Neuron,

Vol. 5, 773-779,

December,

1990, Copyright

0 1990 by Cell Press

Strategic Location of Calcium Channels at Transmitter Release Sites of Frog Neuromuscular Synapses Richard Robitaille, E. M. Adler,* Milton P. Charlton Department of Physiology University of Toronto Toronto, Ontario Canada M5S IA8

and

Summary The localization of Caz+ channels relative to the position of transmitter release sites was investigated at the frog neuromuscular junction (NMJ). Ca2+ channels were labeled with fluorescently tagged oconotoxin CVIA, an irreversible Ca2+ channel ligand, and observed with a confocal laser scanning microscope. The Ca2+ channel labeling almost perfectly matched that of acetylcholine receptors which were labeled with fluorescent a-bung arotoxin. This indicates that groups of Ca2+ channels are localized exclusively at the active zones of the frog NMJ. Cross sections of NMJs showed that Ca2+ channels are clustered on the presynaptic membrane adjacent to the postsynaptic membrane. Introduction Neurotransmitter release is triggered by a transient, stimulus-induced rise in intracellular Ca*+ entering the nerve terminal through channels located in the presynaptic membrane (Katz, 1969; Augustine et al., 1987). Since Ca*+ is believed to diffuse slowly in cytoplasm and is rapidly sequestered (Nasi and Tillotson, 1985; Donahue and Abercrombie, 1988), the amplitude of the Ca2+ transient is critically dependent on the spatial distribution of the Ca*+channels. Clustered Ca2+ channels should create a higher local submembrane Ca*+ concentration than the same number of channelsevenlydispersedovertheentirenerveterminal. This is especially important in the case of neurotransmitter release, which is steeply dependent on the concentration of Caz+ (Augustine et al., 1987). Since the delay between Ca2+ entry and transmitter release is very brief (200 j.rs; Llinas, 1977; Llinas and Heuser, 1977; Llinas et al., 1981) and the diffusion of Ca*+ is slow, the distance between Ca2+ channels and transmitter release sites will also critically influence the Ca2+ environment of the receptors responsible for transmitter release. We investigated the applicability of these concepts of Ca2+ signaling by examining the relative locations of presynaptic Ca 2+ channels and other molecules at transmitter release sites of the frog neuromuscular junction (NMJ). The presynaptic nerve terminal of the *Present chusetts 02129.

address: General

Laboratory Hospital

of Molecular Neurobiology, MassaEast, Charlestown, Massachusetts

frog NMJ is a long structure (several hundred micrometers) characterized by the presence of transmitter release sites, or active zones (AZs), spaced at regular intervals of 1 pm (Couteaux and Pecot-Dechavassine, 1974; Dreyer et al., 1973; Heuser and Reese, 1973; Peper et al., 1974; Ceccarelli et al., 1979). The AZs are located directly across the synaptic cleft from clusters of acetylcholine receptors (AChRs) on the edges of the postjunctional folds of the muscle (Dreyer et al., 1973; see Figure 2C). Freeze-fracture studies have revealed the presence of 10 nm transmembrane particles located only at the AZs (Dreyer et al., 1973; Pumplin, 1983), and it has been suggested that these particles correspond to Ca*+ channels (Pumplin et al., 1981). Therefore, if Ca*+ channels are preferentially located at the AZs, a regular pattern of banding at 1 pm intervals should be apparent when Ca2+ channels are labeled, and this should be congruent with postsynaptic receptor labeling. Conversely, if Ca*+ channels are distributed evenly along the presynaptic nerve terminal, a diffuse pattern of presynaptic staining that does not match the pattern of the AChRs should be observed. We demonstrate that at the frog NMJ, Ca*+channels are exclusively localized at the release sites of the presynaptic nerve terminals. Results To determine their location on the nerve terminal, presynaptic Ca*+ channels were labeled with biotinylated o-conotoxin (o-CgTx; Jones et al., 1989), a specific and irreversible blocker of certain Ca*+ channels (Olivera et al., 1985; Cruz et al., 1988; Gray et al., 1988; Lindgren and Moore, 1989) and of synaptic transmission at the frog NMJ (Kerr and Yoshikami, 1984; Sano et al., 1987). Biotinylated o-CgTxwas purified by highperformance liquid chromatography, and active fractions were applied to frog muscles (Figure 1). Before application of biotinylated o-CgTx, a single nerve stimulus was sufficient to cause an endplate potential large enough to trigger a muscle action potential (Figure IB,). After application of biotinylated o-CgTx (200 pg/mI), the endplate potential rapidly decreased below the threshold for an action potential and, within 20 min, transmitter release was completely blocked (Figure lB2). However, neither spontaneous release nor muscle response to transmitter was blocked by the toxin (Figure 1B3). Because biothylated and native toxins had similar effects (Kerr and Yoshikami, 1984; Sano et al., 1987), it is likely that both toxins bind to the same sites. Preparations treated with biotinylated w-CgTx were exposed to streptavidin-Texas Red to reveal Ca*+ channels. Labeled preparations were viewed with standard phase microscopy to locate endplates, which were then examined in detail with the confocal

NeUr0l-l

774

10

30 Time (min)

50

Figure o-CgTx

1. Purification

and

Physiological

Effects

Muscle=

of Biotinylated

(A) Purification by reverse-phase high-performance liquid chromatography of o-CgTx after biotinylation. The first peak represents unreacted toxin, since its elution time is identical to that of native toxin. The peak marked by an asterisk is the biotinylated co-CgTx fraction that had the most potent effects on transmitter release (see below) and was used for labeling of Caz+ channels. (B) Inhibition of transmitter release by biotinylated o-CgTx. The motor nerve was stimulated and a muscle action potential was recorded (B,). Biotinylated o-CgTx was then applied to the bath at final concentration of 100 ug/ml (approximately 30 JIM). Evoked endplate potentials were blocked in about 20 min fB3. However, spontaneous release of transmitter, seen as miniature endplate potentials, was unaltered (B,), and muscle fibers still contracted in response to direct stimulation (data not shown). (B,) shows three records of miniature endplate potentials obtained 20-21 min after application of biotinylated w-CgTx. Calibration in (B,), 50 mV, 1 ms; in (B2), 2 mV, 1 ms; in (Bj), 2 mV, 100 ms.

laser scanning microscope (Shotton, 1989). Two typical NMJs stained with biotinylated o-CgTx-Streptavidin-Texas Red are illustrated in Figures 2A and 2B. Endplates showed a regular arrangement of bright bands at intervals of about 1 urn (Figures 2A and 2B), suggesting that Ca*+ channels were clustered at the similarly spaced AZs (Figure 2C; Couteaux and PCcotDechavassine, 1974; Dreyer et al., 1973; Heuser and Reese, 1973; Peper et al., 1974; Ceccarelli et al., 1979). Preincubation with native untagged toxin blocked labeling at bands, suggesting that native and tagged toxins compete for a common site and that the pattern of fluorescence seen with the tagged toxin is specific to o-CgTx binding sites. Background staining (Figure 2A) is caused by the nonspecific binding of streptavidin-Texas Red, since this background was present

Figure NMJ

2. Distribution

of w-CgTx

Binding

Sites

along

the

Frog

(A and B] Preparations treated with biotinylated o-CgTx and stained with streptavidin-Texas Red wereviewed with aconfocal laser scanning microscope equipped with the rhodamine filter set and 40x or 60x oil immersion lenses (numerical apertures of 1.3and 1.4, respectively).Theconfocal aperturewasat position 4 (26% of the maximum aperture), 1% of the maximal laser intensity was used, and the photomultiplier tube was set at automatic gain and black level. (A) and (8) illustrate 2 NMJs, and each image is an average of 5 consecutive scans. The images were intensityscaled to the maximal pixel values and sharpened using aconvolution program. The diagram above each figure illustrates the structure of the thread-like NMJ on the cylindrical muscle fiber, and the box indicates the region scanned by the microscope (box length, 67 urn in [A]; 45 urn in [B]). Note the presence of the band pattern at regular intervals of 1.1 urn (SD= 0.06, n = 63). Also note the background staining (mainly in [A]) caused by the nonspecific binding of streptavidin-Texas Red to connective tissue. Bars, IO urn. (C)A diagram of a longitudinal section of the frog NMJ based on electron microscopy (Dreyer et al., 1973; Peper et al., 1974). Note the location of the AZs in relation to the postjunctional folds and AChRs. Abbreviations: ACh-R, acetylcholine receptors; AZ, active zone; PJF, postjunctional fold; SC, Schwann cell; SCF, Schwann cell fingers; SyC, synaptic cleft; V, vesicles).

even when biotinylated w-CgTx had not been applied or when binding of biotinylated w-CgTx was blocked by previous application of the native toxin. To confirm that the w-CgTx labeling was located at synapses, the NMJs were double-labeled to disclose both AChRs (Anderson and Cohen, 1974) and Ca2+ channels. Ca2+ channels were labeled as described above and then the irreversible AChR ligand a-bung-

Ca*+ Channels 775

at Release Sites

arotoxin (a-BuTx) coupled to boron dipyrromethane difluoride (BODIPY; which fluoresces green) was applied to the muscle. Figure 3A shows a typical doublelabeled NMJ where Ca*+ channel label (red) colocalized with the AChR label (green), indicating that the o-CgTx staining was indeed located at the NM]. To verify the presynaptic localization of the w-CgTx label, we treated muscles with collagenase (Betz and Sakmann, 1971) and removed the presynaptic terminals from the NMJs. The appearance of double-labeled synapses was not altered by treatment with collagenase (Peper and McMahan, 1972) and exhibited the usual close registration of a-BuTx and w-CgTx labels as long as synapses remained intact (Figure 3A). However, after nerve terminals were removed by pulling on branches of the motor nerve, only the a-BuTx staining remained at the original sites (Figure 3B). In all five synapses (two muscles) in which removal of the terminal was confirmed by examination with standard phase microscopy, the presynaptic red staining disappeared. This indicates that o-CgTx binding sites are located on the presynaptic terminal. We next investigated the relative location of Ca2+ channels and AChRs at the synapse to determine whether Ca2+ channels are localized at AZs (Dreyer et al., 1973; Heuser and Reese, 1973; Couteaux and P&cot-Dechavassine, 1974; Peper et al., 1974; Matthews-Bellinger and Salpeter, 1978; Ceccarelli et al., 1979). To compare the alignment of presynaptic and postsynaptic structures, NMJs were double-labeled as described above and observed at high magnification. Figure 4 shows part of a double-labeled NMJ that is typical of over 100 observed in eight muscles. Each green band of AChR stain was usually matched by a red band of Caz+ channel stain. Fluorescence from labeled Ca*+ channels and labeled AChRs was thus almost perfectly aligned, suggesting that Ca*+ channels are clustered at AZs opposite the postjunctional folds. Such alignment could be artifactual, since part of the green light emitted by BODIPY passes through the red filter of the photomultiplier tube that detects the red light emitted by Texas Red (bleeding) and could give the impression of a perfect alignment. Color bleed can be reduced by choosing a red filter with a cutoff at longer wavelengths or by subtracting a certain percentage of the green signal from the red signal. We chose instead to exploit the differential bleaching rate of the two fluorescent dyes. When illuminated by the argon laser 514 nm line used in the dual-channel mode of the Bio-Rad MRC-600 microscope, BODIPY bleaches more rapidlythan Texas Red. Figure 5 illustrates a NMJ imaged at low zoom factor (A and B) and at higher zoom factor (C and D) before and after bleaching the central area by repetitive scans. In the central area (dark rectangles in Figure 5B) that was scanned repetitively at high zoom factor, the green bands bleached rapidly and often disappeared but the red bands remained. Figure 5D shows, at high zoom factor, the results of partial bleaching

obtained with fewer scans than used to cause the bleaching in Figure 58. In Figure 5D the green bands are still visible, but they are much dimmer than the unbleached bands shown in Figure 5C. If the red bands had been produced by light emitted from the green bands, then both bands should have bleached at similar rates. On the contrary, Figure 5E shows that during repetitive scans, the green fluorescence declined much faster than the red fluorescence. Therefore little, if any, of the red image was caused by bleeding from thegreen label, and theapparent registration of green and red fluorescence is not artifactual. Furthermore, although a Ca*+ channel band usually corresponded to a postsynaptic receptor band, the shape and size of the band seen with one label was not always exactly the same as that seen with the other label (Figure 4). This occasional mismatch of the green and red images also suggests that the presynaptic red image is not due to color bleed. If bandsof Ca*+channelscovertheentirecircumference of the presynaptic terminal, cross sections of the terminal should reveal an annulus of w-CgTx label. To test this hypothesis, we examined the distribution of nerve terminal Ca2+ channels in cross sections of double-labeled NMJs. Figure 6 shows the Ca*+ channel label (red) located between the nerve terminal and the green band of the AChRs. The Ca*+ channel staining is restricted to the presynaptic membrane facing the muscle fibers. An annular distribution of o-CgTx labeling was not observed in any of the 48 nerve terminal cross sections examined in five muscles. This suggests that Ca*+ channels are distributed exclusively on the surface of the presynaptic terminal where AZs face the muscle cell (Figure 2C) and where the large presynaptic particles are located (Dreyer et al., 1973; Pumplin, 1983). This is consistent with fluorescent Ca*+ indicator imaging studies at the presynaptic terminal of the squid giant synapse. Here, Ca*+ influx was seen to occur only at areas of the terminal facing the postsynaptic neuron, although the distribution of channels between active zones could not be resolved (Augustine et al., 1989). Discussion The linear array of regularly spaced AZs along the frog NMJ has allowed us to determine that o-CgTx labels AZs. The labeling shows a typical band pattern at 1 pm intervals that aligns with AChRs, known to be clustered opposite AZs (Dreyer et al., 1973; Peper et al., 1974). The physiological effects of the biotinylated o-CgTx are consistent with an irIeversible blocker of presynaptic Caz+ channels that has no effect on postsynaptic AChRs. Furthermore, dissociation of the synapse showed that the o-CgTx label is presynaptic. We therefore conclude that w-CgTx labeling discloses presynaptic Ca *+ channels that are clustered at AZs, wheretransmitter is released (Heuserand Reese, 1973; Couteaux and Pecot-Dechavassine, 1974; Ceccarelli et al., 1979).

5A

6

C

D

CaZ+ Channels 777

at Release

Sites

The pattern of Ca*+ channel staining is not likely due to the presence of Schwann cell fingers wrapping around the terminal because these fingers occur at irregular intervals of one to three AZs (Figure 2C; Dreyer et al., 1973; Peper et al., 1974), whereas we see a very good correspondence between presynaptic and postsynaptic labeling at regular intervals. This would not be the case if the streptavidin-Texas Red actually labeled the Schwann cell fingers or if the Schwann cell fingers prevented access of ligand or label to the AZs. Furthermore, intracellular staining of Schwann cells at the frog NMJ shows that they are not regularly spaced and have irregular shapes, unlike the labeled presynaptic areas (McMahan et al., 1972). Because o-CgTx does not affect all types of Ca*+ channels, it may be of little use for labeling Ca*+ channels at synapses of invertebrates (Cruz et al., 1988; Yoshikami et al., 1989; Charlton and Augustine, 1990) or at mammalian NMJs (Sano et al., 1987). However, antibodies found in the serum of patients with Lambert-Eaton syndrome can block certain mammalian Ca*+ channels (Kim and Neher, 1988). It has been shown by electron microscopic immunocytochemistry that these antibodies bind to AZs of mouse NMJs (Fukuokaet,al.,1987a,1987b).Thepresent resultscomplement those obtained with the Lambert-Eaton syn-

Figure

3. Ca*+ Channels

Are

Located

in the Presynaptic

drome antibodies by demonstrating the location of Ca2+ channels in three dimensions (en face view and cross section). However, since the Lambert-Eaton syndrome antibodies disrupt AZs, cause loss of the large membrane particles, and must be applied for a long time (Fukuoka et al., 1987a), this technique may not be as reliable for answering questions about exclusive location of Ca2+ channels as the o-CgTx technique we used. Our present technique allows observation of many AZs simultaneously, and thousands of AZs can be observed in a single muscle where measurements of their dimensions can be easily made. The double-labeling experiments demonstrate that Ca2+ channels are found mainly at AZs and are rare or absent between AZs and on areas of the presynaptic membrane that do not face the muscle fiber. The distribution of o-CgTx binding sites is therefore consistent with the distribution of the IO nm membrane particles and supports the possibility that these particles are Ca2+ channels, but definitive proof must await ultrastructural analysis. The data indicate that there must be many Ca *+ channels per AZ (Pumplin et al., 1981) because the area covered by fluorescence (>I urn per band) is far too large to represent a single channel molecule, which would likely be about IO nm in diameter (Unwin, 1989). The distribution of o-CgTx

Terminal

(AandB)FalsecolorimagesshowingthelabelingofpostsynapticAChRswitha-BuTx-BODIPY(green)andCa2+channelswith biotinylated o-CgTx-streptavidin-Texas Red (red). The green and red images are separated for clarity. (A) A NMJ after incubation in collagenase. The appearance was not altered by the enzyme and is typical of double-labeled NMJs that were not treated with collagenase. Both labels colocalize at the NM]. (B) The same NMJ after the nerve terminals have been pulled away. The shape of the NMJ is slightly altered as a result of some distortion caused by the removal of the nerve. After removal of the nerve terminal, no red staining in the shape of the NM] could be detected even when the red photomultiplier gain was set to maximium. The disappearance of the Ca*+ channel label with removal of the nerve terminal indicates that the Ca2+ channels are located in the presynaptic nerve terminal. Bar, 25 km. Figure

4. Ca2+ Channels

Are

Clustered

Opposite

the AChRs

False color image showing presynaptic Ca ?+ channels labeled with biotinylated w-CgTx-streptavidin-Texas Red (red) and postsynaptic AChRs labeled with a-BuTx-BODIPY (green). The green and red images are separated for clarity. Gain and black level of both photomultiplier tubes were the same, and both confocal apertures were set at 4 (26% of the maximal aperture). The intensity of both images was scaled to the maximal pixel values, and both images were sharpened using a convolution program. The diagram illustrates the structure of this NMJ, and the box indicates the region imaged (length of box, 68 pm]. Note that the patterns of the two labels are very similar and that there is a close registration between them. However, there are a few differences in the pattern where small areas do not overlap (arrows). Bar, 10 pm. Figure

5. Registration

of Red and Green

Bands

Is Not

Caused

by Color

Bleed

(A-D) The contribution to the red image of the green BODIPY fluorescence bleeding through the red low pass filter was evaluated by differential bleaching of BODIPY and Texas Red. (A) and (C) illustrate a NM] before bleaching at low and high zoom, respectively; (B) and (D) illustrate the same NMJ after the bleaching experiment at low and high zoom, respectively. The images obtained before and after the bleaching experiments were sharpened, but not intensity-scaled. The bleached area can be seen as dark rectangles in (B), but little bleaching of bands is evident in the Texas Red image ([B], right). (D) shows the NMJ after partial bleaching with fewer scans than used for bleaching in (B). Note that after partial bleaching, the green bands are still detectable, but are dimmer than the bands in (C). Bars, 25 I.rm (A and B); 10 nm (C and D). (E) Rate of bleaching of the green BODIPY signal (open symbols) and the Texas Red signal (closed symbols) in 3 NMJs. The intensity is the average pixel value for the same area containing 9-11 AZs in green and red images and was determined from successive images, each ofwhich was theaverageof 11 scansThe intensityforeach color has been normalized tothe initial intensifivalue before bleaching. The postsynaptic green label (a-BuTx-BODIPY) was preferentially bleached, leaving the Ca*+ channel labeling intact. Therefore almost none of the red image was contributed directly by the green fluorescence. Figure

6. Cal+ Channels

Are Clustered

in the

Presynaptic

Membrane

Facing

the Muscle

Fiber

This is a false color image in which green and red images have been superimposed. This section. The nerve terminal (NT) was backfilled with Lucifer Yellow. The red Ca2+ channel and the AChRs (green crescent) embedded in the surface of a muscle fiber (M). The Ca2+ membrane adjacent to the postsynaptic membrane where the AChRs are located, Bar, 10

NMJ was double-labeled label is found between channels are confined Pm.

and cut in cross the nerve terminal to the presynaptic

Neuron 778

binding sites on cultured neurons has been reported previously (Jones et al., 1989). The results presented here illustrate aspects of the molecular organization of synapses and exemplify some general principles of rapid Ca2+ signaling. For instance, the location of Ca*+ channels exclusively at AZs is economical both because the minimal number of channels is used and because Ca2+ is admitted to the cell only in close proximityto the exocytotic apparatus. Clustering of channels close to release sites ensures that a large Ca*+ signal is rapidly available to the nearby Ca*+ receptors which initiate transmitter release. Clustering also helps to maximize the Ca*+ signal by increasing the probability that Ca*+ from adjacent channels will overlap. Exclusion of Ca*+ channels from other areas minimizes the entry of Ca*+ that would not participate in phasic exocytosis but that would still have to be pumped out of the cell. Such details of channel location can aid calculation of Ca*+ transient magnitude (Chad and Eckert, 1984; Simon and Llinas, 1985; Zucker and Fogelson, 1986) and therefore may be useful in determining the nature of the Ca*+ receptors involved in transmitter release (Augustine et al., 1987; Smith and Augustine, 1988). Experimental

Procedures

Synthetico-CgTx GVIA (2 mglml; Peninsula Laboratory, Belmont, CA) in 0.1 M bicarbonate buffer (pH 8.5) was combined with biotin (long arm) NHS (Vector Laboratory, Burlingame, CA) freshly dissolved in dimethylformamide (1 mg/ml) at a I:10 molar ratio for5 min at room temperature. The reaction was terminated by adding02596 ethanolamine.The productwas passed through a Bio-Rad RP-318 column (0.8 mllmin) in a linear gradient of acetonitrile (100/o-30%) dissolved in 0.1% trifluoroacetic acid. The eluent was monitored by a spectrophotometer at 214 nm, and fractions were collected. Electrophysiological recordings were performed on isolated frog (Rana pipiens) cutaneous pectoris muscle and associated motor nerve. Transmitter release was detected by measuring endplate potentials evoked by nerve stimulation using standard electrophysiological recording techniques and glass microelectrodes. Ringer’s solution contained 120 mM NaCI, 2 mM KCI, 1 mM NaHCOs, 1.8 mM CaCI,, 5 mM HEPES @H 7.2). Ca2+ channels were labeled as follows: Isolated cutaneous pectoris muscles were stretched and pinned at 110% of rest length and were incubated in biotinylated w-CgTx (100 ug/ml diluted in frog Ringer’ssolution)for5-6 hr, rinsed in frog Ringer’s solution for 45 min, and fixed with 3% formaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 60 min. The muscles were rinsed overnight in PB at 4OC and then incubated for 2 hr in streptavidin coupled to Texas Red (250 ug/ml in PB; Molecular Probes, Eugene, OR). After rinsing for 30 min in PB, the muscles were mounted on a microscope slide. Streptavidin forms a strong bond with the biotin coupled to w-CgTx. In control experiments this entire procedure was preceded by a 4 hr incubation with native o-CgTx at a concentration of 50 ug/ml in frog Ringer’s solution. The NMJs were observed using a Bio-Rad MRC-6OOconfocal microscope equipped with a rhodamine filter set. For double-labeling experiments, the same preparationwas incubated with a-BuTx coupled to BODIPY (a-BuTx-BODIPY; 25 uglml in PB for 45 min; Molecular Probes) after images of single labeling with w-CgTx were obtained. Double-labeled NMJs were viewed with the confocal laser scanning microscope using the dual wavelength mode, which allowed fluorescence from both dyesto bedetected simultaneouslywith twodifferent photomultiplier tubes. Thedual wavelength configuration uses the 514 nm line from the argon ion laser, a band pass filter (514-550 nm) to

select the green emitted light detected by one photomultiplier tube, and a low pass filter (590 nm cutoff) to select the red emitted light detected by the second photomultiplier tube. Red and green images can be displayed separately (Figures 3,4, and 5) or superimposed (Figure 6). In some experiments (Figure 3 only) presynaptic nerve terminals were enzymatically dissociated from the muscles fibers by treatment with collagenase (Betz and Sakmann, 1971; Peper and McMahan, 1972). NMJs were double-labeled as described above, and the muscle was incubated in a solution of 600 U/ml collagenase (Worthlegton Biochemical Corp., Freehold, NJ) at room temperature for 60 min. The treated preparation was observed with the confocal laser scanning microscope, and images of NMJs were made (Figure 3A). Secondary branches of the motor nerve were then pulled away with fine forceps, and the same NMJs were imaged again (Figure 3B). Contamination (bleeding) of the red signal (Texas Red) by the green signal (BODIPY) was assessed by testing the differential bleaching of the the two labels. A NMJ was initially scanned at low zoom factor (1.5x) as a control before bleaching and again after bleaching. A smaller area of the junction was illuminated at a larger zoom factor (3x) for 99 consecutive scans to cause bleaching of the central area. The localization of Ca2+ channels on the circumference of the nerve terminal was investigated using cross sections of NMJs. Specific staining of the nerve terminals was achieved by backfilling the nerve with Lucifer Yellow (20 mM for 36 hr; Molecular Probes) as described by Mulloney (1973). The NMJs were then double-labeled as described above and observed with theconfocal laser scanning microscopetoverifythequalityofthestaining. The muscles were then soaked in a solution of 10% glycerol (in 0.1 M PB) for 60 min and frozen in an embedding m.edium (OCT, Miles Lab., Naperville, IL). Cross sections (24 urn) were made with a cryotome and collected on microscope slides. The sections were observed with the confocal laser scanning microscope using the same dual-wavelength filters as above. Acknowledgments We thank Dr. H. L. Atwood for critical reading of the manuscript and Craig Mizzen and Babak Jahromi for technical assistance. We thank Dr. A. Jorgensen for helpful discussions. This work was supported by a Medical Research Council grant (to M. P. C.). E. M. A. was supported by the University of Toronto. R. R. was supported by a Medical Research Council Postdoctoral Fellowship. We thank the Ontario Laser and Lightwave Research Center (University of Toronto) for use of the Bio-Rad 600 microscope. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement in accordance with 18 USC Section 1734 solely to indicate this fact. Received

August

8, 1990; revised

October

12, 1990.

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