Fig. 1. Filopodia form at presynaptic boutons during synaptic activity and show varied morphology and distributions of SVs and endosomes.

(A) Time-lapse series of an axon coexpressing GPI-EGFP and SypmOr2 during stimulation with 600 APs (40 Hz). White arrow, filopodium. Scale bar, 1 μm. (B) Percentage of SypmOr2-responsive boutons forming filopodia. Line is the mean, and error bar is SEM. (C) Percentage of filopodia-forming boutons displaying SypmOr2 responses and percentage of filopodia displaying SypmOr2 responses. Lines are the median, and error bars are interquartile range (IQR). (D) Plot of normalized intensity values along the line shown in (A). (E) Time-lapse series of an axon coexpressing GPI-EGFP and SypmOr2 during stimulation with 600 APs (5 Hz). White arrow, filopodium. Scale bar, 1 μm. (F) Plot of normalized intensity values along the line shown in (E). (G) Primary hippocampal neurons were stimulated with 600 APs (40 Hz) and then subjected to high-pressure freezing (HPF) and freeze substitution (FS). 2D view of a filopodium split into 200-nm segments from the tip to the parent axon. (H) 3D reconstruction of a filopodium showing segments, endosomes (brown), SVs (cyan), main axon filaments (magenta), and mitochondria (dark green) from 2D tracings of electron micrographs. (I) Normalized SV densities within the first four segments from the tip. Boxes are IQR, and whiskers show minimum to maximum. P values, Kruskal-Wallis with Dunn’s test. (J) Normalized endosome fractions within the first four segments from the tip. Boxes are IQR, and whiskers show minimum to maximum. P values, Kruskal-Wallis with Dunn’s test. (K) Representative 2D and 3D images of each filopodium and parent axon from three image stacks. Segments are 200 nm long and provide the scale, except for filopodium 10 (2D), for which the scale matches filopodium 11. Sample sizes and exact P values are shown in the plots. Summary statistics are provided in table S3.