Article Figures & Data
Figures
- Figure 1.
FM4-64 spots appear during 100 Hz stimulation. A, Average FM4-64 fluorescence measured versus time in resting terminals. FM4-64 (3 μm) was added at t = 0. Filled circles indicate measurements and the solid line a single-exponential best fit to the data (time constant = 87 s). Experiments for FM4-64 spot data were started ∼120 s after dye application. B, Left, NMJ in the presence of FM4-64, which shows outline of nerve terminal before stimulation. Middle, Same NMJ after 30 s, 100 Hz nerve stimulation; fluorescent spots have appeared. Right, Difference between middle and left panels revealing FM4-64 spots; the terminal is outlined. Scale bar, 2 μm. C, The change in fluorescence intensity for spots compared with the whole terminal as a fraction of initial fluorescence is plotted versus time. During the time indicated by the gray bar, 100 Hz stimulation was delivered.
- Figure 2.
FM4-64 spots and spH spots have similar properties. A, Top, Typical difference in spH or FM4-64 fluorescence intensity after a 30 s, 100 Hz stimulation (different preparations). Bottom, Spots identified in the top images by the automatic spot-finding program (see Materials and Methods). Scale bar, 3 μm. B, Cumulative probability plot of spH spot sizes (solid black line) and FM4-64 spot sizes (solid gray line). For comparison to the overall synaptic vesicle population, the synaptic vesicle distribution was assessed after loading with FM dye (dotted line). Inset, A typical FM dye-loaded terminal. The average spot sizes were 0.92 ± 0.05, 0.98 ± 0.06, and 2.07 ± 1.10 μm2 for spH spots, FM4-64 spots, and FM dye load, respectively. The dashed line represents the spot size distribution after randomly mixing terminal pixel locations. Data are averaged from 298 spH spots, 220 FM4-64 spots, and 466 FM dye load spots. Scale bar, 3 μm. C, Same plot as Figure 1C with the rise in FM4-64 spot fluorescence shown (filled circles). For comparison, peak spH spot fluorescence was normalized to match the peak FM4-64 spot fluorescence and plotted (open circles). The mean time to half maximum was 14.5 ± 1.3 s and 13.3 ± 0.6 s for spH and FM4-64 spots, respectively (p = 0.38, Student's t test). The gray bar indicates 100 Hz stimulation.
- Figure 3.
FM4-64 spots persist well after spH endocytosis and reacidification. A, Raw change in fluorescence (in arbitrary units) is plotted versus time for either FM4-64 spots (black line) or nonspot areas of the terminal (gray line; arrow marks end of stimulus train). Fluorescence in the FM4-64 spots dispersed slowly after stimulation (train duration indicated by gray bar), particularly when compared with the time course of synaptic vesicle endocytosis and reacidification (spH fluorescence, dotted line). Fluorescence in FM4-64 spots was estimated to match nonspot area fluorescence ∼17 min after stimulation. Data are averaged from five different terminals. For clarity, error bars are shown for every fifth data point. B, spH labels 35% of the vesicles labeled by FM4-64 at the end of 100 Hz stimulation. Inset, Electrophysiological recording of EPPs during 100 Hz stimulation. The vertical calibration bar indicates the peak amplitude of the first EPP, and the horizontal calibration bar indicates 2 s. Main plot, Quanta (m) are plotted versus shock number. The upper solid line indicates the summed quantal release from EPPs. The lower line indicates the “surface vesicles” or those expected to be visible with spH fluorescence based on a simple model using the spH recovery time constant (see Materials and Methods). Actual FM4-64 fluorescence was scaled to match EPP data (filled circles). The gray circles show the average spH fluorescence rise during stimulation after scaling to the theoretical surface vesicle number (lower solid line).
- Figure 4.
Images show close proximity of spH and FM4-64 spots. Images showing the fluorescence change in response to a 30 s, 100 Hz stimulation measured for both spH (first column) and FM4-64 (0.75 μm, second column). The overlay (third column) shows considerably colocalization (yellow). We also number other areas of interest: an spH spot flanked by two FM4-64 spots (spot labeled 1), an spH spot without a nearby FM4-64 spot (spot labeled 2), and an FM4-64 spot without a nearby spH spot (spot labeled 3). The fourth column shows the spot outlines for spH (green) and FM4-64 (red). Scale bar, 3 μm.
- Figure 5.
Quantification of spH and FM4-64 spot overlap. A, Cumulative probability plot of the nearest-neighbor distances for FM4-64 spots visible after a 30 s, 100 Hz stimulation (open circles). The automatic spot detection prevents any two similar spot types from existing within ∼1 μm of each other (pixel size = 270 nm, vertical gray line). Spots placed randomly in the terminal (1000 iterations) have an average distribution indicated by the solid gray line with 95% of all random trials falling between the dashed gray lines. Data represent 105 spots from five different terminals. B, Cumulative probability plot of the nearest-neighbor distances for FM4-64 spots to spH spots (open circles). Spots centered within one pixel distance lie to the left of the vertical gray line (270 nm). Spots placed randomly in the terminal 1000 times have an average distribution indicated by the dark gray line with 95% of all random trials falling within the dashed gray lines. C, For each fluorescent label, the total fluorescence change in each pixel was determined, then pixels were sorted based on their relative fluorescence changes during the 30 s, 100 Hz train. The x-axis shows the percentage of those sorted pixels (for example, 10% represents the 10% of all terminal pixels with the largest fluorescence change during stimulation). The overlap in pixel locations for spH and FM4-64 fluorescence is plotted from 0% to 100% (black line with error bars). Overlap is considerably more than by random chance (dashed line). Some of the overlap is lost if the FM4-64 channel is shifted by one pixel in both the x and y directions (solid gray line), suggesting that the original alignment between the two channels was optimal.
- Figure 6.
Images show little overlap of spH and FM4-64 spots after moderate stimulation. Images showing the fluorescence change in response to a 30 s, 40 Hz stimulation measured for both spH (first column) and FM4-64 (0.75 μm, second column). The overlay (third column) shows mostly greens and reds with little colocalization (yellow). The fourth column shows spot outlines for spH (green) and FM4-64 (red). Scale bar, 3 μm.
- Figure 7.
Quantification of spH and FM4-64 spot overlap after moderate stimulation. Plots similar to Figure 5. A, Cumulative probability plot of the nearest-neighbor distances for spH spots identified after a 30 s, 40 Hz stimulation (open circles). Data represent 132 spots from four different terminals. Spots placed randomly in the terminal 1000 times have an average distribution indicated by the dark gray line with 95% of all random trials falling within the dashed gray lines. Pixel size is indicated by the vertical gray line. B, Cumulative probability plot of the nearest-neighbor distances for spH spots to FM4-64 spots (open circles). Spots placed randomly in the terminal 1000 times have an average distribution indicated by the dark gray line with 95% of all random trials falling within the dashed gray lines. Pixel size is indicated by the vertical gray line. C, The overlap in sorted pixel locations for spH and FM4-64 fluorescence is plotted from 0% to 100% of the pixels (black line with error bars). Overlap is only modestly more than expected by random chance (dashed gray line).
Additional Files
Supplemental Data
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- supplemental material - Supplemental Figure
