1Graduate Program in Neuroscience; 2Department of Psychiatry; and 3Department of Physiology, Kinsmen Laboratory, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Prange, Oliver and
Timothy H. Murphy.
Analysis of multiquantal transmitter release from single cultured
cortical neuron terminals. Application of single synapse recording methods indicates that the amplitude of postsynaptic responses of single CNS synapses can vary greatly among repeated stimuli. To determine whether this observation could be attributed to
synapses releasing a variable number of transmitter quanta, we assessed
the prevalence of multiquantal transmitter release in primary cultures
of cortical neurons with the action potential (AP)-dependent
presynaptic turnover of the styryl dye FM1-43 (Betz and Bewick
1992, 1993
; Betz et al. 1996
). It was assumed
that if a high proportion of vesicles within a terminal were loaded with FM1-43 the amount of dye released per stimulus would be
proportional to the number of quanta released and/or the probability of
release at a terminal. To rule out differences in the amount of release (between terminals) caused by release probability or incomplete loading
of terminals, conditions were chosen to maximize both release
probability and terminal loading. Three-dimensional reconstruction of
terminals was employed to ensure that bouton fluorescence was accurately measured. Analysis of the relationship between the loading
of terminals and release indicated that presumed larger terminals
(>FM1-43 uptake) release a greater amount of dye per stimulus than
smaller terminals, suggesting multiquantal release. The distribution of
release amounts across terminals was significantly skewed toward higher
values, with 13-17% of synaptic terminals apparently releasing
multiple quanta per AP. In conclusion, our data suggest that most
synaptic terminals release a relatively constant amount of transmitter
per stimulus; however, a subset of terminals releases amounts of
FM1-43 that are greater than that expected from a unimodal release process.
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INTRODUCTION |
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The action potential (AP)-dependent uptake and
release of the styryl dye FM1-43 was used to evaluate the behavior of
single CNS synaptic terminals (Murthy and Stevens 1998;
Murthy et al. 1997
; Ryan and Smith 1995
;
Ryan et al. 1996
) and neuromuscular junction endplates
(Betz and Bewick 1992
; Betz et al. 1992
).
These studies exploited the ability of released synaptic vesicles to recycle (Heuser and Reese 1973
) and be loaded with dye.
Rigorous control studies by the Betz laboratory established that
FM1-43 is localized to synaptic vesicles and that its turnover
correlates well with more direct capacitance measurements of release
(Henkel et al. 1996
; Smith and Betz
1996
). Use of FM1-43 in CNS studies indicates that the rate of
dye release from loaded terminals is a reliable indicator of synaptic
strength (Isaacson and Hille 1997
; Murthy et al.
1997
; Ryan et al. 1996
). Furthermore,
application of FM1-43 uptake and release to CNS neurons were used to
provide data for statistical analyses of transmitter release
(Murthy et al. 1997
, 1998
; Ryan et al.
1997
). These studies suggest a multimodal release process in
CNS neurons in which individual quantal peaks can be identified in data
from FM1-43 labeling of vesicles. It was argued that these peaks
reflect single-vesicle release events. One assumption made in the study
by Murthy and Stevens (1998)
and Murthy et al. (1997)
was that CNS
terminals release at most one vesicle per AP stimulus. Therefore if
terminals were restricted to releasing a single vesicle apparent
differences in the amount of FM1-43 turnover between terminals would
reflect release probability (Murthy et al. 1997
). We
extended these studies and further tested these assumptions in primary
cultures of cortical neurons by using conditions under which maximal
loading of terminals was achieved, where release probability was high,
and where analysis procedures were used to assure that bouton
fluorescence was accurately measured. Our results indicate that
multiquantal release occurs yet is restricted to a small but
potentially significant fraction of cultured cortical neuron synapses
(<20%).
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METHODS |
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Embryonic cortical neurons and glial cells (from day 18 rat fetuses) were grown 3-4 wk in vitro on poly-D-lysine-coated glass coverslips before use in imaging experiments. Coverslips were cut into two pieces, placed into a customized perfusion chamber (volume ~500 µl), and fixed by platinum weights to prevent drifting. Continuous perfusion was supplied by a Hanks balanced saline solution (HBSS) medium containing (in mM) 137 NaCl, 5.0 KCl, 0.34 Na2HPO4 (7H2O), 10.0 Na+-HEPES buffer, 1.0 NaHCO3, and 22.0 glucose at pH 7.4 and ~315 mosm. CaCl2 and MgSO4 were altered as indicated. To stimulate activity-dependent synaptic uptake (loading) and release (unloading) of FM1-43, constant current stimulation (30 mA) was delivered via two platinum electrodes fixed on opposite sides of the perfusion chamber (distance ~8 mm). This field stimulation reliably induced AP generation in single neurons (see Fig. 1). All experiments were conducted at room temperature (~23°C).
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Bouton loading was achieved by application of 1,200 stimuli at 10 Hz in
the presence of 10 µM FM1-43; CaCl2 and
MgSO4 were supplemented at 2.5 and 1.0 mM, respectively. We
expected that this number of stimuli would result in complete loading
of vesicle pools, as FM1-43 experiments (Liu and Tsien
1995; Murthy et al. 1997
) and ultrastructural
analysis (Harris and Sultan 1995
) estimate that, on
average, cortical synapses contain <500 synaptic vesicles. During
AP-evoked FM1-43 loading, synaptic activity was blocked with a
cocktail of glutamate receptor blockers
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 3 µM) and
2-amino-5-phosphono-valeric acid (D,L-APV; 60 µM). The time of FM1-43 exposure coinciding with AP trains was ~2
min and continued for 60 s after the last AP to allow for
completion of endocytosis (Ryan and Smith 1995
). After
bouton loading, the preparation was washed for 15 min. The washing
medium was CaCl2-free and contained 5 mM MgSO4
to minimize synaptic FM1-43 release attributed to spontaneous APs and
miniature synaptic activity. Bouton unloading was performed in a medium
expected to result in maximal release probability (5 mM
CaCl2 and 1 mM MgSO4). CNQX (3 µM) and APV
(60 µM) were also supplemented to block recurrent synaptic stimulation.
Confocal imaging with a Bio-Rad MRC 600 system attached to a Zeiss upright (Axioskop) microscope with an Olympus 0.9 NA '60 water immersion objective was used for all experiments. Laser intensity was attenuated to 1%, and the confocal pinhole was set to 3.5 (Bio-Rad units). To improve signal-to-noise properties, the slow scan mode (without averaging) was used. Corrections in image intensity were made for field inhomogeneity (signal attenuation on edges) associated with high NA objectives by dividing data sets by a control image (carboxyfluorescein solution).
For data acquisition a field of 128 × 128 µm (400 × 400 pixels) was scanned every 3 s during baseline and periods of AP trains (see Fig. 3). Imaging during the first 45 s (baseline) was used to calculate signal-to-noise properties at each synapse and was followed by continuous 1-Hz field stimulation (21 s) to determine synaptic FM1-43 unloading responses. After this first train stimulus, a second baseline without stimulation (30 s) was established to allow reloading of the readily releasable pool of vesicles. Finally, a second 1-Hz train (21 s) consisting of either 21 paired (10-ms interpulse interval; n = 18 experiments) or unpaired stimuli (n = 4 experiments) was applied, and presynaptic FM-43 fluorescence intensity was monitored. This acquisition was followed by a 10-Hz stimulus train (120 s) to determine the total amount of FM1-43 fluorescence that was releasable by APs (Fig. 3B). A (vertical) z series of 13 consecutive confocal images (spaced at 0.54 µm) over the area of interest was acquired for each experiment with a computer-controlled focus motor. Each bouton's fluorescence intensity in the focal plane was corrected based on its relative position within the confocal z section (Fig. 2). Additionally, boutons contaminated by signals from stained structures above or below their focal plane were eliminated from further analysis (e.g., Fig. 2, boutons 2 and 3).
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Confocal images were exported as byte arrays by removal of data headers and analyzed with custom routines written with the IDL (Research Systems, Boulder, CO) programming language. For each experiment, 300 putative synaptic boutons were analyzed, and fluorescence changes over time were averaged over ~3.7 µm2 at each site. Nonreleasable FM1-43 fluorescence (defined as background fluorescence remaining after the 1,200-pulse stimulus train) was subtracted at each bouton before further analyses were performed. FM1-43 release in response to AP-inducing field stimulation was averaged over the 21-s stimulus train (7 images), and an automated response criterion was used to select responsive from nonresponsive putative boutons (Fig. 3A). To be considered for further analysis, boutons had to meet the following criteria: 1) the decrease in FM1-43 fluorescence in response to two 1-Hz trains of stimulation had to be >2.5 SD of the baseline fluorescence, and 2) the baseline variation (SD) had to be <10% of the bouton's total releasable fluorescence. For experiments in which the effect of changes in [Ca2+]o on paired-pulse modulation (PPM) were examined, the first selection criterion was modified (the decrease in FM1-43 fluorescence in response to the paired 1-Hz train stimulation alone had to be >2.5 SD of the baseline fluorescence). This more strict criterion was used to counteract effects of low [Ca2+]o on the signal-to-noise properties of FM1-43 release values caused by low release probability and to more accurately measure PPM by avoiding selection of boutons exhibiting high release during both stimulus trains.
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To determine the degree of PPM at each bouton, the relative FM1-43 release (normalized to baseline period 2) during the second (paired) stimulus train was divided by the relative FM1-43 release (normalized to baseline period 1) during the first (unpaired) stimulus train (Figs. 3, A and B, and 4B). Analysis was restricted to boutons with PPM values between 0 and 10 (98% of all sites). For control experiments, both stimulus trains consisted of unpaired stimuli (Fig. 4A), and the FM1-43 release ratio was calculated accordingly (relative FM1-43 release during second/first stimulus train).
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Whole cell, current-clamp experiments (Hamill et al.
1981) were conducted with an Axon Instruments Axopatch 200B
amplifier and 7 M
electrodes pulled from 1.5-mm glass capillaries.
The patch pipettes were filled with a solution containing (in mM) 0.05-0.3 fluo-3 K+ salt, 122 K+MeSO4, 20 NaCl, 5 Mg-ATP, 0.3 GTP, and 10 HEPES (pH 7.2); in some cases EDTA was substituted for fluo-3 as a
Ca2+ buffer.
For statistical testing of normality, the Kolmogorov-Smirnov test was used. Nonparametric tests were applied for comparisons of medians (Mann-Whitney test). For correlation analysis, the nonparametric Spearman test was used over the Pearson test when it resulted in a better fit to a linear model. One-way analysis of variance (ANOVA) was used to confirm that data acquired in different experiments could be pooled.
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RESULTS |
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Activity-dependent turnover of the synaptic vesicle probe FM1-43
was used in primary cultures of cortical neurons to estimate the number
of transmitter quanta released from a single terminal in response to AP
stimulation. To ensure that FM1-43 bouton loading and release were
accurately measured, several control experiments were performed. By
using whole cell, current-clamp recordings it was established that
electric field stimulation (1 ms; 30 mA) reliably resulted in single
APs (n = 8 cells; data not shown). Paired field stimuli
(10-ms interpulse interval; 5 mM Ca2+, 1 mM
Mg2+) applied every 1 s for 50 s also reliably
induced AP pairs (Fig. 1A; n = 4 cells).
Additionally, high-frequency trains of APs (
600 APs at 10 Hz) were
reliably delivered without failure of APs (Fig. 1B;
n = 4 cells). Thus whole cell, current-clamp records
suggest that the number of field stimuli given is indicative of the
number of APs produced. For studies in which field stimuli were
employed, a stimulus intensity of ~50% above threshold was used to
ensure that most terminals were responsive. This threshold was
established three ways: direct measurement of the relationship between
FM1-43 release and field voltage, current-clamp recordings, and the
use of fluo-3-loaded cultures (data not shown) as described by others (Ryan and Smith 1995
).
The goal of our experiments was to quantitate the amount of FM1-43
released from terminals to determine if single or multiple vesicles
were released per AP. Stained boutons could potentially be out of focus
and were also subject to signal contamination from neighboring boutons
or other stained structures. Therefore we corrected each bouton's
fluorescence intensity based on its relative position within the
vertical (z-) axis of the specimen (Fig. 2; see
METHODS) and excluded boutons contaminated by signals from
stained structures above or below their focal plane (e.g., Fig. 2,
boutons 2 and 3). To establish whether presumed boutons generated a
significant FM1-43 unloading response during field stimulation,
automated software procedures were used that selected responsive
boutons based on their individual signal-to-noise properties (Fig.
3A; see METHODS). FM1-43 fluorescence that
could not be released during a 10-Hz field stimulation train (1,200 APs; Fig. 3B) was subtracted at each site to ensure that
fluorescence values obtained accurately represent a pool of releasable
synaptic vesicles (Murthy et al. 1997; Ryan and
Smith 1995
). Analysis of the relative release rates indicated
that on average little depression occurred over the 21 stimuli applied
at 1 Hz (Fig. 3C).
We assumed that the amount of FM1-43 release from a single bouton is
proportional to release probability, the fraction of the vesicular pool
loaded with FM1-43, and the number of quanta released with a single AP
(see Eqs. 1 and 2 and DISCUSSION).
Because release probability (Prel.) is dependent
on [Ca2+]o (Dodge and Rahamimoff
1967; Katz and Miledi 1968
; Mintz et al.
1995
), we elevated [Ca2+]o to 5 mM to
maximize Prel. and thus ensure that potential
differences in FM1-43 release reflect the number of quanta released
and not Prel.. To confirm a high release
probability under these conditions, we used paired-pulse stimulation
(Castro-Alamancos and Connors 1997
; Debanne et
al. 1996
; Dobrunz and Stevens 1997
;
Stevens and Wang 1995
). In all experiments
(n = 18) we applied a second paired stimulus train (21 paired stimuli at 1 Hz; interpulse interval 10 ms) after the initial
1-Hz stimulus train and calculated the degree of PPM at each bouton
(n = 1,772) by dividing the relative FM1-43
fluorescence change (% change) observed during the second train by
that observed during the first train (Figs. 3B and
4B). To allow sufficient time for reloading the readily
releasable pool of synaptic vesicles (Dobrunz and Stevens
1997
; Stevens and Sullivan 1998
; Stevens
and Tsujimoto 1995
), we established a second baseline period
(30 s) between the two stimulus trains. As a control we conducted
experiments (n = 4) in which both first and second stimulus trains were comprised of only single stimuli. In these control
experiments, the ratios of the FM1-43 fluorescence changes during
stimulus train 2 versus stimulus train 1 were distributed around a
median of 0.96 (n = 448 boutons; Fig. 4A).
The slightly higher mean release ratio of 1.13 ± 0.83 was
attributed to only 5% of values (outside the median release ratio ± 2 SD); exclusion of these potentially spurious values resulted in a
mean release ratio of 1.00 ± 0.52. This result demonstrated a
constant rate of FM1-43 release at single boutons in response to
identical consecutive stimulation protocols. This control is important
as it establishes that the system is stable and exhibits little
run-down or facilitation.
The values for PPM (1 Hz paired/1 Hz single stimuli) at single synaptic
boutons were distributed around a median of 1.36 (mean 1.77 ± 1.46; n = 1,772; Fig. 4B). A PPM ratio of
2.0 would be predicted if both the first and second stimulus were
successful. Comparison of the release ratios obtained with the two
different stimulation paradigms indicated greater release with paired
stimuli than with single stimuli (Mann-Whitney test: P < 104). However, the majority of synapses (>75%)
showed PPM values <2, confirming that most synapses in these cultured
preparations possess a high Prel. in 5 mM
[Ca2+]o. Additional control experiments
(n = 4; 1 mM [Ca2+]o)
indicated that evoked FM1-43 could be elevated by 60% (difference in
median release rate; Mann-Whitney test: P
<10
4) when increasing [Ca2+]o
from 1 to 5 mM (n = 51/691 boutons). Paralleling this
increase in FM1-43 release rates, we found a significant decrease in
PPM (25% difference in median PPM; Mann-Whitney test: P
<0.05) when increasing [Ca2+]o from 1 to 5 mM. These findings are in agreement with studies that show maximal
Prel. and paired-pulse depression in cortical and hippocampal synapses in 5 mM [Ca2+]o
(Castro-Alamancos and Connors 1997
). To further rule out
that differences in FM1-43 release amounts between synaptic boutons were caused by differences in Prel., we
restricted further analyses to boutons with a high initial
Prel. (PPM ratio <2; n = 1,292 boutons).
To measure the AP-induced FM1-43 release from single boutons, we
averaged the change in FM1-43 fluorescence over seven images (recorded
every 3 s) during 1-Hz stimulation (21 stimuli; Fig. 3,
B and C). Data were pooled data from 18 separate
experiments that were conducted under comparable conditions (constant
confocal gain and pinhole setting, laser intensity, and FM1-43
concentration). To determine that these experiments were comparable, we
conducted a one-way ANOVA on both baseline variation and FM1-43
release data. This analysis demonstrated that for the 18 experiments
analyzed both the stimulated FM1-43 release and the baseline noise
values were drawn from the same distributions and thus could be pooled (one-way ANOVA: P <0.05). Similarly, one-way ANOVA found
that the FM1-43 release ratios obtained in four control experiments were also drawn from the same distribution and thus could be pooled (P <0.05). In the analysis of the pooled data we found that
the relative FM1-43 fluorescence decrease over the 21 s of
stimulation was linear (linear regression: r = 1.0;
P < 10
5; average of n = 1,292 boutons; Fig. 3C), indicating that on average little
depression of release occurred during this period. Within this
population of synapses we found that the degree of FM1-43 unloading
during the 21 APs train varied considerably (mean 5.80 ± 3.21 pixel value). Accordingly, we also found a high CV (SD/mean: 0.44) for
FM1-43 unloading after subtraction of the baseline variance. Furthermore, we found that the amount of FM1-43 loading into boutons after 1,200 APs, a measure of the vesicular pool and thus synapse size
(Henkel et al. 1996
), exhibits a similar high degree of
variability (mean 39.7 ± 18.4 pixel value; CV baseline variance
subtracted: 0.46). When comparing the amount of FM1-43 loading with
the amount of FM1-43 fluorescence released per AP we observed a
significant positive correlation (r = 0.65;
P <10
5; n = 1,292 boutons)
between these two parameters (Fig.
5A). However no significant
correlation (r =
0.03; P = 0.50) was
found between the amount of FM1-43 unloading during 21 APs and the
degree of baseline variation measured over an identical time period
(Fig. 5B). Hence factors such as FM1-43 bleaching or dye
loss from nonvesicular pools contributed little to the observed
positive correlation between synaptic FM1-43 loading and release.
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As our data indicated that the amount of FM1-43 released per AP and
the total FM1-43 loading of boutons were not constant (Fig.
5A), we analyzed the distribution of FM1-43 release amounts across boutons. This distribution demonstrated a positive skew when
data sets comprising most boutons (PPM <10; skew = 2.17 pixel value; n = 1,772 boutons) or only those boutons with
high Prel. (PPM <2; skew = 2.43 pixel
value; n = 1,292 boutons) were examined (Fig.
6, A and B). Hence
the positive skew toward higher FM1-43 release amounts was not
attributed to sites with low initial Prel. (480 sites with PPM >2). Furthermore, the degree of PPM (a measure of
Prel.) contributed little to the observed
positive skew in FM1-43 release amounts at synaptic boutons with high
Prel. (PPM <2; n = 1,292 boutons) as we find a poor correlation (r = 0.25) between the amount of FM1-43 release and the degree of PPM at single
sites (Fig. 6D). In contrast, when considering the full range of PPM values (0-10; n = 1,772 boutons) we find
a better correlation (r =
0.54) between PPM and
FM1-43 release amounts, mostly caused by boutons with low initial
release amounts (Fig. 6C).
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As we observed that data sets with a broad range of PPM values (<10)
could contain boutons with low initial Prel.
(high PPM values), we restricted further analysis to data sets in which boutons possessed PPM values of <2 (Fig. 6B). To examine
how the distribution of the FM1-43 release amounts compared with the
noise within this data set (caused by bleaching, background dye loss, and instrument noise), we calculated the FM1-43 fluorescence variation at each bouton during a baseline period identical to the stimulus train
duration (Fig. 6B; baseline noise as shaded histogram). The
baseline noise data were not positively skewed (median: 0.07 pixel
value; mean:
0.11 ± 1.93 pixel value; skew:
0.13 pixel value)
and hence could not account for the positive skew observed in the of
FM1-43 release amounts. Additionally, the negative skew in the
baseline noise distribution was attributed to only 0.2% (3/1,292) of
the values; exclusion of these values resulted in as normally
distributed baseline noise population (Kolmogorov-Smirnov test vs. a
Gaussian distribution: P >0.05). Comparison between the
distributions of evoked release amounts and baseline noise indicated
that the variation in evoked release amounts between boutons was
significantly greater than that expected from baseline variation alone
(Kolmogorov-Smirnov test: P <10
5).
To estimate the fraction of boutons exhibiting multiquantal release, we used two different methods: determination of the number of release values that 1) cause the positive skew of the distribution and 2) are outside the median release value +2 SD of the baseline noise. By using the first approach, we found that the skew in the distribution of evoked FM1-43 release could be attributed to 17% (n = 221/1,292) of all boutons with the highest FM1-43 release amounts (Fig. 6B), as exclusion of these boutons from the analysis resulted in a population that was not different from the baseline noise distribution (Kolmogorov-Smirnov test vs. baseline noise: P >0.05). By using the second approach, we found that 13% (n = 167/1,292) of the values were greater than the median release value +2 SD of the baseline noise. With the use of a computer simulation that modeled the signal-to-noise properties of our system, we observed that the Kolmogorov-Smirnov test could reliably detect a skewed population (n = 1,292 release values) attributed to even <10% of boutons exhibiting multiquantal release. Thus during stimulation most of the boutons released FM1-43 amounts that could be described by a single gaussian peak, whereas release amounts of the remaining sites (13-17%, depending on the analysis) were outside a unimodal distribution and were apparently the result of multiquantal release.
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DISCUSSION |
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We observed a significant positive correlation between loading of
single boutons (with FM1-43) and the amount of FM1-43 they release in
response to APs generated with field stimuli (Fig. 5A). A
similar result was previously observed by Betz and Bewick (1993) at the
neuromuscular junction. These authors found that despite variability in
the intensity of single FM1-43 spots most sites appeared to release a
relatively constant percentage of loaded FM1-43 per stimulus. Their
conclusion was that larger release sites release proportionally more
transmitter. Studies also indicate a similar relationship in central
neurons as Isaacson and Hille (1997)
and Ryan et al. (1997)
reported
that, whereas the percentage of FM1-43 release from loaded terminals
varies little, the size of terminals varies greatly. These observations
indicated that CNS synaptic terminals do not release a constant amount
of transmitter per impulse. Ryan et al. (1997)
, with FM1-43
endocytosis in response to single APs, reported that multiquantal
release may occur from hippocampal terminals. However, it is possible
that the apparent differences in the number of quanta released may
reflect differences in release probability among boutons
(Hessler et al. 1993
; Murthy and Stevens
1998
; Murthy et al. 1997
; Rosenmund et
al. 1993
) or conceivably incomplete loading of vesicle pools.
Murthy et al. (1997)
reported that larger terminals, those with greater
FM1-43 loading, had a higher release probability. An assumption made by Murthy and Stevens (1998)
and Murthy et al. (1997)
and (for some
experiments) by Ryan et al. (1997)
was that endocytosis and exocytosis
were matched so that the degree of FM1-43 loading (endocytosis) would
be a measure of release probability. We extended these findings by
directly measuring FM1-43 release (and not endocytosis) with conditions in which two complicating variables, vesicle pool loading and release probability, were fixed at saturating levels. Additionally, to prevent synaptic depression we used brief and low-number stimulation protocols (21 APs at 1 Hz) that result in the release of only a small
fraction (on average 15%) of the dye-loaded vesicle pool. As vesicle
repriming (availability of vesicles once released) was estimated to
have a t1/2 of ~20-30 s (Ryan
et al. 1993
, 1996
), we did not expect significant dilution by
unlabeled vesicles during our dye unloading measurements. By using this
experimental approach, we confirm the findings of Korn et al. (1993)
,
Trussell et al. (1993)
, Tong and Jahr (1994)
, Vincent and Marty (1996)
, Ryan et al. (1997)
, and Auger et al. (1998)
, which indicate that multiquantal transmitter release can occur from single synaptic terminals, albeit at a relatively small proportion of terminals (<20%). Presumably multiquantal release could account for a portion of the apparent variability in the amplitude of synaptic responses that
are recorded from single boutons (Forti et al. 1997
;
Liu and Tsien 1995
; Murphy et al. 1995
).
Our observation that, under conditions of high release probability,
presumed larger boutons (greater FM1-43 loading) release more FM1-43
per impulse than smaller terminals suggests multiquantal release. As
larger boutons possess greater release site areas and more docked
vesicles (Schikorksi and Stevens 1997) they provide a
conceivable anatomic basis for multivesicular release. Fitting the
amount of FM1-43 released versus bouton loading to a model where
bouton volume was proportional to release site area failed to describe
our data. This model would predict a curve with a slope proportional to
bouton radius
1 (bouton area/bouton volume;
r2/4/3
r3 = 0.75r
1). The
data were better described by a simple linear relationship between
bouton loading and the amount of release. However, analysis of the
correlation indicates that a relatively large fraction of the observed
variance in release amounts (42%; R2 = 0.42;
Fig. 5A) could be accounted for by a linear relationship with bouton loading.
As synaptic boutons possess a variety of parameters that control the
rate of evoked vesicular release (excitability, probability of release,
amount of release, stimulation induced facilitation, or depression), we
used different controls to confirm that differences in the rate of
FM1-43 unloading among boutons would reflect differences in the amount
of vesicular release and not other parameters of synaptic variability.
First, we chose stimulus parameters that result in a linear rate of
FM1-43 release during a train of APs (Fig. 3C). This
apparent linear rate of release was confirmed by the analysis of
consecutive images of FM1-43-loaded boutons as described by Isaacson
and Hille (1997). Second, we used conditions expected to result in
maximal release probability at all boutons and excluded boutons from
the analysis that showed the potential of further increase of release
probability (Figs. 4 and 6). Additionally, factors such as AP
propagation failure were unlikely to account for the variability in
release amounts among terminals (Allen and Stevens 1994
;
Mackenzie et al. 1996
)
Consistent with the idea of multiquantal release, analysis of the
distribution of release amounts demonstrated a significant skew toward
higher release amounts, which was in excess of the system noise. Making
the assumption that the peak with the smallest amplitude reflects the
fluorescence value of a single vesicle, we would expect that most
terminals on average release one vesicle and that a subpopulation of
boutons (<20%) releases two or more vesicles per stimulus.
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(1) |
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(2) |
The apparent linear relationship between release amount and the bouton
size would suggest a process in which terminals can regulate the number
of quanta released based on their size. However, this is not a strict
relationship as additional synaptic parameters (other than terminal
size) could contribute to the skew in FM1-43 unloading of boutons.
Structural analysis of forebrain excitatory synapses suggests that a
significant proportion, 10-20%, contains multiple release sites
(reviewed by Edwards 1995). From our data it is not
possible to determine whether terminals that release a larger amount of
FM1-43 per impulse contain multiple release sites. Furthermore, in
using mass cultures of neurons we assume that all neurons regardless of
phenotype (i.e., glutamatergic or GABAergic) load a similar amount of
FM1-43 into their terminals. This is most likely the case because the
dye is loaded passively (Betz et al. 1996
) into vesicles
that possess similar sizes in different phenotypes of CNS synaptic
terminals (Hamori et al. 1990
). Nevertheless, regardless
of the previous caveats, our data obtained by analysis of FM1-43
loading and unloading suggest that CNS terminals can release multiple
quanta, adding caution to interpretation of experiments that apply
quantal analysis to CNS synapses.
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ACKNOWLEDGMENTS |
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We thank P. Mackenzie for preparation of cultures, assistance with some experiments, and comments on the manuscript.
T. H. Murphy is a Medical Research Council (MRC), EJLB, and Sloan Research Scholar. O. Prange is supported by a scholarship of the Deutscher Akademischer Austauschdienst. This work was supported by funds from the EJLB Foundation and by an operating grant from MRC Canada.
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FOOTNOTES |
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Address for reprint requests: T. H. Murphy, Kinsmen Laboratory, 4N1-2255 Wesbrook Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 August 1998; accepted in final form 14 December 1998.
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