Article Figures & Data
Figures
- Figure 1.
A, Representative amperometric data from a single PC12 cell. The arrow under the trace represents the time of stimulus (100 mm K+) application. B, Examples of individual amperometric current transients. The trace on the left has no discernable foot signal; that on the right is preceded by a foot. C, A schematic diagram illustrating the flux of neurotransmitter through the fusion pore. The stages of fusion are numerically coordinated with the associated regions of the amperometric traces in B. D, E, Representative TEM images of PC12 cell dense-core vesicles fusing with the plasma membrane on stimulation with 100 mm K+. Dark arrows indicate vesicles that appear to be undergoing exocytosis. Scale bars, 50 nm.
- Figure 2.
A, Summary of mean foot frequency ratio values created at individual PC12 cells (see Materials and Methods). For the ratios presented, an average of 117 ± 14 and 87 ± 21 amperometric values were used to determine before and after means, respectively. Error bars represent the mean ± SEM of foot frequency ratio values for the different experimental conditions (control, n = 6 cells; l-DOPA, n = 6; reserpine, n = 5). Because the mean foot frequency ratio values approximate 1 for control cells, it is clear that the incubation time itself did not have a significant effect on foot frequencies. B, Plot of foot frequencies calculated in this work as compared with foot frequencies reported in the literature for various-sized vesicles. Foot frequencies for PC12 cells were calculated by taking samples of mean values for different cells [reserpine-treated cells are denoted “R” in plot (n = 5; foot frequency of 52 ± 4%), saline-treated (control) cells are denoted “C” (n = 6; foot frequency of 34 ± 3%), and l-DOPA-treated cells are denoted “D” (n = 6; foot frequency of 24 ± 1%)]. Literature values are plotted for bovine adrenal chromaffin cells (denoted “A” in plot) (Chow et al., 1992), mouse mast cells (denoted “M” in plot) (Alvarez de Toledo et al., 1993), and beige mouse mast cells (denoted “BM” in plot) (Alvarez de Toledo et al., 1993), which have average vesicular radii of 125 nm (Neher, 1993), 400 nm (Breckenridge and Almers, 1987), and 1350 nm (Breckenridge and Almers, 1987), respectively. **p < 0.01 and ***p < 0.001 versus control, respectively (t test).
- Figure 3.
Histograms of raw spike characteristics are heavily skewed to the right, so normalized histograms from log-transformed values were created (Pothos et al., 1998b; Colliver et al., 2000b). A, B, A comparison of the Gaussian distributions for the log transform of the number of molecules released for those spikes with feet, and without, in the control condition (n = 6 cells) (A) and after a 90 min incubation in 100 μm l-DOPA (n = 6 cells) (B). Although a shoulder appears to be present in the distribution of control cells with versus without feet, there is not a significant shift in the Gaussian distributions under either set of conditions presented here in either control or l-DOPA-treated cells.
- Figure 4.
Both the number of molecules released through the exocytotic fusion pore and the time course of said release are dependent on vesicular size. Calculations for vesicular volume are as described in the Materials and Methods. A, Averaged amperometric current transients for one reserpine-treated (n = 28 before; n = 37 after), saline-treated (n = 85 before; n = 73 after), or l-DOPA-treated (n = 73 before; n = 41 after) PC12 cell before (black) and after (gray) the 90 min incubation period. The scale bar is the same for all three averaged transients. The traces appear without the typical shelf-like feature of a foot. This is primarily a consequence of the averaging of the amperometric traces, which exhibit variable foot geometries. B, Mean foot area values shown as a function of vesicle volume. Under control conditions, a cellular average of 5695 ± 751 molecules were released through the fusion pore (before full fusion) per event. C, Mean foot duration values (the time lapse between the onset of the foot and the inflection point between the foot and the full fusion event) shown as a function of vesicle volume. Under control conditions, the average cellular time course for release through the fusion pore was 1.3 ± 0.1 msec. For the data plotted, all amperometric foot signals were pooled to determine the mean foot area and duration values for reserpine, control, and l-DOPA-treated conditions, respectively. Error bars represent the mean ± SEM of the foot characteristic values for the different experimental conditions (control, n = 6; l-DOPA, n = 6; reserpine, n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control, respectively (t test).
- Figure 5.
Percentage of the total contents released during the foot as a function of vesicle radius (calculations for vesicular radii are as described in Materials and Methods). This percentage was obtained by dividing the foot charge by the charge of the total amperometric signal (foot plus spike). The percentage values for each experimental condition examined were then averaged (reserpine, n = 5; control, n = 12; l-DOPA, n = 6); 97, 517, and 182 amperometric spikes were used for each of the points, respectively. By extending the x-axis, the data have been extrapolated to predict that a synaptic vesicle with a radius of 20 nm would release 9% of its total contents through the fusion pore (Δ).
