Article Figures & Data
Figures
- Figure 1.
No disparity in action potential-evoked poststimulation loading for FM1-43 and FM2-10. A, Granule neuron cultures were loaded with either FM1-43 or FM2-10 using trains of 200 (10 Hz), 400 (40 Hz), or 800 (80 Hz) action potentials. Dyes were loaded during stimulation at S1, but washout was delayed for 5 min at S2. Unloading was stimulated by 30 s stimuli of 50 mm KCl except for cultures loaded with 200 action potentials, for which two sequential trains of 400 action potentials (40 Hz) were used. B, C, Time course of the average response for nerve terminals loaded with either FM1-43 (B) or FM2-10 (C) during action potential stimulation at 80 Hz is displayed. D, Mean ΔS2/ΔS1 ratio ± SEM is displayed for each stimulation condition. All experiments were performed four times except those with 200 action potentials (n = 3). No significant difference between FM2-10 and FM1-43 loading was observed during any stimulation protocol (Student's t test).
- Figure 2.
Uptake of large dextrans only occurs during strong action potential stimulation. Left, Representative fields in which tetramethylrhodamine-dextran (50 μm) was applied to cultures during trains of 200 (A; 10 Hz), 400 (C; 40 Hz), or 800 (E; 80 Hz) action potentials and then immediately washed away. Right, Fields in which dextran was applied to cultures for 2 min immediately after trains of 200 (B; 10 Hz), 400 (D; 40 Hz), or 800 (F; 80 Hz) action potentials. Scale bar, 15 μm. Mean number of dextran puncta per field ± SEM is displayed for either during (G; open bars) or after (H; hatched bars) stimulation. Dotted line illustrates puncta number per field attributable to intrinsic background fluorescence in the absence of dextran. All experiments were performed three times. ***p < 0.001, one-way ANOVA.
- Figure 3.
HRP labels bulk endosomes when applied during strong action potential stimulation. A–F, HRP was applied to cultures during trains of 200 (A; 10 Hz), 400 (C; 40 Hz), or 800 (E; 80 Hz) action potentials and then immediately fixed. Alternatively, HRP was applied to cultures for 5 min immediately after trains of 200 (B; 10 Hz), 400 (D; 40 Hz), or 800 (F; 80 Hz) action potentials and then fixed. Representative electron micrographs are displayed. Black arrows indicate HRP-labeled endosomal structures, whereas white arrows indicate HRP-labeled SVs. Scale bars, 100 nm. Mean number ± SEM of either HRP-labeled (solid bars) or clear (open bars) endosomes (G) or SVs (I) generated during stimulation is displayed (200 action potentials, n = 17 nerve terminals; 400, n = 26; 800, n = 53). Mean number ± SEM of either HRP-labeled (solid bars) or clear (open bars) endosomes (H) or SVs (J) generated after stimulation is displayed per nerve terminal (200 action potentials, n = 18 nerve terminals; 400, n = 39; 800, n = 38). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA for HRP structures.
