Fig. 3. Presynaptic filopodia form kinapses with postsynaptic targets in cultured neurons and are present in human cortex.

(A) Representative overlay of a hippocampal neuron cotransfected with GPI-EGFP and Bsn-RFP, stimulated with 600 APs (40 Hz), fixed, and imaged. White arrows, filopodia colocalizing with Bsn-RFP. Scale bar, 1 μm. (B) Overlay image of a neuron transfected with GPI-EGFP and live-labeled with Cy3-conjugated anti-vGAT antibody. White arrows, colocalizing filopodia. Scale bar, 1 μm. (C) Overlay image of a neuron transfected with GPI-EGFP and live-labeled with Oyster550-conjugated anti-GluA antibody. White arrows, colocalizing filopodia. Scale bar, 1 μm. (D to F) Percentages of filopodia-forming boutons and filopodia colocalizing with Bsn-RFP (D), vGAT-Cy3 (E), and GluA-Oyster550 (F). Lines are the median, and error bars are IQR. (G) Confocal time-lapse series showing kinapses between filopodia and GluA-labeled postsynapses during 600-AP (40-Hz) stimulation. White arrows indicate filopodia establishing kinapses. Scale bar, 1 μm. (H) Quantification of GluA–GPI-EGFP colocalizing objects at rest versus at maximum filopodial extension. Bars are the mean. P values, paired t test. (I) Quantification of GluA-GPI-EGFP overlap volume at rest and at maximum filopodial extension. Bars are the mean. P values, paired t test. (J) Representative 2D views (left) and 3D reconstructions (middle and right) of synaptic boutons with putative filopodia from human cerebral cortex. White arrows, filopodia. Middle: 3D reconstructions of synaptic boutons with filopodia showing SVs (red), endosomes (brown), mitochondria (blue), and endoplasmic reticulum (yellow) from 2D tracings of electron micrographs. Right: 3D reconstructions of the same filopodia forming contacts with dendritic spines (a and b) or dendritic shafts (c and d) and thereby enhancing connectivity to a single (a and d) or multiple neurons (b and c). Scale bars, 500 nm.