Confocal spot detection of stained synaptic vesicles in resting boutons reveals macroscopic fluctuations reflecting vesicle movement. (a) Fluorescence image of an exemplar hippocampal bouton 20 days in vitro. stained with 15 μM FM 1–43 by electric field stimulation (120 APs, 10 Hz). Scale bar is 1 μm. After dye washout (10 min), a fluorescence image was taken and a single identified bouton was placed in the center of the laser beam. (b) A z scan through the bouton was performed for positioning in the focal plane. (c) Exemplar spot confocal fluorescence time course of a resting bouton (shaded) measured for 10 min (sampling rate 2 Hz, with 6 ms illumination of the sample per interval). Large up- and downward steps in fluorescence can be seen signifying synaptic vesicle movement in and out of the detection volume. For comparison, the time series of a bouton in a preparation fixed with 4% paraformaldehyde (black). Note the slight decrease in the fluorescent signal that either arises from slight “stage drift” or slight concerted movement of fluorescent vesicles on a very slow timescale (bleaching was minimal under these measurement conditions). (d) Plotted is the variance of 229 individual recordings from control cells versus their mean. Data were fit with a line yielding an intercept of −0.02 ± 0.27 and a slope of b = 0.103 ± 0.018, i.e., a variance-over-mean ratio of ∼0.1 for control cells.

Movement of synaptic vesicles is controlled by MLCK. (a) and (b) Exemplar time series of boutons stained with FM 1–43 (15 μm) by 120 APs (at 10 Hz) before (a) and after application of 15 μM ML7 (the data were already high-pass filtered at 0.05 Hz). (c) Simulated fluorescence time series for control vesicles. (d) Simulated fluorescence time series for ML-7. (e) Respective averaged power spectra for control and in the presence of 15 μM ML-7. For power spectral analysis of fluorescence traces, the average value was subtracted and the data were high-pass filtered at 0.05 Hz to remove slow trends in the data that could arise from stage drift or slow movements of boutons. (f) Shown are the same PSDs as in the left panel, but overlaid are results from simulations with diffusion coefficient D = 5 × 10⁻⁵ μm²/s and a cage size of 50 nm (blue circles). Mean fluorescence for this simulation is 11.2 kcps and the variance 0.96 kcps² (as expected for control cells, see Fig. 1). Simulation for the same cage size but with diffusion coefficient doubled to D = 1 × 10⁻⁴ μm²/s is shown in gray (rhombs). The PSD is shifted upwards, and the variance increases to 2.02 kcps². The mean fluorescence is 11.3 kcps for this simulation. Red circles show the PSD of experiments after ML-7 treatment (cf. left panel). A simulation with D = 5 × 10⁻⁶ μm²/s (10-fold lower than control) and a cage size of 50 nm (orange circles) mimics experimental ML-7 data quite well. The mean fluorescence for this simulation was 11.5 kcps and the variance 0.17 kcps².

OA treatment increases observed fluctuations. (a) Respective ensemble-averaged power spectra after application of 5 μM OA reveal a large increase in vesicle mobility compared to control conditions. (b) Exemplar time series of individual resting boutons after application of 5 μM OA to the bathing medium. OA was applied for at least 25 min. (c) Exemplar simulated fluorescence time series for OA vesicles with two populations with different diffusion coefficients (cf. panel e, PSD with blue circles). (d) In the presence of 5 μM OA, increasing concentrations of LB (preincubated for 1 h) substantially increase vesicle mobility in a dose-dependent manner: control; 5 μM OA; 5 μM OA + 1 μM LB; 5 μM OA + 10 μM LB, 5 μM OA + 25 μM LB. (e) PSD of experiments after OA treatment in red. Superimposed (gray rhombs) is a simulation with D = 5 × 10⁻⁴ μm²/s and a cage size of 30 nm with mean = 12.7 kcps and variance = 1.80 kcps². Shown in blue circles is a simulation where 75% of the vesicles were modeled using control parameters (D = 5 × 10⁻⁵ μm²/s, cage size = 50 nm), but 25% had a 100-fold higher diffusion coefficient (5 × 10⁻³ μm²/s) and a cage size of 50 nm. The mean intensity for this simulation is 12.0 kcps, and the variance is 2.17 kcps². Experimental PSD (black squares) is shown for comparison.

Schematic of model for simulating confined synaptic vesicle movement. A sketch of the confocal detection volume (r_xy = 170 nm, structural parameter ∼6) is overlaid on the outline of a typical synapse with Ø ∼ 1 μm. Since the detection volume is extending in the z direction by approximately a factor of 1.8, only movements in the x, y plane will significantly contribute to fluctuations. Vesicles were simulated as nonextended fluorescent objects and placed at random positions in a square of 500 nm edge length. Since the detection volume has an e⁻² radius of 170 nm, vesicles outside this square do not contribute significantly to overall fluorescence. Vesicles were allowed to undergo a random walk (shaded lines) within a confined region (for illustration purposes, three confined regions are shown as dashed ellipses). The space constant of the grid was 1 nm.

Determination of single vesicle fluorescence contribution in FFS measurements. (a and b) Fluorescent puncta with similar appearance as 40-nm fluorescent beads are visible after very week stimulation with 5 APs at 5 Hz during a 10 s application of 15 μM FM 1−43. Scale bars are 1 μm (a). FM dye fluorescence is lost after applying 3 × 600 APs (b). At the beginning of every experiment, a z stack of the region of interest was obtained. The fluorescence (in counts) of a punctum was determined as the intensity difference between images in the plane of best focus before and after a destaining stimulus of 3 × 600 APs in a circular region of 13 pixels (1 pixel = 58 nm). The existence of a functional synapse was confirmed by a subsequent round of strong stimulation (600 APs at 20 Hz during 60 s application of 15 μm FM 1–43), which resulted in a bright fluorescent spot (c). The fluorescence from this spot could be released with 3 × 600 APs (d). Note that these images were acquired at ∼12 times lower laser intensity than in a and b. Scale bars are 1 μm. (e) Histogram of fluorescence intensities of puncta (determined as difference between a and b) for 5 APs shows a quantal distribution, which could be fit with a sum of three Gaussians (black line) according to the model of Murthy and Stevens (31) (the individual Gaussians for peak 2 and 3 are also shown in dashed lines): with peak spacing μ, amplitude of each peak A, and residual background fluorescence r. The coefficient of variation for measurements c_m = 0.08 was estimated from repeated measurements of fluorescent puncta; the coefficient of variation of vesicle surface area c_v has been estimated by Murthy and Stevens to be 0.2 (31). The residual fluorescence after the destaining stimulus was found to be r = 40 counts on average. The fit yields a single vesicle fluorescence intensity of μ = 96.8 ± 3.2 counts. (f) Fluorescence intensity (ΔF) histogram of fully loaded boutons (loaded with 3 × 600 APs at 20 Hz during 180 s of dye application and destained with 3 × 600 APs) yields an average intensity of 5137 ± 122 counts. Thus a fully stained bouton contains ∼52 stained vesicles, which is in good agreement with findings in the literature (30–32). Note that some boutons failed to take up dye during the 5 AP stimulus (see (a) and (c)). (g) Fluorescence intensity (ΔF) histogram of boutons stained with 120 APs (at 10 Hz, 1 min dye application) as in the FFS experiments. The average bouton intensity was found to be 2353 ± 206 counts corresponding to ∼24 labeled vesicles. (h) Fluorescence intensity histogram of 40-nm fluorescent beads settled on a cell layer (plotted is ΔF determined by subtracting the background). Images were taken as in a. A single Gaussian fit yields an average intensity of 48.6 ± 1.2 counts. A single vesicle (see a and d) is thus ∼2-fold brighter than a bead. (i) Autocorrelation of beads diffusing freely over a cell layer (measuring conditions as in confocal spot detection experiments). A fit with a three-dimensional diffusion model gives a diffusion time of τ_D = 1.65 ms and N = 8.96 beads, corresponding to 4.44 ± 0.06 kcps per bead (cpb).

Acute staurosporine treatment and disruption of actin or tubulin alone does not affect vesicle mobility. (a) Averaged power spectra for control conditions and for acute application of 1 μM staurosporine. (b) Averaged power spectra for control conditions and after 1 h preincubation (at 37°C) 1 μM staurosporine (lower trace). (c) Averaged PSD for control conditions and application of 10 μM CD. (d) Averaged PSD for control conditions and application of 25 μM LB.