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. 2026 Oct 28;12(44):eaei2661. doi: 10.1126/sciadv.aei2661

Fig. 5. NHE inhibition suppresses presynaptic filopodia formation and promotes asynchronous neurotransmitter release.

Fig. 5.

(A) Confocal time-lapse series showing filopodia formation (white arrows) in neurons transfected with GPI-EGFP and treated for 30 min with vehicle, SMIFH2, EIPA, and amiloride or acutely with dynasore for 2 to 4 min. Neurons were stimulated with 600 APs (40 Hz). Scale bar, 1 μm. (B) Normalized number of filopodia per 10-μm axon length. Lines are the median, and error bars are IQR. P values, Kruskal-Wallis with Dunn’s test. (C) Normalized filopodial length. Lines are the median, and error bars are IQR. P values, Kruskal-Wallis with Dunn’s test. (D) Quantification of filopodial shape. Lines are the median, and error bars are IQR. P values, Kruskal-Wallis with Dunn’s test. (E) Ratio of stable to transient filopodia. Lines are the mean, and error bars are SEM. P values, two-way ANOVA with Sidak’s test. (F to L) Synaptic transmission at CA3-CA1 synapses assessed by whole-cell patch-clamp recordings in wild-type mouse brain slices with (blue) or without (black) 50 μM EIPA for 30 min. (F) Schematic of recording configuration. (G) Input-output plot of stimulation intensity versus EPSC amplitude. Symbols are the mean, and error bars are SEM. P values, two-way ANOVA with Sidak’s test. (H) Representative EPSC traces during 600-AP, 40-Hz stimulation. (I) Synaptic rundown during 600-AP, 40-Hz stimulation. Circles are the mean; error bars are SEM. P values, mixed-effects model with Sidak’s test. (J) Representative traces of asynchronous release events occurring after high-frequency stimulation in control and EIPA-treated conditions. (K) Analysis of posttrain asynchronous release frequency. Bars are the mean, and error bars are SEM. P values, unpaired t test. (L) Amplitude of posttrain asynchronous events. Bars are the mean, and error bars are SEM. P values, unpaired t test.