Fig. 2. Presynaptic filopodia formation is coupled to neuronal activity in cultures and acute brain slices.

(A) Axon coexpressing GPI-Apple and GCaMP6f after APV/CNQX treatment. Scale bar, 1 μm. (B) GCaMP6f responses and filopodia in APV/CNQX-treated cultures. Lines are the median. (C) Axon coexpressing GPI-Apple and GCaMP6f after 4-AP treatment. White arrows, filopodia and calcium responses. Scale bar, 1 μm. (D) Event-triggered GCaMP6f fluorescence aligned to T-max. Line is mean; shading is SEM. (E to H) GCaMP6f fluorescence during the 55-s pre–T-max window (E and F) and at T-max (G and H), relative to the recording mean. (E and G) Recording-mean ΔF/F₀ versus maximum pre–T-max ΔF/F₀ (E) or T-max ΔF/F₀ (G). Points above the dashed line indicate above-mean calcium. (F and H) Maximum pre–T-max ΔF/F₀ (F) or ΔF/F₀ at T-max (H) relative to the recording-mean. Bars are the mean, and error bars are SEM. P values, paired t test. (I) Peak–to–T-max lag intervals in 4-AP–treated neurons. (J) Stable-to-transient filopodia ratio after 4-AP treatment. Bars are the mean, and error bars are SEM. P values, unpaired t test. (K) Number of filopodia at boutons during basal activity, 4-AP treatment, or 4-AP following EGTA AM preincubation. Bars are the mean, and error bars are SEM. P values, mixed-effects model with Sidak’s test. (L) Thy1-GFPM cortical region with sparse EGFP expression. Boxes, axons (pink) and dendrites (yellow). Scale bars, 100 μm (large image) and 2 μm (inset). (M) Time-lapse images of Fura Red-loaded neurons treated with 4-AP (top two) or APV/CNQX (bottom). White arrows, filopodia; yellow arrows, filopodia emerging during Fura Red fluorescence decreases depicted in kymographs. Scale bars, 1 μm. (N) Number of filopodia formed along axons in slices. Lines are the median, and error bars are IQR. P values, Kruskal-Wallis with Dunn’s test. (O) Stable-to-transient filopodia ratio in 4-AP–treated slices. Bars are the mean, and error bars are SEM. P values, unpaired t test.