Vesicle-Associated Proteins and Quantal Release at Single Active Zones of Amphibian (Bufo marinus) Motor-Nerve Terminals

G. T. Macleod, J.-B. Gan, and M. R. Bennett

The Neurobiology Laboratory, Department of Physiology and Institute for Biomedical Research, University of Sydney, New South Wales 2006, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1B subunit (Abalpha 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1B subunit (Abalpha 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 alpha 1B subunit (Abalpha 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 Abalpha 1B 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 MOmega . 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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Fig. 1. Placement of electrodes with respect to FM1-43 blobs on a terminal branch. Terminal branches first were stained with FM1-43 and the electrode tips of 3 microelectrodes manipulated into place relative to each other in the same plane of focus. Stained nerve terminal then was raised into the plane of focus from below. A: electrodes manipulated into position above the terminal branch (bright-field). B: FM1-43-stained terminal imaged prior to the placement of microelectrodes (epi-fluorescence). C: superimposed images from A and B. D: microelectrodes in their final position for recording at the surface of the muscle (bright-field). E: same as in D but viewed using epi-fluorescence. F: superimposed images from D and E.

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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Fig. 2. Algorithms for determining the source of a quantal event and its amplitude. A: recordings of a single miniature endplate potential (MEPP) by 3 electrodes (a-c) positioned around an FM1-43 blob on a terminal branch; the ratio of their amplitudes is 0.7 is to 1 is to 1.4, respectively; this yields a ratio of distances from the electrodes to the source of the MEPP of 1.4 is to 1 is to 0.7, respectively, assuming that the MEPP amplitude recorded at each microelectrode is inversely proportional to its distance from the source of the MEPP. B: 1 of the 2 possible locations for the source of the MEPP at the intersection of the THREE circles shown with radii in the ratio of 1.4 is to 1 is to 0.7 centred on the electrodes (the axes, x and y, define the spatial dimensions in the plane of the 3 electrode tips). C: other possible location at the intersection of the 3 circles the radii of which are also in the ratio of 1.4 is to 1 is to 0.7. D: another approach for determining the source of the MEPP: this involves calculating the locus of possible source locations determined by consideration of the MEPPs recorded by just 2 microelectrodes in turn; the loci based on amplitudes from microelectrodes a and b is given by the lower circle; microelectrodes b and c giving the upper circle; the intersection of these 2 sets of loci gives 2 solutions to the problem in the same way as does the approach illustrated in B and C. Further criteria must be used to decide which of the TWO solutions in B and C or D is correct. Assumption that the event amplitude recorded at each microelectrode is inversely proportional to the distance from the source of the event allows an estimate to be made of the amplitude of an event as it would be measured at a distance of 1 µm from the site of its generation.

The computerized algorithm provided two theoretical solutions for each event. An absolute amplitude was calculated for each of the solutions. As the objective was to generate spatial maps of quantal release independently of information about the location of elements of the terminal branch, a procedure by which to identify the correct solution was developed based on amplitude. If we accept the mathematical solutions closest to the center of the three electrodes for every event, the amplitudes associated with these solutions will be the smallest amplitudes. Not all of the solutions in this group will be correct as some of the events detected will have been events with a large amplitude outside the triangle defined by the electrodes. However, the correct amplitude will be determined for all events that occurred within the electrodes, some of which will be large-amplitude events. The rejection criterion is as follows: if the amplitude associated with one of the mathematical solutions is >2 SD above the mean of the smallest amplitudes, that solution can be rejected as being improbable; if the other solution has an amplitude that is below this value, it can be accepted (see Figs. 7F and 9Fa). If neither or both amplitudes are above this value, both solutions, and hence the event, is rejected. It is important to remember that most of the events, because of the small radius of detection of each electrode, are likely to have occurred within the electrodes.

The validity of this procedure was tested by using a fourth microelectrode to record the quantal events intracellularly at a location within 20 µm of the three extracellular electrodes (Fig. 9E). Figure 9Fa shows a plot of the amplitude of the solution calculated to be spatially furthest from the microelectrodes (solution 2) against the amplitude of the solution that is closest (solution 1). The two arrows indicate the same value on each axis, in this case 0.575 mV, which is 2 SD above the mean of the amplitudes of solution 1; the threshold used to reject solution 2 for those events in the top left quadrant (delineated by the arrows). Figure 9Fc shows a plot of the amplitude of each miniature EPP (MEPP) recorded with the intracellular electrode against the amplitude calculated for the same event recorded with the extracellular electrodes and accepted in Fb as being the most probable solution. These amplitudes were correlated with a coefficient of 0.87 (Fig. 9Fc). Variance about the line of best fit in the direction of the ordinate is generally within ±2 SD (SD = 146 µV) of the noise of the intracellular trace. In this example, the exclusion of one of the solution pair on the basis of being physiologically improbable is an exacting test of the rejection criterion as the more distant solution is on average not greatly improbable (compare the 2nd solutions in Fig. 9Fa with those in Fig. 7F).

All solutions were plotted relative to one electrode defined as the origin in the rectangular coordinate system. All movements of the preparation and electrode tips during the period of recording were corrected for by the computerized algorithm. Only events within the triangulation region at the time of the event were accepted in experiments where drift occurred. The video record was used to confirm that electrode or preparation drift was uniform over the recording period. Numerous simulated data sets were processed to test the ability of the algorithm to reproduce two-dimensional maps, calculate the correct amplitude for events, and correct for terminal and electrode drift. The effect of electrical noise on the scatter of current generation sites and amplitude variance was simulated (Fig. 3) based on the electrode configuration and electrical measurements made in the experiment in Fig. 7. The simulation demonstrated that although the spatial distribution of the origins of quanta determined by the algorithm is affected by electrical noise, the variance of the amplitudes is unaffected. With active zone centers separated by 1 µm, the clusters of release sites associated with each active zone are quite distinct as demonstrated by Fig. 3Ca where the individual current generation sites are projected onto the abscissa. When active zone centers are separated by 0.5 µm (inner rows of exocytotic sites only 0.36 µm apart), the discrimination of two clusters in the presence of commonly observed electrical noise levels can only be made if there are very large numbers of events (Fig. 3Cc). The variation in the amplitude of uniquantal EPPs estimated by the algorithm is identical to that of the parent population before electrical noise was added in the simulation.



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Fig. 3. Simulated effect of electrical noise on the location and size of uniquantal EEPs. A: amplitude distributions of simulated uniquantal EEPs, electrical noise, and the resulting amplitudes calculated by the algorithm. Aa: Gaussian distribution of uniquantal EEP amplitudes at a distance of 1 µm from their point of origin (mean 0.59 mV and CV 0.28). Ab: amplitude distribution of the uniquantal EEPs generated in Aa, randomly assigned 10 apiece to the 80 sites of exocytosis in Ba and measured by an electrode placed at the origin of the coordinate system in Ba (mean 0.18 mV). Ac: Gaussian distribution of electrical noise (SD 0.01 mV). Ad: amplitude distribution of the uniquantal EEPs calculated (at a distance of 1 µm) by the algorithm from the simulated event amplitudes measured at each electrode after noise had been added (mean 0.59 mV and CV 0.28). B: plots of defined sites of exocytosis (a) and the sites of postsynaptic quantal currents calculated by the algorithm when noise is added in a simulation (b). Ba: 2 parallel active zones with sites of exocytosis running parallel with the center of each active zone ridge and exactly 70 nm distant. Twenty sites of exocytosis are regularly spaced at 100-nm intervals on each side of each active zone. Active zones are 2 µm in length, and the centers are 1 µm apart and are placed centrally within the triangle formed by the tips of 3 electrodes (open circle ). Location of each event, calculated by the algorithm, once electrical noise is added to the event amplitude measurement calculated for each electrode, is plotted in Bb (distribution of amplitudes shown in Ad). C: effect of reducing the distance between active zones on the ability to resolve individual active zones. Ca: distribution of event locations projected onto the abscissa from the simulation plot in Bb. Simulation process was repeated for active zones separated by 0.75 (b) and 0.5 µm (c).

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1B subunit of N-type calcium channels (Abalpha 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 Abalpha 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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Fig. 4. Distribution of vesicle-associated proteins along motor-nerve terminal branches. A and B: distribution of AbSV2 and AbS staining respectively along a 20-µm length of terminal branch. C and D: distribution of AbSV2 and Abalpha 1B. E and F: distribution of AbSV2 and AbS25. G and H: fluorescence from a terminal filled with rhodamine-dextran and the distribution of FM1-43 in the same terminal branch after being stained with FM1-43 subsequent to the capture of the image in H; calibration bar is 5 µm and applies to images A-H.



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Fig. 5. Relationship between the distribution of staining due to antibody AbSV2 and antibodies AbS, Abalpha 1B, and AbS25 as well as FM1-43 staining and rhodamine-dextran filling along terminal branches. A: relationship between the average intensity of fluorescence due to AbS and AbSV2 in contiguous 0.2 µm sections along a single 76-µm branch; the correlation coefficient (r) is 0.89. B: relationship between the intensity of fluorescence due to Abalpha 1B and AbSV2 at contiguous 0.1-µm sections along a single 40-µm branch; r = 0.72. C: relationship between the intensity of fluorescence due to AbS25 and AbSV2 in contiguous 0.2-µm sections along a single 47-µm branch; r = 0.75. D: relationship between the size of FM1-43 blobs along a single 35-µm branch, and the average diameter of the terminal branch at these sites as determined by the width of the rhodamine fluorescence there; r = 0.62. E: histograms of the distances between FM1-43 blobs and between AbSV2 blobs along separate terminal branches; a: 771 distances measured from 28 terminals, mean = 1.50 ± 0.56 SD; b: 567 distances measured from 26 terminals, mean = 1.53 ± 0.63 SD. FM1-43 in Ea and AbSV2 labeling in Eb was viewed using an inverted Leica 4D laser scanning confocal microscope (Ar-Kr laser) with an oil immersion 16/0.5 objective.

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, down-arrow ) 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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Fig. 6. Spatial and amplitude-frequency distributions of MEPPs originating from FM1-43-stained terminal sites, recorded with microelectrode placements on a line. A: terminal branch with 9 approximately circular blobs of FM1-43 together with the positions of the 3 microelectrode tips (); Each electrode is no more than 4 µm from another and placed on a line parallel to that of the line of FM1-43 blobs and <4 µm from the line of blobs (calibration of the image is given by the abscissa in B). B: plot of the average FM1-43 intensity of all pixels in an 8-µm strip (60 × 1 pixel) running across and perpendicular to the long axis of the terminal; the peaks of intensity are therefore aligned with the middle of the FM1-43 blobs in A; 0 on the abscissa indicates a position equidistant between the positions of the 2 outer microelectrodes. C: spatial distributions of the sources of the MEPPs along each half micron of the terminal in A; the modes of the histograms are aligned with the modes of the peaks of FM1-43 intensity given in B with the exception of the histogram between 4 and 6 µm; again, 0 on the abscissa is defined as in B. D: diagram showing the spatial arrangement of 3 externally placed microelectrodes with their tips along a line parallel to the long axis of an FM1-43-stained terminal. E, a-d: amplitude-frequency histograms for the MEPPs that correspond to each of the 4 FM1-43 blobs closest to the central electrode.

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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Fig. 7. Spatial and amplitude-frequency distributions of MEPPs originating from FM1-43 blobs, recorded with microelectrode placements in a triangle. A: 4 FM1-43 blobs of a terminal branch, together with the positions of the 3 microelectrode tips (; calibration of the image is given by the abscissa in B). B: plot of the average FM1-43 intensity of all pixels in an 6-µm strip (45 × 1 pixel) running across and perpendicular to the line of the 4 vesicle blobs; the peaks of intensity are therefore aligned with the middle of the FM1-43 blobs in A. C: spatial distribution of the current sources in relation to the position of the electrode tips (); axes measure distance relative to the bottom left corner of the image in A; , locations of MEPPs that are >0.6 mV in amplitude; open circle , locations of MEPPs with an amplitude <0.6 mV. D: spatial distributions of the sources of the MEPPs along each half micrometer of the terminal in A; principal mode of the histogram is aligned with a mode of the peaks of FM1-43 intensity given in B. E: diagram illustrating the placement of the tips of 3 extracellular microelectrodes at the corners of an equilateral triangle straddling an FM1-43-stained terminal. F: plot of the amplitude of the solution calculated to be spatially furthest from the microelectrodes (solution 2) against the amplitude of the solution that is closest (solution 1). left-arrow  and down-arrow , same number on each axis, in this case 0.914 mV, which is 2 SD above the mean of the amplitudes of solution 1; the threshold used to reject solution 2 for those events in the top left (defined by left-arrow  and down-arrow ). Events in other quadrants were rejected outright (see METHODS). G: amplitude-frequency histogram of all MEPPs for which solution 2 could be rejected on the basis that the amplitude associated with the locus of the current source was improbably large, these are the same MEPPs that are spatially represented in C; , corresponds to ; , corresponds to open circle .



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Fig. 8. Relationship between the quantity of FM1-43 fluorescence at a release site and the spontaneous and evoked transmitter release from the site. A: 4 examples (a-d), each from a separate terminal branch, of the relationship between the quantity of FM1-43 fluorescence in each of a number of contiguous FM1-43 blobs and the number of MEPPs detected at each of the blobs, as determined by the 3 electrodes in a line technique. Periods of recording are 472, 183, 190, and 61 s, respectively. B: 4 examples (a-d) of the relationship between the quantity of FM1-43 fluorescence and the number of MEPPs () and the number of EPPs (open circle ) at each of a number of contiguous blobs as determined by the triangulation technique. MEPPs were collected at the same time as the motor nerve was stimulated at 0.5 Hz. Periods of recording are 728, 724, 219, and 605 s, respectively.

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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Fig. 9. Spatial and amplitude-frequency distributions of MEPPs originating from a FM1-43-stained terminal sites, recorded with 3 extracellular microelectrodes in a triangle and 1 intracellular microelectrode. A: FM1-43 blob that is straddled by the tips of the 3 extracellular electrodes () that are ~5 µm apart (calibration of the image is given by the abscissa in B). B: plot of the average FM1-43 intensity of all pixels in a 6-µm strip (45 × 1 pixel) running vertically through the image in A. C: spatial distribution of the current sources in relation to the position of the microelectrode tips; open circle , MEPPs <0.35 mV; , MEPPs >0.35 mV. D: spatial distribution of the sources of the MEPPs along each half micron of the terminal in A; the mode of the histogram is aligned with the mode of the peak of FM1-43 intensity in B. E: arrangement of a 4th electrode (intracellular) within 20 µm of the array of 3 extracellular electrodes. Fa: plot of the amplitude of the solution calculated to be spatially furthest from the microelectrodes (solution 2) against the amplitude of the solution that is closest (solution 1). down-arrow  and left-arrow , same value on each axis, in this case 0.575 mV, which is 2 SD above the mean of the amplitudes of solution 1. Fb: amplitude-frequency histogram of all MEPPs for which solution 2 could be rejected and are the same MEPPs that are spatially represented in C. Fc: plot of the amplitude of each MEPP recorded with the intracellular electrode against the amplitude calculated for the same event recorded with the extracellular electrodes and accepted in Fb as being the most probable solution.

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 (open circle ) 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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Fig. 10. Spatial and amplitude-frequency distributions of MEPPs and EPPs originating from FM1-43 blobs, recorded with microelectrode placements in a triangle. A: set of FM1-43 blobs of a terminal branch, together with the positions of the 3 microelectrode tips (; calibration of the image is given by the abscissa in B). B: plot of the average FM1-43 intensity of all pixels in a 7-µm strip (52 × 1 pixel) running across and perpendicular to the long axis of the terminal branch in A (vertical). Ca: spatial distribution of the MEPP current sources in relation to the position of the electrode tips; , locations of MEPPs that are >0.4 mV in amplitude; open circle , location of MEPPs with an amplitude <0.4 mV. C: spatial distribution of the EPP current sources in relation to the position of the electrode tips;  and open circle , correspond, respectively, to the same amplitude ranges as described for Ca. Da: spatial distribution of the current sources of the MEPPs along each half micrometer of the terminal in A; the modes of the histograms are aligned with the modes of the peaks of FM1-43 intensity given in B. Db: spatial distribution of the current sources of the EPPs along each half micrometer of the terminal in A. Modes of the histograms generally are aligned with the modes of the peaks of FM1-43 intensity given in B. E: diagram illustrating the configuration of electrodes. F: amplitude-frequency distributions of MEPPs (a) and of EPPs (b) for all events accepted during the 728-s recording; frequency of stimulation was 0.47 Hz.

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).



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Fig. 11. Intensity of FM1-43 staining along the entire length of terminal branches of motor-nerve terminals. A: simple terminal branch ~40-µm-long stained with FM1-43. P and D indicate the most proximal and most distal points of the branch, respectively. B: profile of the intensity of the FM1-43 staining along the branch at intervals of 0.13 µm. C: average amount of FM1-43 fluorescence per blob in each one-tenth of the terminal length along the branch in A. D: average distance between the midpoint of the blobs in each one-tenth of the terminal length for the branch in A. E: complex of what is perhaps 2 parallel terminal branches, probably in the same synaptic gutter, stained with FM1-43. P and D are defined as in A. F- H: same as B-D, respectively; no allowance has been made for the fact that there may be 2 terminal branches present. Calibration in A is given by the abscissa in B and the abscissa in F gives the scale for E.

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).



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Fig. 12. Average changes in the size of FM1-43 blobs and distances between blobs along terminal branches of different length. A and B: average fluorescence per blob and the distance between the midpoints of these blobs, respectively, for simple terminal branches (Fig. 11A) of length 0-40 µm (), 40-80 µm (open circle ), and 80-120 µm (black-down-triangle ). Terminals in each length class were divided into 10 contiguous sections, and both average distance between clusters and fluorescence per blob were calculated for each one-tenth before pooling with other terminals in the same length class. Each point is the mean of >= 6 terminal branches, with the SE indicated by the vertical bars. C and D: same as A and B, except for complex terminals (like that illustrated in Fig. 11E).

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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Fig. 13. Distribution of sizes of FM1-43 blobs and their distance apart along nerve terminal branches. A: frequency distribution of the average size of FM1-43 blobs for terminal branches of the different length categories indicated (the size of the blobs for each branch were normalized to the largest blob in the branch before the pooling). B: frequency distribution of blobs sizes over all terminal branches (, number of terminal branches studied). C: frequency distribution of the average distance between the centers of FM1-43 blobs along terminals of the different length categories indicated. D: frequency distribution of the distances between the centers of FM1-43 blobs for all terminal lengths (, number of distances >5 µm).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha 1B subunit of the N-type calcium channel (Sheng et al. 1994). Antibodies to syntaxin, SNAP-25 and the alpha 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 alpha -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 alpha 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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ABSTRACT
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