The Neurobiology Laboratory, Department of Physiology and Institute for Biomedical Research, University of Sydney, New South Wales 2006, Australia
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ABSTRACT |
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Macleod, G. T.,
J.-B. Gan, and
M. R. Bennett.
Vesicle-Associated Proteins and Quantal Release at Single Active
Zones of Amphibian (Bufo marinus) Motor-Nerve Terminals.
J. Neurophysiol. 82: 1133-1146, 1999.
A study was made to determine the disposition of
vesicle-associated proteins (syntaxin, SV2, SNAP-25) and calcium
channels with respect to the spatial extent of spontaneous and evoked
quantal release within regions of amphibian motor-nerve terminal
branches delineated by FM1-43 stained vesicle clusters (blobs).
Discrete concentrations of vesicles revealed ~2 µm apart along the
length of terminal branches through FM1-43 staining were identical in size and spacing to those identified along terminal branches with SV2
antibody (AbSV2). Fluorescent antibodies to syntaxin 1 (AbS), SNAP-25 (AbS25) and the calcium channel 1B subunit (Ab
1B) were found in relatively high concentrations coincident with the AbSV2 blobs. Three extracellular recording electrodes were placed in the
vicinity of individual FM1-43 blobs, and an algorithm was used to
determine the spatial origin of miniature endplate potentials (MEPPs)
and EPPs together with their relative amplitudes. MEPPs and EPPs
originated throughout the region stained by FM1-43 but not elsewhere;
amplitude-frequency distributions of MEPPs and EPPs were similar for
all FM1-43 blobs with average coefficients of variation of no less than
0.28. A linear relationship existed between the size of an FM1-43 blob,
measured as the integrated extent of FM1-43 staining of a blob, and the
frequency of MEPPs as well as the probability of EPPs from the blob.
There was a proximo-distal gradient in the size of FM1-43 blobs along
the length of single terminal branches, suggesting a gradient in
release probability along the branches. The frequency distribution of the distances between blobs was approximately Gaussian, whereas the
frequency distribution of the size of blobs was highly skewed and was
best fitted with a gamma distribution. It is concluded that there are
correlations among the extent of labeling of SNAP-25, syntaxin and
calcium channels at a release site, the store of vesicles to be found
there, and the probability of spontaneous and evoked quantal release.
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INTRODUCTION |
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Birks et al. (1960)
noted, in an ultrastructural study of the amphibian motor-endplate,
that "one often sees the vesicles concentrated in certain
well-defined areas focused on a dense zone of the axon membrane
directly opposite a postsynaptic fold," which they referred to as
"special zones of the axon membrane." Couteaux and
Pecot-Dechavassine (1970)
later called these "active zones," a term now used to designate the release sites for quantal transmission at all synapses (Schikorski and Stevens
1997
). In the earliest description of quantal transmission, the
question was posed as to whether all the several hundred quantal
release sites at a motor-nerve terminal have the same probability to
secrete a quantum of transmitter (del Castillo and Katz
1954
). With the development of the concept of the active zone,
this became equivalent to asking if all the active zones have the same
probability for secretion on arrival of a nerve impulse. This assumes
that the active zone identified using structural criteria is equivalent to a quantal release site identified using functional criteria, an
equivalence that might be referred to as the active-zone hypothesis.
Postsynaptic folds occur at intervals of ~1 µm apart at motor-nerve
terminals when observed at the ultrastructural level (Dreyer et
al. 1973), a measure that has been vindicated many times with the subsequent development of iodinated
-bungarotoxin staining of
the lips of the postsynaptic folds, where the high concentration of
nicotinic receptors are located (Fertuck and Salpeter
1976
; Matthews-Bellinger and Salpeter 1978
).
However, this might not provide a good measure of the position of the
active zones as postsynaptic folds frequently occur unaccompanied by a
dense zone with its concentration of vesicles and indeed often have a
finger of Schwann cell process intercalated between the presynaptic
membrane and the postjunctional fold (see, for example, Plates 1 and 5 in Birks et al. 1960
; Figs. 2 and 3 in Kashapova
et al. 1991
). In this case, only the concentration of vesicles
together with perhaps molecular markers of the position of the dense
zone gives an unequivocal measure of the sites of the active zones,
particularly as postsynaptic folds always occur when these presynaptic
components of the active zone are present (see, for example,
Birks et al. 1960
). In the present work, it is shown
that when these criteria are used, the position of the active zones is
on average ~2 µm rather than ~1 µm ascertained on the basis of
the position of the postsynaptic folds. To determine then if the active
zone is equivalent to the quantal release site, a technique is required that can resolve the spatial distribution of extracellular currents generated by a quantal release to <1 µm. Using two extracellular electrodes placed ~10 µm apart and parallel to the long axis of a
terminal branch, del Castillo and Katz (1956)
were able
to show that discrete sites of quantal release occur on such branches. This approach gave estimates of how far apart these release sites occur
of ~0.5-2.3 µm (Wernig 1976
), although there was no
attempt to ascertain if they had the same probability for secretion.
Experiments subsequently have been performed in which a single
extracellular electrode is placed at different positions along a
terminal branch for the purposes of determining if different sets of
release sites within the recording distance of the electrode possess
the same probability for secretion (Bennett and Lavidis 1979,
1982
): very different probabilities were observed, with in
general more proximal recording sites having a higher probability than
more distal recording sites (Bennett et al. 1986
). Given that active zones occur at fairly regular intervals of ~1 µm or so
along most of the length of terminal branches (Dreyer et al. 1973
), these observations indicate that active zones do not
have a uniform probability for secretion of quanta. Indeed a detailed study of the statistics of quantal release recorded with an
extracellular electrode from visualized terminal branches indicates
that low probability for release sites are mixed in among relatively
high probability for release sites along entire terminal branches, although there is still a general proximo-distal decline in the average
probability for secretion (Bennett and Lavidis 1989
). However, neither single nor double extracellular electrode techniques give sufficient spatial resolution to reliably provide information concerning quantal release from single active zones.
This difficulty recently has been overcome by using three extracellular
recording electrodes to resolve the sites of quantal release to within
a few hundred manometers (Zefirov et al. 1990a). Such an
approach has shown that while most quantal release occurs along a line
at right angles to the longitudinal axis of terminal branches, some
does occur to either side of the line; furthermore quantal release
declines from the centers of the lines toward both ends (Zefirov
et al. 1990b
). This technique has established that spontaneous
quantal release can be recorded from all sites although only a
proportion of these have a sufficiently high probability for secretion
to participate in evoked release; furthermore sites at the distal end
of terminal branches have on average a lower probability for secretion
than do those at the proximal end (Zefirov and Cheranov
1995
). Such work confirms the observations made with a single
extracellular electrode (Bennett 1996
) and has extended it to map the release probability within the active zone itself.
The active zone hypothesis, which equates the active zone
identified on structural grounds with a functional release site, has
not yet been established. However, techniques are now available to do
this, as it is now possible to stain the vesicles "concentrated in
certain well-defined areas focused on a dense zone" using styryl dyes
such as FM1-43 to localize the center of the zones (Betz et al.
1992). Three extracellular electrodes then can be positioned around the FM1-43-stained zones to record the sites of quantal transmission with respect to the zones. Furthermore plasma membrane proteins of the soluble NSF-attachment protein receptor (SNARE) complex, namely syntaxin and SNAP-25, are likely to be very closely associated with the "dense zone" (Sudhof
1995
), so that the relationship between the
FM1-43-stained vesicles and the active zone now also can be checked.
Finally in the quantal hypothesis, the amplitude of the quantum
generated between active zones and within an active zone has been taken
to be the same (del Castillo and Katz 1954
). Whether
this is the case now can be ascertained using suitable algorithms in
conjunction with the three electrode technique. An analysis of the
active zone hypothesis and of the quantal hypothesis is provided in the
present work.
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METHODS |
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Experimental animals
All experiments were performed on the iliofibularis muscle of
the toad Bufo marinus. Animals were killed by double
pithing. As the motor nerve terminal in these animals can change its
properties with different seasons, it is important to specify the
months in the Southern Hemisphere in which the experiments were carried out (Bennett et al. 1991). Labeling with antibodies to
syntaxin 1 (AbS), SNAP-25 (AbS25), and the calcium channel
1B
subunit (Ab
1B) was done in March/April, July/August, and
August/September, respectively. Rhodamine dextran loading was performed
in July/August. Preparations for the comparison of FM1-43 and SV2
staining (Fig. 5E) were stained in December and March
(FM1-43) and November and February (SV2). Figures 6 and 7 were done in
January. Figures 9 and 10 were done in June and August, respectively.
Figure 11, A-D and E-H, were done in June and
July, respectively. Data in Figs. 12 and 13 were collected in June,
July, and August.
Antibodies and fluorescent probes
Anti-mouse SV2 antibody (AbSV2), which is specific for the
synaptic vesicle proteoglycan SV2 (Buckley and Kelly
1985), kindly was provided by Dr. Buckley (Harvard University).
Polyclonal antibodies to syntaxin 1 (AbS) and the calcium channel
1B
subunit (Ab
1B) were purchased from Alomone Labs. The polyclonal
antibody to SNAP-25 kindly was provided by Dr. Patanow (Pennsylvania
State University College of Medicine). Cy2 and Cy3 conjugates of donkey
anti-mouse and donkey anti-rabbit IgG adsorbed against cospecific IgGs,
were purchased from Jackson Immunoresearch (West Grove, PA).
Rhodamine-B-conjugated dextran (10,000 MW) (rhodamine dextran) and
N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)s-tyryl) pyridinium dibromide (FM1-43) were purchased from Molecular Probes.
Immunohistochemistry
The iliofibularis muscle and nerve were dissected intact from
animals between 50 and 70 mm in length and pinned on a silicone elastomer (Sylgard) bed in an organ bath. Preparations were fixed with
4% paraformaldehyde in 0.1 M phosphate-buffered saline [PBS; containing (in mM) 15.2 KH2PO4, 84.8 K2HPO4, and 150 NaCl, pH 7.4] at 4°C for 1 h then rinsed in PBS. Single fibers were
teased from the muscle and then placed in 0.1% dimethyl sulfoxide
(DMSO) in phosphate-buffered horse serum [100 ml PBS, 2 ml normal
horse serum (NHS), 0.1 ml Triton X-100, 1 g Bovine Serum Albumin]
for 30 min at room temperature to permeabilize the cell membranes. Fibers then were washed in PBS for 10 min (3 times), and immersed in
20% NHS in PBS for 1 h to block nonspecific binding sites in the
tissue. Fibers were rinsed in PBS for 10 min (3 times) then incubated
with primary antibodies AbSV2 and AbS, AbS25 or Ab1B at dilutions of
1:100, 1:40, 1:200, and 1:50, respectively, in PBS at 4°C for
16 h.
Fibers then were rinsed in PBS 10 min (4 times) followed by incubation
for 1 h with the secondary fluorescent antibodies at room
temperature (anti-mouse Cy2, 1:200; anti-rabbit Cy3, 1:500). Fibers
were rinsed in PBS for 10 min (3 times) then mounted in a 20% dilution
of Sigma glycerol/gelatin (GG-1) mixture, cover slipped, and sealed.
All double-labeled preparations were viewed on a Leica TCS NT UV laser
confocal microscope system with the pinhole set at a constant value to
ensure consistent illumination intensity between samples. Unless
otherwise indicated all other imaging was done using standard
fluorescence microscopy.
Loading with FM1-43 and rhodamine dextran
Motor-nerve terminals were labeled with FM1-43 (Betz et
al. 1992) by bathing the preparation in 2 µM FM1-43 in a
modified Ringer solution (53.7 mM NaCl and 60 mM KCl) for 5 min. The
preparation was washed for a minimum of 30 min before images were
captured. Betz et al. (1993)
drew the distinction
between terminal branches composed of a "linear array of nearly
circular fluorescent spots" and those containing "oval shaped spots
which sometimes appeared as distinct pairs located transversely across
the terminal width." Both terminal types were observed in the
iliofibularis of Bufo. Loading of terminal axons with
rhodamine dextran was achieved by cutting the nerve trunk close to the
point at which it enters the muscle and exposing the severed end to a
mixture containing 40 mM rhodamine dextran and 2.5% Triton X in
Ringer. The nerve was left exposed to the rhodamine dextran solution at
room temperature for between 6 and 16 h but viewed no sooner than
16 h after loading commenced. The muscle was washed continually
with fresh Ringer. [Ca2+]o was 1.8 mM when
applying FM1-43 and when loading with rhodamine dextran.
Standard fluorescence microscopy and image processing
Trans-illumination using a 50 W incandescent light source was
used alternately with epi-illumination from a 100 W mercury arc lamp.
An Olympus Fluorescein filter set was used to excite FM1-43
fluorescence, which was observed using a WV-BP310 Panasonic camera
fitted to a BHT Olympus microscope with an Olympus ×40 water immersion
objective (0.7 NA). Photo damage was minimized by stopping down the
aperture iris diaphragm and inserting a 50% NDF in the excitation
light path. These optics were used for both electrophysiology and
collecting images for quantification of FM1-43 and rhodamine dextran
staining patterns. Images were acquired using a Scion Corp LG3
frame-grabber by averaging 32 frames at a rate of approximately eight
frames per second. Pixellation of images was constant at 7.45 pixels
per micrometer. Before quantification the background was subtracted to
give a low but nonzero average value, and the remaining pixel values
were linearly stretched to give a range between 0 and 255 (Betz
and Bewick 1993). Images were collected of terminal branches
120 µm in length. In some instances, sharp focus images of an
entire branch could only be achieved by composing a montage of the
branch from images taken in different focal planes. Serial images were
adjusted to allow for bleaching. Measurements were made of the distance
between discrete blobs and the quantity of fluorescence of each blob. A
fluorescent blob was recognized as being discrete if its pixels were
separated from adjacent blobs with high-intensity value pixels by a
trough in values of >2 SD of the background noise, thus defining the
minimum distance between blobs as the distance between the centers of
two pixels separated by a single pixel (2 × 1/7.45 µm = 0.26 µm). The quantity of fluorescence for each blob was calculated
as the sum of all pixel values along a section of terminal branch,
between two contiguous troughs either side of a discrete blob, after
subtracting the average local background level from each pixel. For
characterization of these measures along the entire length of terminal
branches, each branch was divided into 10 contiguous sections, allowing
the measures from branches of different lengths to be pooled within
each length class.
Electrophysiology
The organ bath was constantly perfused at a rate of 3 ml/min with frog Ringer: which contained (in mM) 111.2 NaCl, 2.5 KCl, 1.5 NaH2PO4, 16.3 NaHCO3, 7.8 glucose, and 1.2 MgCl2 bubbled with a gas mixture of 95% O2-5% CO2. [Ca2+]o was 0.4 mM for all electrophysiological experiments and 1.8 mM for all procedures involving labeling with FM1-43. Temperature was maintained at ~20°C.
Electrophysiological recordings were made using either three or four
microelectrodes in various configurations. The tips of extracellular
electrodes were heat polished to a final inner diameter of 0.5-1.5
µm and then filled with 2 M NaCl. The intracellular electrode was
filled with 3 M KCl and yielded a resistance of ~20 M.
Microelectrodes tips were placed either at the points of a rough
equilateral triangle no more than 8 µm apart while straddling a nerve
terminal branch or in a straight line spaced no more than 4 µm apart,
parallel to the long axis of a terminal branch and no more than 4 µm
distant. Placements of the extracellular electrodes observed through
trans-illumination were made relative to a superimposed image of the
FM1-43 stained nerve terminal and their final positions relative to the
FM1-43 blobs were checked using epi-fluorescence (see Fig.
1). When the intracellular electrode was
used, the muscle cell was impaled once all extracellular electrodes were in place and both trans-illumination and epi-fluorescence were
used to record any relative movements. A video record of the muscle
surface and electrodes was made throughout the period of
electrophysiological recording using a low level of trans-illumination. A 10-nA negative current was injected through each electrode while in
recording position both before and after recording to check that the
tip resistances had remained the same. At some sites a slow,
negative-going deflection of the trace appeared consistently on all
extracellular traces after the stimulus artifact with a latency similar
to the evoked endplate potentials (EPPs). This deviation, which at some
sites had an average amplitude
50 µV, could be observed even when
no event was detected with the intracellular electrode. No attempt was
made to quantify the EPP probability at these sites. A separate
Axoclamp-2A amplifier was used for each electrode. Data were collected
using a MacLab/4 s data-acquisition system, low-pass filtered at 5 kHz
and digitized at 20 kHz. All negative-going events that were
discernible by eye were measured using Igor Pro. For a set of
amplitudes to be accepted as corresponding to the same quantal event,
they were required to occur within 0.5 ms of each other and all
amplitudes were required to be >2 SD of the noise amplitude.
Recordings were rejected for any one of the following reasons; changes
in the bright-field or fluorescence appearance of the terminal;
bursting behavior, although a consistently high level of spontaneous
release was accepted; movement of either an electrode tip or the
terminal branch by >1µm during the period of recording.
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Determination of current source locations
TRIANGULAR CONFIGURATION OF RECORDING ELECTRODES.
Data on the relative positions of the microelectrode tips and the
recorded amplitude of events from each electrode allows the calculation
of coordinates for the postsynaptic site of current generation for each
event relative to the electrode tips. For the triangular configuration
of electrodes, Zefirov et al. (1990a) previously derived
a system of equations that provide for a geometric construction of two
overlapping circles in a rectangular coordinate system the points of
intersection of which define two mathematical solutions for each event
(Fig. 2D). The equations
(11 in Zefirov et al. 1990a
) were built into an Excel
spreadsheet that automatically calculated the equations of the circles
and solved for the points of intersection from the data for each event.
On the basis of the experimental determination that voltage attenuates
as the reciprocal of the distance from the site of quantal current
generation (see Zefirov et al. 1990a
), the amplitude of
each event, at a nominal distance of 1 µm from its postsynaptic site
of origin, could be calculated by multiplying the recorded amplitude by
the calculated distance.
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Linear configuration of recording electrodes
The configuration of three electrodes along a straight line allowed data to be gathered from a longer section of the terminal branch than the three electrodes in a triangle technique but precluded the generation of two-dimensional maps. The line of electrode tips was laid parallel to the midline of the terminal branch, and the distance between the two lines was carefully monitored. The ratio of measured amplitudes from the outer two electrodes gave an inverse ratio of distances which provided for two mathematical solutions for each event along the midline of the terminal branch. One solution invariably fell in a zone between lines projected from the adjacent outer two electrodes (see Fig. 6, A-C), the other solution fell well outside this zone. Again, the amplitude of each event solution could be calculated at a nominal distance of 1 µm from its origin by multiplying the recorded amplitude by the calculated distance. For the process of determining the correct solution for each event, amplitude attenuation was calculated between the coordinates of each spatial solution and the coordinates of the central electrode. This provided an estimate of the amplitude that the central electrode would detect for each theoretical solution. The solution with the estimated amplitude closest to that measured by the central electrode was accepted. Inability to discriminate between the true and false solution was very rare.
The trigonometric construction that allows the origin of these events to be plotted on the midline of the terminal branch, based on the ratio of the measured amplitudes, assumes that the midline of the terminal branch is parallel to the line defined by the outer two electrodes. To the extent that there is a local deviation of the terminal branch from a straight line, the events detected at such locations will receive spatial solutions that are translated along the axis of the terminal branch.
The projection of all solutions onto a one-dimensional line also can
introduce error into amplitude estimates. As some terminal branches may
be 4 µm in diameter, the amplitudes calculated for events that
arise
2 µm either side of the midline of the terminal will have a
margin of error. To the extent that the smallest events are
underestimated and the largest events are overestimated, the variance
of the resulting amplitudes will be inflated. In contrast, when data
are collected with the electrode tips in a triangular configuration,
the ratio of measured amplitudes determines the exact location of the
origin of the events in two dimensions. The amplitudes associated with
these unconstrained true solutions are likely to yield a better
estimate of the underlying variance of event amplitudes.
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RESULTS |
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Vesicle-associated proteins at motor-nerve terminals in relation to FM1-43 blobs
Fluorochrome conjugated antibodies against the vesicle-associated
proteins syntaxin (AbS), SNAP-25 (AbS25), and SV2 (AbSV2) as well as
the 1B subunit of N-type calcium channels (Ab
1B) were used to
determine the distribution of these proteins along the length of
terminal branches at relatively high resolution using laser confocal
microscopy. AbSV2 staining was observed in relatively discrete blobs of
different diameter, the centers of which were separated by between 1 and 4 µm (Fig. 4A). Counter staining the same sections with AbS showed a continuous distribution along the entire length of the axolemma, with spots of high
concentration at about every 1-4 µm (Fig. 4B). The sites
of high concentration of staining due to AbS were aligned with those of
AbSV2 (compare Fig. 4, A and B). The correlation
between the intensity of AbS and that of AbSV2 staining is quantified
in Fig. 5A. Each point in Fig.
5A represents the average intensity value of a 1-pixel-thick slice taken perpendicular to the long axis of the terminal in the AbS
image compared with the intensity value from the same section through
the AbSV2 filter image. High points of AbS25 staining along terminal
branches also were aligned with high points of AbSV2 staining on the
same sections (compare Fig. 4, F with E), with
the intensities of each of these antibodies well correlated along the
length of terminal branches (Fig. 5C). Staining of the same
sections with Ab
1B and AbSV2 showed that these also possessed similar peaks along the length of terminal branches (compare Fig. 4,
D with C), although the correlation between these
peaks was not as good as that for the other antibodies (see Fig.
5B). This distribution of vesicle-associated proteins was
compared with that of regions of synaptic-vesicle endocytosis
visualized by depolarizing terminals for 5 min in a high
[K+] Ringer solution in the presence of the
FM1-43. Blobs of variable-size stain along terminal branches were
observed after this process, with the centers of these blobs also
separated by distances of between 1 and 4 µm, similar to the
distances between AbSV2 blobs observed in the fixed preparations (see
Fig. 5E, a and b).
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A comparison also was made between the extent of FM1-43 staining along the length of terminal branches and the diameter of the branches by first loading the branches with rhodamine coupled to dextran by orthograde transport into the terminals and then stimulating them in the presence of FM1-43 as described above. The extent of FM1-43 staining was well correlated with the diameter of the terminal branch at positions where the blobs of FM1-43 were detected (Fig. 5D; P < 0.01 for each of the 6 terminals branches examined), suggesting that the blob size reflects the size of vesicle clusters that extend across the whole width of the terminal.
Spontaneous potentials recorded with three extracellular electrodes in relation to FM1-43 blobs
Sets of extracellular recording electrodes were next used to
determine the amplitude of MEPPs at their site of origin as well as the
spatial distribution of these sites with respect to the blobs of FM1-43
stain along between 4- and 8-µm lengths of terminal branches. Two
different configurations of three extracellular recording electrodes
were used. In one of these, three electrodes were placed <4 µm apart
and on a line parallel to the FM1-43 stained elements of the nerve
terminal branch (Fig. 6A). In
this case, the spatial distribution of the origins of MEPPs (Fig.
6C) could be compared directly with the spatial distribution
of FM1-43 staining intensity along the terminal (Fig. 6B).
Within the field of measurement of MEPPs by the three electrodes there
was very good agreement between the peak position of the MEPP sources
and that of the peak FM1-43 staining (compare Fig. 6, C with
B). Furthermore the area over which the MEPPs originated
coincided with the area of FM1-43 stain for each blob of stain. An
exception sometimes occurs, such as that of the fourth intensity peak
from the right in Fig. 6B, which has a peak
source of MEPPs associated with it that does not fall on the peak of
FM1-43 intensity. As discussed in METHODS, this kind of
effect can be attributed to local deviations of the terminal elements
from a straight line parallel to the electrode line, such as the kink
in the terminal that coincides with the local mismatch between MEPP
sources and FM1-43 staining. Amplitude-frequency histograms of the
MEPPs for the four FM1-43 blobs that are closest to the three
electrodes gave approximately Gaussian distributions with similar means
and coefficients of variation. These are, respectively, for the
distributions from left to right in Fig. 6E, 0.77 mV and 0.38, 0.63 mV and 0.43, 0.56 mV and 0.33, and 0.50 mV and 0.36. In all,
20 FM1-43 blobs or part blobs were examined from six terminal branches,
revealing an average coefficient of variation of 0.344 ± 0.063 (SD). Correlation between the quantity of FM1-43 stain in a blob and
the frequency of MEPPs arising at that site was tested. The integrated
amount of FM1-43 fluorescence was measured for each blob or part blob
within the line of the two outer microelectrodes (Fig. 6,
A-C, ) and plotted against the number of MEPPs that arose within the length of terminal branch associated with each blob.
Of the six experiments performed using this electrode configuration, four examples (those with the most number of quantal events) are given
in Fig. 8A (a-d) with correlation coefficients
of 0.96, 0.75, 0.91, and 0.93, respectively (Fig. 6 is represented in
Fig. 8Aa).
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The other configuration of three extracellular electrodes used involved
arranging the electrodes at the corners of an equilateral triangle of
side ~5 µm, straddling one or two FM1-43 blobs of the terminal
branch (Fig. 7A). In this
case, application of the three-electrode algorithm indicated that the
source of the largest number of MEPPs was in register with the FM1-43
blobs within the triangular area contained by the three electrodes
(compare Fig. 7, C with A). MEPPs originated from
the entire region of the blob in the approximate center of the triangle
and were not confined to any particular part of the blob, such as a
line, with the frequency of MEPPs following the intensity of FM1-43
staining (Fig. 7, A and C). Comparison between
the spatial distribution of MEPPs projected onto a line at the base of
the triangle in Fig. 7C, given in Fig. 7D, and
the intensity of FM1-43 staining projected onto this line, given in
Fig. 7B, shows that the frequency distribution of MEPPs follows that of the FM1-43 intensity. As the current amplitude of a
quantal event decays rapidly with distance from its source, FM1-43
fluorescence (A) and events (C) outside the
triangle, defined as the line that circumscribes the three electrode
tips, were not considered in the quantification of fluorescence and
event numbers for comparison in Fig. 8.
In all, such an analysis was performed on six sets of recordings from
FM1-43 blobs of six terminal branches. In each recording, using the
three electrodes in a triangle technique, the MEPP frequency was
proportional to the quantity of FM1-43 staining of the terminal from
which the MEPPs arose, as shown for the four experiments in Fig. 8
(B, a-d; ).
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The algorithm next was used to determine the amplitude of the MEPPs at a fixed distance of 1 µm from their sources so as to remove the effects of current decay with distance of the microelectrode from the current source. An amplitude is calculated for each of the two theoretically possible MEPP current sources, as shown in Fig. 7F, the criterion discussed in the METHODS was used to reject the least probable solution. The amplitude-frequency histogram of MEPPs accepted in this way for the events located within the triangle is shown in Fig. 7G. These MEPPs follow an approximately Gaussian distribution with mean of 0.59 mV and a coefficient of variation of 0.27. There is no tendency for MEPPs of a particular amplitude to come from particular sites on the terminal branch (compare Fig. 7, C with A). This was the case for all of the 13 experiments using either configuration of the electrodes.
In all, using the three electrodes in a triangle technique, 16 FM1-43 blobs or part blobs were examined from seven terminal branches, revealing an average coefficient of variation of 0.280 ± 0.078 (SD). The discrepancy between this value and the value arrived at using the three electrodes in a line technique (0.344 ± 0.063, n = 20), although not significant, may result from the inferior accuracy of the latter technique as emphasized in the METHODS.
A variation on the configuration of three extracellular electrodes at the corners of an equilateral triangle was performed by introducing a fourth electrode to record intracellularly (Fig. 9E). This modification allowed simultaneous intracellular measurement of the same events detected by the extracellular electrodes to test the procedure we employ to distinguish which theoretical solutions are acceptable (see METHODS). In the example shown, a dumbbell-shaped FM1-43 blob with peaks 1.5 µm apart within the triangular array of electrodes (Fig. 9A) gave rise to a discrete spatial distribution of MEPPs that was aligned with the spatial extent of the blobs (Fig. 9C). This was confirmed by considering the projection of the FM1-43 blob onto the base of the triangle (Fig. 9B) and comparing this with the frequency of MEPPs from sites that were projected onto this base (Fig. 9D): the two were well aligned. The amplitude-frequency histogram of these MEPPs determined from the algorithm was Gaussian (Fig. 9Fb), with a mean of 0.367 ± SD 0.102 mV (CV = 0.278; n = 50). This was compared with the histogram of all MEPPs recorded with the intracellular electrode, which was also Gaussian with a mean of 0.985 ± 0.401 mV (CV = 0.407; n = 127); the variance of the MEPPs arising from all the release sites of the motor-nerve terminal being slightly greater than that of the MEPPs arising from the two FM1-43 blobs.
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Evoked potentials recorded with extracellular electrodes in relation to FM1-43 blobs
A study next was made of the spatial origins of single quantal
EPPs in relation to FM1-43 blobs arising at stained motor-nerve terminals. Three extracellular electrodes were again arranged in a
triangular array about a terminal branch (Fig.
10A), and in the example
shown, these electrodes are ~5 µm apart and straddle almost one
complete FM1-43 blob and part of another (Fig. 10B). The
spatial distribution of MEPPs in the plane of the triangle (Fig.
10C) is much the same as that of the spatial extent of the FM1-43 blobs within the triangle (Fig. 10A). This has been
quantified in the usual way by comparing the projections of the
intensity of FM1-43 staining onto the base of the triangle as shown in
Fig. 10B, with that of the spatial-frequency distribution of
MEPPs on this line, given in Fig. 10Da: the frequency is
correlated with the extent of staining. Next, the EPPs that occurred
during stimulation at 0.47 Hz for 724 s, were measured and the
algorithm applied to determine their origins and amplitudes as for the
MEPPs. The EPPs also were aligned in the two-dimensional plane of the
triangle with the FM1-43 blobs (compare Fig. 10, Cb with
A), so that when these were projected onto the base of the
triangle, the one-dimensional frequency distribution of the EPPs (Fig.
10Db) was similar to that of the intensity of FM1-43 stain
in the blobs (Fig. 10B). Similar comparisons were made for
all of the five experiments in which EPPs were collected and compared
with the intensity of stain in the FM1-43 blobs. The relationship
between EPP probability and quantity of FM1-43 in the blobs is shown
for four of these experiments (a-d) in Fig. 8B
() and although the number of blobs for each experiment is low there
is a good level of correlation in each. Data from Fig. 10 is
represented in Fig. 8Bb.
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Spatial distribution of FM1-43 blobs along terminal branches
This correlation between the quantity of FM1-43 fluorescence from each blob and the probability of evoked secretion suggests that FM1-43 staining along an entire long terminal branch during stimulation, when examined at an appropriate spatial resolution, should give a measure of the relative probability of evoked release for all release sites along the length of a branch. Figure 11A shows a simple motor-nerve terminal, possessing a branch ~40 µm long, after stimulation for 5 min in a high-[K+] Ringer solution in the presence of FM1-43. By eye the intensity of staining near the proximal end of the terminal is higher than at the distal end, although some very intensely stained sites occur in the middle of the branches (Fig. 11B). This result is shown quantitatively in Fig. 11C, at the level of resolution of 10ths of the branch length, indicating that the intensity falls off rapidly over the distal half of the branch. This method of measurement does not show up the fluctuations in FM1-43 intensity associated with the size (quantity of FM1-43 fluorescence) of the individual blobs (seen in Fig. 11B). The distance between the middle of adjacent blobs was next measured along the terminal branch and was shown to be approximately constant at ~2 µm (Fig. 11D).
|
In 50% of terminal branches on some muscles parallel sets of FM1-43
blobs were observed (Fig. 11E). These branches may be of the
kind previously described by Betz, Mayo, and Bewick
(1992)
in which vesicle accumulations tend to occur in two
clusters in transverse sections through terminal branches (see their
Fig. 8). An alternative possibility, that two branches were in the same
synaptic gutter seems less likely because of the high frequency with
which these parallel patterns of blobs were observed compared with the
relatively low polyneuronal innervation of these mature muscle fibers
(Malik and Bennett 1987
). As a consequence of the alignment of the blobs (Fig. 11E), measurements of the
fluorescent profile along the branch tended to give substantially
larger readings than for branches that did not possess such adjacent
blobs (compare Fig. 11, F with B) although the
quantity of fluorescence per single blob did not vary much along a
branch until the second half of the branch was reached when it
decreased in the proximo-distal direction along the branch (Fig.
11G). If the distance between blobs was measured for those
terminals that possessed parallel blob alignments, then this remained
the same at ~2 µm (Fig. 11H), consistent with the
interpretation that the blobs are aligned.
This kind of analysis was performed on all terminals for which FM1-43 staining was carried out, and these branches divided into three different categories according to their length, namely 0-40, 40-80, and 80-120 µm. Pooling the results for each category showed that in general the size of FM1-43 blobs decreased in the proximo-distal direction along terminal branches over the distal half of the terminal, whether these branches possessed parallel blob alignments or not (Fig. 12, A and C). Comparison of the profiles (Fig. 12, A and C) has been facilitated by stretching all values such that the highest value in the profile for each length class became the value 1. In each of these different length of branch categories the average distance between blobs was ~2 µm, and this did not vary along the proximo-distal length of the terminals branches whether they possessed parallel blob alignments or not (Fig. 12, B and D).
|
If the probability for quantal secretion is proportional to the size of FM1-43 blobs, then it is of interest to determine the frequency distribution of blob sizes over a large number of terminal branches. Although the average size of blobs for different branches did not vary much (Fig. 13A), the frequency histogram of individual blob sizes was very positively skewed, more like a gamma distribution than a Gaussian (Fig. 13B). This was not the case for the distance between blobs, which was more Gaussian, with a slight positive skew, giving an average distance between blobs of 1.89 µm and a median distance of 1.66 µm (Fig. 13D).
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DISCUSSION |
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Active zones and vesicle-associated proteins
The active zone has been defined in terms of a circumscribed
presynaptic density around which synaptic vesicles are clustered, facing openings of the postsynaptic junctional folds (Couteaux and Pecot-Dechavassine 1970; Dreyer et al.
1973
). The position of these postjunctional folds now can be
determined with considerable accuracy using
-bungarotoxin labeling
because of the relatively high concentration of nicotinic receptors on
the lips of the folds (Fertuck and Salpeter 1976
;
Matthews-Bellinger and Salpeter 1978
), allowing staining
with the toxin to identify the spatial layout of the folds. In
addition, the synaptic vesicle keratin sulfate proteoglycan SV2 is
found ubiquitously throughout the nervous system (Bajjalieh et
al. 1993
; Scranton et al. 1993
), so that the
clusters of synaptic vesicles at the active zone can be identified with
antibodies to SV2. Furthermore styryl dyes such as FM1-43 can be used
to label all of the synaptic vesicles clustered at an active zone
following a suitable protocol of stimulation (Henkel et al.
1996
). Thus the approximate middle of the cluster of vesicles should give information about the position of the active zone using
either SV2 antibodies or the styryl dyes. The present work confirms
that FM1-43 stains the set of vesicles labeled with SV2 antibody. The
average distance between the centers of the vesicle clusters stained
with FM1-43 was about the same as that when these were identified with SV2.
The localization at the presynaptic membrane of synaptic
vesicle-associated proteins that belong to the target SNARE complex proteins regulating secretion, namely syntaxin and SNAP-25
(Sollner et al. 1993), may be used to define the regions
of regulated secretion. The high concentrations of the SNAREs at
specific localizations is likely to define where vesicles in general
bind and fuse (Walch-Solimena et al. 1995
). The
isolation of a complex called the secretosome (Bennett
1996
), that includes syntaxin, which is an absolute requirement for secretion (Schulze et al. 1995
), and N-type calcium
channels (Leveque et al. 1994
; O'Connor et al.
1993
), indicates a close relationship between the
voltage-dependent channel that admits calcium ions and the SNAREs.
Indeed it appears that the C-terminal one-third end of syntaxin, which
is anchored in the presynaptic membrane, interacts with the
1B
subunit of the N-type calcium channel (Sheng et al.
1994
). Antibodies to syntaxin, SNAP-25 and the
1B subunit
therefore may be used to identify the position of regulated transmitter
secretion. These suggestions have been examined directly in frog
motor-nerve terminals using immunohistochemistry. Although it is known
that syntaxin 1 and SNAP-25 can be found along the whole axolemma, in
nonsynaptic as well as synaptic regions (Garcia et al.
1995
; Sesack and Snyder 1995
), relatively high concentrations of these are found opposite the high concentrations of
-bungarotoxin-labeled nicotinic receptors (Garcia et al.
1995
). In the present work, the high points of concentration of
syntaxin and SNAP-25, and to a lesser extent
1B label, were aligned
with the high points of concentration of the SV2 label. This suggests that the highest concentration of the core complex is located opposite
the region where the vesicle concentration is at its highest. However,
staining for proteins of the SNARE complex was no less restricted in
its spatial extent than the staining for clusters of vesicles,
indicated by both SV2 and FM1-43 staining. It seems likely then that
the region over which secretion occurs is defined by the juxtaposition
of synaptic vesicles with high concentrations of proteins belonging to
the SNARE complex.
Active zones and quantal secretion
The three extracellular electrode technique, together with FM1-43
staining of the active zone vesicles and a suitable algorithm, has
allowed a comparison to be made of both the origin of quanta in small
elements of the terminal as well as the amplitude of the quanta. Both
spontaneous and evoked quantal release were shown to originate
exclusively from the regions of the nerve terminal that contained
FM1-43 blobs, so that all release was from within these regions and
there was no release between such regions (compare with
Cherki-Vakil et al. 1995). Spontaneous quanta released,
whether at high or low frequencies, originated throughout these regions with good correlations between the frequency of this release within any
one region and the density of FM1-43 staining within the region. Given
that vesicles are concentrated at active zones, these results indicate
that the highest spontaneous release occurs at the middle of the zones
but not exclusively so, with a drop in frequency occurring with
distance from the middle of the zones in all directions. However,
Zefirov and Cheranov (1995)
have shown that sites
delineated by the spatial layout of the origins of spontaneous quanta
are much shorter toward the ends of terminal branches. There may be differences in quantal size between adjacent zones at the end of
terminal branches that would explain the slight diminution in size of
quanta observed here together with the large diminution in frequency
with two-electrode measurements (Robitaille and Tremblay 1989
; Tremblay et al. 1984
). It is interesting
in this regard that staining nerve terminal branches with
rhodamine-dextran showed that there was a decrease in the width of the
terminal branches in the proximo-distal direction, so that active zones
probably decrease in length along the branches.
Amplitudes of spontaneous quantal releases from single FM1-43 stained
sites were approximately distributed as a Gaussian. Adjacent sites
showed about the same mean amplitude of quantal sizes and a coefficient
of variation of 0.28 (electrodes in a triangle), which is about the
same as that first determined by Fatt and Katz (1952)
(30%) from their records of the intracellularly recorded spontaneous
quantal releases. There was no tendency for quanta of a particular
amplitude to be generated from a particular position in the
FM1-43-stained sites. This is interesting as it might be expected that
quantal size should decrease toward the ends of the active zone, where
the number of postsynaptic receptors available for binding with ACh
declines (Fertuck and Salpeter 1976
). These observations
on the amplitude of spontaneous quanta indicate that the variance in
the quantal size does not originate from differences between zones or
within zones, at least in the final proximo-distal third of terminal
branches where these measurements were made. Quantal variance most
likely arises at this junction as a consequence of differences in the
amount of ACh packaged into vesicles.
The observations on evoked quantal release are not as quantitatively
convincing as those on the spontaneous quanta because of the smaller
numbers of the former that were collected compared with the latter.
However, the results were qualitatively similar. The origins of evoked
quantal releases with respect to the FM1-43-stained regions were
similar to those observed for the spontaneous quanta: evoked quanta
possessed similar amplitude distributions as those of the spontaneous
quanta both for different regions of FM1-43 staining as well as within
a particular region of staining. All evoked quanta originated within
FM1-43-stained regions of the terminal branches and could be found
throughout these regions. The probability of evoked quantal release
from adjacent active zones was correlated with the relative size of the
zones as delineated by the extent of FM1-43 staining and the spatial
layout of the origins of the spontaneous quanta. Zefirov and
Cheranov (1995) and Zefirov et al. (1990b
, 1995
)
also observed this correlation between the spatial extent of the
origins of spontaneous quantal releases and the probability of evoked
quantal releases as well as the fact that adjacent active zones could
have quite different probabilities for evoked quantal release. However,
in the present work, there was little evidence for evoked release to
occur along a line, release originating from throughout the FM1-43
blob, although higher in the central and more intensely stained regions
of the blob. This difference between the present work and that of
Zefirov and Cheranov (1995)
and Zefirov et al.
(1990b
, 1995
) might arise as their observations of release,
being largely confined to a line, principally were made in the proximal
portion of the terminal branches, whereas ours were made primarily in
the final one-third of the branches. Perhaps a more plausible
explanation is that the terminals that Zefirov and his colleagues have
chosen possess vesicle blobs that are more in the form of elongated
ellipses (such as those in Fig. 7A of Betz et al.
1992
) rather than circles (as in the present work and in Fig.
7C of Betz et al. 1992
). The present work
directly confirms the observations made with a single extracellular
electrode on evoked quantal release recorded in different calcium
concentrations, namely that there are large differences in the
probability of quantal release at adjacent release sites
(Bennett and Lavidis 1986
).
It has been argued on theoretical grounds that the probability of
evoked release at an active zone should be correlated with the extent
of FM1-43 labeling of the vesicle pool there (Macleod et al.
1998). In the present work, a correlation has been found between the probability of evoked quantal release and the extent of
FM1-43 staining at a release site. This explains why correlations have
been found between the extent of quantal release from an entire
terminal and the cumulative active zone length of the terminal, if the
latter is proportional to the size of the FM1-43-stained release sites
(Propst and Ko 1987
; Propst et al. 1986
).
Given this correlation between evoked quantal release and FM1-43
staining, the latter could be used to determine the probability profile of releases sites along the whole length of all the terminal branches of a motor-nerve terminal. Such a decline in the probability of quantal
release in the proximo-distal direction confirms previous studies made
with single extracellular electrodes of release at consecutive sites
along entire terminal branches (Bennett et al. 1986
,
1989
) as well as with two intracellular electrodes placed at
either end of terminal branches (d'Alonzo and Grinnell
1985
; see, however, van der Kloot and Naves
1996
).
Active zones, quanta, and the secretosome hypothesis
The discovery of nonuniformity in the probability for secretion of
quanta at different sites along amphibian motor-nerve terminal branches
(Bennett and Lavidis 1979) has provided a preparation for the elucidation of the factors that control the probability of
secretion at release sites. In the present work, the quantal hypothesis
has been shown to hold for single release sites delineated by FM1-43
staining, but the active zone hypothesis has not been supported by the
observation that both spontaneous and evoked release occur over a
region that is substantially larger than that of the zone defined on
ultrastructural grounds (see INTRODUCTION). Although
freeze-fracture studies have not examined the ultrastructure of the
toad active zone, transmission electron microscopy shows that it is
found opposite invaginations of the postjunctional membrane at similar
intervals along the length of terminal branches as in the frog as well
as possessing similar dimensions to that in frog (Bennett et al.
1989
; Everett and Edwards 1997
). The spatial distribution of the release site is given by the extent of FM1-43 staining and therefore is determined by the number of synaptic vesicles
at the release site. The probability of evoked quantal release is
proportional to the size of the release site given by the spatial
layout of the origins of the spontaneous quantal releases as well as by
the spatial arrangement of FM1-43 labeling and that of the
vesicle-associated proteins (syntaxin and SNAP-25) together with the
N-type calcium channels. These correlations suggest that the size of
the vesicle cluster at a release site is matched by the size of the
area of membrane that possesses secretosomes. If secretosomes act
independently of each other, then the probability of individual
secretosomes secreting a quantum on the arrival of an action potential
may be determined by stochastic modeling of quantal transmission at
active zones (Bennett et al. 1997
). Given this
probability (p) and the number of secretosomes in the active
zone (n), it is a simple matter to apply the binomial equation to obtain the number of quanta released from an active zone
under an impulse. The present work supports this model by showing that
the probability of quantal secretion from a site is proportional to the
number of synaptic vesicles found there and that these are associated
with a high-density of the SNARE proteins and N-type calcium channels
that are part of the secretosome.
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ACKNOWLEDGMENTS |
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We thank Dr. Lynne Cottee for assistance with the
immunohistochemistry and confocal microscopy and Dr. Keith Brain for
assessment of the algorithm derived by Zefirov et al.
(1990a) and construction of the computerized spreadsheets.
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FOOTNOTES |
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Address reprint requests to M. R. Bennett.
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 24 December 1998; accepted in final form 14 May 1999.
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REFERENCES |
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