°C. In six separate preparations,
Fstim/Frest was
0.95 ± 0.07 (Table 1). Because these preparations did not move,
we were able to capture images within 1 sec after the completion of the
stimulus train. Moreover, we recorded separate images during the test
stimulus train at 32°C and observed no increase of Ca Crimson
fluorescence in the synaptic boutons (data not shown). Thus, at no time
during or after the test stimulus train did we detect any increase of Ca Crimson fluorescence at nerve endings of csp mutants at
the nonpermissive temperature. These results suggest that Ca entry is
severely compromised in these mutants at elevated temperature (compared
with room temperature or wild-type controls at 32°C) (Table 1).
Finally, the results in Table 3 show that resting Ca Crimson
fluorescence is essentially unchanged between 22°C and 32°C (the
ratio is close to 1), which indicates that there was no
temperature-dependent change of resting Ca ion homeostasis in these
mutants.
The TS failure of evoked neurotransmitter release in csp
mutants was reversible with cooling (Umbach et al., 1994; Umbach and
Gundersen, 1997) and so was the effect on stimulus-dependent Ca ion
entry. Thus, when the same preparation of Figure 3a-d was cooled to 22°C, we observed a recovery of the Ca Crimson response to
nerve stimulation (Fig. 3e,f, Table 2). As summarized
in Table 1,
Fstim/Frest of
preparations after a challenge at 32°C was similar to the ratio
obtained before warming (1.26 ± 0.20 vs 1.18 ± 0.11). These
results show that csp mutant preparations retained the
ability to respond to nerve stimulation and that the absence of a
change of cytosolic Ca at 32°C reflected a physiological deficit in
these organisms.
shibire mutant Drosophila display a TS failure of
stimulus-evoked neurotransmitter release that has been correlated with
a loss of synaptic vesicles (Poodry and Edgar, 1979; Kosaka and Ikeda,
1983). We reasoned that if a regulatory link between synaptic vesicles
and presynaptic Ca channels normally existed (Mastrogiacomo et al.,
1994; Umbach et al., 1995), this link might cease to function when
synaptic vesicles were depleted. To assess this possibility, we
monitored presynaptic Ca ion activity of shibire mutant
larvae at permissive and nonpermissive temperatures. As indicated (Fig. 4a,b, Table 2),
shibire mutants displayed an enhanced fluorescence intensity
of Ca Crimson in response to nerve impulses that was reminiscent of
wild-type controls at 22°C (Figs. 1, 2). Overall, Fstim/Frest for
shibire larvae at 22°C was indistinguishable from controls
(Table 1). These results imply that Ca entry was unaffected in these
mutants at the permissive temperature.
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Figure 4.
Stimulus dependence of Ca Crimson fluorescence at
a shibire mutant neuromuscular junction. Nerve impulses
evoke an increase of Ca Crimson fluorescence at 22°C (a,
b). After warming to 32°C and depleting the nerve terminals
of releasable quanta, the same stimulation protocol fails to raise Ca
Crimson fluorescence (c, d). Note that the 5 min
recovery period that we used after the initial stimulus train was
insufficient for the resting fluorescence of Ca Crimson to return to
the same level as in a. Partial recovery of the
stimulus-dependent increase of Ca Crimson fluorescence is detected 10 min after the preparation was cooled from 32 to 22°C (e,
f).
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We next warmed the shibire mutant preparations to 32°C for
10 min and stimulated the motor nerve (at 10 Hz for 1-2 min) until paralysis (failure of muscle twitch) was complete. This conditioning train of impulses elicited a significant increase (relative to resting
terminals) in the intensity of Ca Crimson fluorescence measured at the
onset of paralysis
(Fstim/Frest = 1.29 ± 0.10 for the same four preparations of Table 1). Thus, we
were able to detect changes of presynaptic Ca ion activity during the
period of time when shibire nerve terminals were being
depleted of synaptic vesicles. This was an important observation,
because we were concerned that as vesicle depletion proceeded, it might
have increased the volume of shibire nerve terminals
(thereby diluting the Ca Crimson), and this could have compromised our
ability to detect changes of Ca ion activity. Instead, our data argue
that nerve terminal volume did not change appreciably in
shibire mutants under these conditions. A similar conclusion
was drawn by Koenig and Ikeda (1989), who showed that there was no
detectable expansion of the plasma membrane of shibire nerve
terminals at the nonpermissive temperature.
After we induced paralysis in the shibire mutant
larvae at 32°C, we were then interested in the response of the nerve
terminals to nerve stimulation. Thus, after a rest period (5 min) to
allow cytosolic Ca to recover to the basal level, we delivered the
standard train of nerve impulses (10 Hz for 2 min) and recorded
presynaptic Ca Crimson fluorescence. In this situation, we detected no
increase of Ca Crimson fluorescence in images taken within 1 sec after the stimulation protocol (Fig. 4c,d, Tables 1, 2)
(moreover, images captured during this stimulus train also showed no
significant change of Ca Crimson fluorescence; data not shown). The
difference between
Fstim/Frest at
22°C and 32°C (Table 1) was highly significant for these mutants
(p < 0.01 by the paired Student's t
test). These data suggest that Ca ion entry was inhibited, and that
presynaptic Ca channels failed to operate in shibire
mutants, after they were paralyzed at 32°C. As before, we also
verified that the resting fluorescence of Ca Crimson at 32°C was not
abnormally high relative to 22°C (Table 3), because potential dye
saturation would have compromised our ability to detect changes of
cytosolic Ca. Instead, all of our results (Fig. 4, Tables 1, 2) point
to a block of the stimulus-dependent change of cytosolic Ca ion
activity in paralyzed shibire mutants.
Finally, to verify the integrity of the shibire preparations
subsequent to the 32°C challenge, we cooled the larvae back to 22°C
and saw a partial recovery of stimulus-dependent changes of presynaptic
Ca ion activity (Fig. 4e,f, Table 2). We consistently found that the ratio,
Fstim/Frest,
after recovery from the 32°C challenge in shibire, was
less than the ratio before 32°C (Table 1). Concomitantly, we observed
that the rate of failure of excitatory junctional potentials (in
response to 10 Hz stimulation for 2 min), before 32°C challenge was
<4%, whereas afterward in two of three preparations it exceeded 50%.
Thus, within the time period that we were testing for recovery of
responses in shibire mutant larvae, there was a parallel
decline of both the Ca signal and quantal transmitter release elicited
by 10 Hz stimulation.
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DISCUSSION |
These investigations were undertaken to test the hypothesis
that the TS abolition of neurotransmitter release in csp
mutant Drosophila reflects a failure of stimulus-dependent
Ca ion entry at nerve endings. Previous work had shown that evoked, but
not spontaneous, quantal secretion of neurotransmitter ceased in these mutants at the nonpermissive temperature (Umbach et al., 1994; Umbach
and Gundersen, 1997). Moreover, because action potential conduction and
postsynaptic sensitivity to glutamate remained unaltered in
csp mutants at elevated temperatures, we had concluded that
there was a failure in the process that couples action
potential-dependent depolarization to the synchronous release of
neurotransmitter at csp nerve endings (Umbach et al., 1994).
A recent extension of these studies revealed that agents that raised
cytosolic Ca ion activity independently of voltage-gated Ca channels
were effective at promoting quantal transmitter release in
csp mutants at the nonpermissive temperature (Umbach and
Gundersen, 1997). However, treatments that relied on the opening of
presynaptic Ca channels were ineffective at overcoming the secretory
block in csp mutants (Umbach and Gundersen, 1997). Although
these latter results were compatible with the hypothesis that there was
a defect in the entry of Ca ions at nerve endings of csp
mutant Drosophila, we could not formally exclude other
explanations (for instance, our data did not eliminate the possibility
that there was a shift in the affinity for Ca ions of the Ca sensor
that regulates exocytosis at nerve endings (Umbach and Gundersen,
1997). The current investigations provide independent support for the
idea that presynaptic Ca channels fail to operate normally in
csp mutant Drosophila. Thus, our results indicate
that there is a parallel blockade of evoked neurotransmitter release
and stimulus-dependent changes of presynaptic ionized Ca in paralyzed
csp and shibire mutant Drosophila at
32°C. Because there is no precedent for the involvement of the
csp and shibire gene products in presynaptic Ca
buffering (and there are significant, stimulus-dependent changes of
presynaptic Ca in these mutants at permissive temperatures), we
interpret the lack of change of nerve terminal Ca in the mutant
organisms at 32°C as reflecting a decline of Ca entry. These
observations have important implications for nerve terminal and csp
function.
Among the implications of this study is that csps participate in
a regulatory interaction involving presynaptic Ca channels. Because
csps are synaptic vesicle proteins (Mastrogiacomo et al., 1994; for
review, see Buchner and Gundersen, 1997), we infer that this reaction
occurs during or subsequent to the docking of a synaptic vesicle in the
vicinity of presynaptic Ca channels. Obviously, many questions remain
to be answered about the biochemical and biophysical mechanisms of this
csp-Ca channel link. However, one conclusion, which is inescapable, is
that this interaction is not essential for the survival of
Drosophila. Thus, we know that mutants lacking the
csp gene are viable; however, they die prematurely (Zinsmaier et al., 1994). Hence, it appears that this csp-Ca channel link helps to sustain normal presynaptic function but that compensatory mechanisms can supplant the loss of csps in csp mutant
Drosophila. We propose that it is the temperature-dependent
failure of this "compensatory machinery" that underlies the TS
blockade of transmitter release in the csp mutants. Future
investigations of the mechanism of the csp-Ca channel link should
provide insights into these compensatory pathways and their thermal
sensitivity in the csp mutants.
As outlined above (and previously) (Umbach et al., 1995), it is
reasonable to propose that csps interact with Ca channels during or
after the docking of a synaptic vesicle at the plasma membrane.
Assuming that this is the case, it is also plausible that this
interaction ceases to operate at nerve endings that are depleted of
synaptic vesicles. It is in this context that shibire mutant
Drosophila offer a unique opportunity to test the hypothesis that synaptic vesicles (and vesicle-associated proteins, such as csps) participate in modulating nerve ending Ca channels. This
is because point mutations in the shibire gene, which
encodes dynamin (Chen et al., 1991; van der Bliek and Meyerowitz,
1991), lead to a temperature-dependent block of synaptic vesicle
recycling in these organisms. This failure of vesicle recycling
produces a TS block of synaptic transmission and a depletion of
synaptic vesicles (Poodry and Edgar, 1979; Kosaka and Ikeda, 1983).
Thus, we tested the status of Ca ion influx at nerve endings of
shibire mutant Drosophila at a time when synaptic
transmission was completely blocked. Our results indicate that Ca ion
influx at nerve endings is conditionally attenuated in these
shibire mutants. Ca influx is normal at the permissive
temperature, but it is blocked at elevated temperatures. These
observations support the hypothesis that synaptic vesicles participate
in a physiological modulation of presynaptic Ca channels. On the basis
of the results with the csp mutants, we infer that csps are
part of this vesicle-mediated signaling process. Presumably, the
purpose of this vesicle-channel link is to confine Ca ion entry to
sites at the nerve ending where a vesicle is poised to discharge its
contents. In other words, our results predict that Ca ion entry at
nerve endings should be restricted to those sites where synaptic
vesicles (with their associated csps) are appropriately docked and
primed for exocytosis.
An important goal for the future is to relate the current observations
to work of others that has documented a considerable degree of
variation among cell types in the size of the Ca domains that regulate
secretion (for review, see Schweizer et al., 1995; Stanley, 1997). For
instance, work of Borst and Sakmann (1996) led to the conclusion that
an influx of >10,000 Ca ions from as many as 60 Ca channels
contributes to the triggering of individual exocytotic events at a
calyciform synapse in the rat auditory system. At the other end of the
spectrum, work of Stanley (1993) has suggested that single exocytotic
events may be induced by the influx of far fewer Ca ions (~200).
Because we have estimated that a single synaptic vesicle harbors
appreciably fewer than 60 csp molecules (Mastrogiacomo et al., 1994),
the stoichiometry of the csp-Ca channel link becomes an issue. In
other words, to reconcile our results with those of Borst and Sakmann
(1996), it is necessary to assume that a single csp can alter the
function of more than one presynaptic Ca channel (or that there is
cooperation among vesicles in the modulation of these channels). At the
same time, it is easier to accommodate a link between csps and the smaller number of release-triggering Ca channels that is suggested by
the work of Stanley (1993). Resolution of these issues should considerably improve our understanding of presynaptic secretory dynamics.
| |
FOOTNOTES |
Received Dec. 2, 1997; revised Jan. 30, 1998; accepted Feb. 18, 1998.
This work was supported by National Institutes of Health Grant NS31934
(J.A.U.) and a Grant-in-aid and a research travel grant from the
Japanese Ministry of Education (Y.K.). We thank Drs. H. Kuromi and A. Ueda for help with the Ca imaging and Dr. A. Grinnell for helpful
comments on this manuscript.
Correspondence should be addressed to Dr. Joy A. Umbach, The Department
of Molecular and Medical Pharmacology and The Crump Institute for
Biological Imaging, University of California Los Angeles School of
Medicine, Los Angeles, CA 90095.
| |
REFERENCES |
-
Borst JGG,
Sakmann B
(1996)
Calcium influx and transmitter release in a fast CNS synapse.
Nature
383:431-434[Medline].
-
Buchner E,
Gundersen CB
(1997)
The DNA-J-like cysteine string protein and exocytotic neurotransmitter release.
Trends Neurosci
20:223-227[Web of Science][Medline].
-
Chen MS,
Obar RA,
Schroeder CC,
Austin TW,
Poodry CA,
Wadsworth SC,
Vallee RB
(1991)
Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis.
Nature
351:583-586[Medline].
-
Grigliatti TA,
Hall L,
Rosenbluth R,
Suzuki DT
(1973)
Temperature-sensitive mutations in Drosophila melanogaster XIV. Selection of immobile adults.
Mol Gen Genet
120:107-114[Web of Science][Medline].
-
Gundersen CB,
Umbach JA
(1992)
Suppression cloning of the cDNA encoding a candidate presynaptic calcium channel subunit of Torpedo.
Neuron
9:527-537[Web of Science][Medline].
-
Jan L,
Jan YN
(1976)
Properties of the larval neuromuscular junction of Drosophila melanogaster.
J Physiol (Lond)
262:189-214[Abstract/Free Full Text].
-
Koenig JH,
Ikeda K
(1989)
Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockade of membrane retrieval.
J Neurosci
9:3844-3860[Abstract].
-
Kosaka T,
Ikeda K
(1983)
Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila.
J Neurobiol
14:207-225[Web of Science][Medline].
-
Mastrogiacomo A,
Parsons SM,
Zampighi GA,
Jenden DJ,
Umbach JA,
Gundersen CB
(1994)
Cysteine string proteins: a potential link between synaptic vesicles and presynaptic calcium channels.
Science
263:981-982[Abstract/Free Full Text].
-
Poodry CA,
Edgar L
(1979)
Reversible alterations in the neuromuscular junctions of Drosophila melanogaster bearing a temperature-sensitive mutation, shibire.
J Cell Biol
81:520-527[Abstract/Free Full Text].
-
Schweizer FE,
Betz H,
Augustine GJ
(1995)
From vesicle docking to endocytosis: intermediate reactions of exocytosis.
Neuron
14:689-696[Web of Science][Medline].
-
Stanley EF
(1993)
Single calcium channels and acetylcholine release at a presynaptic nerve terminal.
Neuron
11:1007-1011[Web of Science][Medline].
-
Stanley EF
(1997)
The calcium channel and the organization of the presynaptic transmitter release face.
Trends Neurosci
20:404-409[Web of Science][Medline].
-
Umbach JA,
Gundersen CB
(1997)
Evidence that cysteine string proteins regulate an early step in the Ca2+-dependent secretion of neurotransmitter at Drosophila neuromuscular junctions.
J Neurosci
17:7203-7209[Abstract/Free Full Text].
-
Umbach JA,
Zinsmaier KE,
Eberle KK,
Buchner E,
Benzer S,
Gundersen CB
(1994)
Presynaptic dysfunction in Drosophila csp mutants.
Neuron
13:899-908[Web of Science][Medline].
-
Umbach JA,
Mastrogiacomo A,
Gundersen CB
(1995)
Cysteine string proteins and presynaptic function.
J Physiol (Paris)
89:95-101[Web of Science][Medline].
-
Umbach JA, Grasso A, Zurcher SD, Kornblum HI, Mastrogiacomo A,
Gundersen CB (1998) Electrical and optical monitoring of
-latrotoxin action at Drosophila neuromuscular junctions.
Neuroscience, in press.
-
van der Bliek A,
Meyerowitz EM
(1991)
Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic.
Nature
351:411-414[Medline].
-
Zinsmaier KE,
Hofbauer A,
Heimbeck G,
Pflugfelder GO,
Buchner S,
Buchner E
(1990)
A cysteine-string protein is expressed in retina and brain of Drosophila.
J Neurogenet
7:15-29[Web of Science][Medline].
-
Zinsmaier KE,
Eberle KK,
Buchner E,
Walter K,
Benzer S
(1994)
Paralysis and early death in cysteine string protein mutants of Drosophila.
Science
263:977-980[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1893233-08$05.00/0
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Top
Abstract
Introduction
