Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43

Abstract

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Fig. 1.

Okadaic acid promoted recovery from photobleaching in resting nerve terminals. A, Control nerve terminal before bleaching (top), immediately after bleaching (middle; the bleached region is marked with anarrowhead), and 28 min after bleaching (bottom). The bleached regions in the middle and bottom are almost identical, indicating that little or no dye moved from the neighboring, unbleached regions into the bleached region.Inset, The brightness (y-axis, not normalized) of the bleached spot is plotted for each of the three panels.Circles are from controls (A); squaresare from okadaic acid–treated terminals (B). Data represent average brightness of three concentric circles (radii 0.2, 0.4, and 1.0 μm) centered on the bleached regions. Note that each region began at the same brightness and was bleached by a similar amount. Only the okadaic acid–treated preparation showed significant recovery.B, Nerve terminal treated with okadaic acid. These images are like those in A, except that okadaic acid (5 μm) was added 40 min before bleaching (and was present throughout the experiment), and the last image was acquired only 5.4 min after bleaching. It is clear that significant recovery from photobleaching occurred relatively rapidly. Scale bar, 2 μm.C, Quantification of photobleach recovery. Barsshow mean percent recovery (+1 SEM) from photobleaching for various treatments. In each case, measurements were made 25–30 min after photobleaching. The control and okadaic acid (OA) results are like those illustrated in Figures 1 and 2. The third column (CD–OA) shows that CD (20 μm for 20 min) did not block the recovery from photobleaching induced by okadaic acid. The last two columns(Ssp–OA and Calmid–OA) show that Ssp (2 μm for 60 min) and calmidozolium (Calmid) (10 μm for 30 min) each blocked the action of okadaic acid.

Fig. 2.

Nerve stimulation did not promote recovery from photobleaching. A, Image of nerve terminal immediately before photobleaching. B, Same terminal immediately after bleaching. Note the bleached center in the spot marked with anarrowhead. C, Same terminal after repetitive nerve stimulation (10 Hz for 5 min). All of the spots destained as a result of the nerve stimulation. The bleached spot is less evident as a result of the surrounding destaining. Scale bar, 4 μm. D, Brightness of pixels along a line drawn through the center of several adjacent spots for each image in A–C(A, solid line; B, dotted line; C, dashed line). The bleached region is shaded. Note that nerve stimulation did not cause the bleached region to grow brighter, indicating that no net movement of dye into the bleached region occurred during the stimulation period.

Fig. 3.

Averaged results from six experiments confirmed that no significant recovery from photobleaching occurred during nerve stimulation. Each filled symbol shows the average brightness of pixels lying a fixed distance from the center of the bleached region [distance = 0.5 μm (squares), 1.0 μm (triangles), and 1.8 μm (diamonds)]. Theopen circles show the average brightness in several control regions, far away from the bleached spot. Pixels closest to the center of the bleached region (filled squares) were most completely bleached, whereas at slightly farther distances (trianglesand diamonds), less bleaching occurred. A 5 min rest period followed the bleach, and then the nerve was stimulated continuously at 10 or 20 Hz while additional images were acquired. The center of the bleached region (filled squares) showed no significant recovery. Brightnesses were calculated as the average brightness of pixels lying along concentric circles centered on the bleached region (which was approximately circular). Vertical lines show −1 SEM for the center of the bleached regions; other SEs were of similar magnitude and are omitted for clarity.

Fig. 7.

Partially bleached regions can be destained completely by nerve stimulation. The profile of brightness through a single fluorescent spot is plotted as in Figure 6D. Thesolid line shows the profile before photobleaching. The extent of photobleaching was reduced by shortening the duration of exposure to the laser. In this case, the fluorescent spot was bleached (shaded region) by only 30–40% (dotted line). Then the nerve was stimulated (10 Hz for 5 min). The spot destained completely (dashed line). This shows that vesicles located in the center of a bleached region still can undergo destaining.

Fig. 4.

Botulinum toxin blocked destaining of frog motor nerve terminals preloaded with FM1-43. Preparations were stained with FM1-43, and then one (B) was exposed to botulinum toxin (type D) (10 nm) for 4 hr. Then the prestimulation images (top) were acquired. Next, the nerves to each preparation were stimulated (10 Hz for 10 min), and images again were taken (bottom). The control terminal (A) destained during the nerve stimulation, whereas the botulinum toxin–poisoned terminal (B) did not. In the control panels, a myelinated axon is visible in the upper left corner. Scale bar, 5 μm.

Fig. 5.

Botulinum toxin did not block the actions of black widow spider venom or okadaic acid. A, These preparations were stained with FM1-43, and then one (right) was poisoned with botulinum toxin (type C) (10 nmfor 3 hr). The control images (top) show fluorescent spots characteristic of normal staining. Then black widow spider venom (0.3 glands/ml) was applied for 40 min, and the images (bottom) were acquired. It is clear that black widow spider venom caused destaining in control and botulinum-poisoned terminals alike. Scale bar, 10 μm. B, The control image (top) shows a terminal in a preparation that was stained with FM1-43 and then poisoned with botulinum toxin (type C) (10 nm for 3 hr). Then 5 μm okadaic acid was applied for 120 min, and the terminal was reimaged (bottom). The blurring of fluorescent spots is clearly apparent. This is an extreme example of the okadaic acid effect, probably resulting from the prolonged exposure to okadaic acid. In general, blurring of dye spots could be seen after exposure to okadaic acid for 30–40 min. Scale bars, 10 μm.

Fig. 6.

Fluorescence photobleach recovery did not occur in botulinum toxin–poisoned preparations. A, A nerve terminal stained with FM1-43 and poisoned with botulinum toxin before photobleaching. B, The same terminal immediately after photobleaching (arrowheads mark bleached regions).C, After repetitive nerve stimulation (30 Hz for 5 min). Little or no change occurred as a result of the nerve stimulation. Scale bar, 5 μm. D, Profile of brightness along a line passing through the bleached region for the images inA–C. E, Averaged results from six different experiments with centers of bleached regions aligned atx = 0. Solid line shows mean +1 SEM before bleaching. Results obtained immediately after bleaching and after repetitive nerve stimulation are shown with dotted anddashed lines, respectively. Error bars, which were about the same length as before bleaching, are omitted from these two lines for clarity.

Fig. 8.

Photobleached regions exhibit normal functional properties, including restaining and destaining with FM1-43.A, Images of a normal terminal (1) that was bleached (2, arrowhead), restained with FM1-43 (3), and then destained by nerve stimulation (30 Hz for 5 min) (4). The restaining and destaining of the previously bleached region were indistinguishable from control regions. Scale bar, 10 μm. B, Brightness profiles of the images in Figure8A.