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. 2020 Sep 30;37(2):145–165. doi: 10.1007/s12264-020-00581-9

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — Enhanced spatiotemporal dynamics of neuronal surface ASIC1a underlying BDNF treatment. A–C Cultured rat cortical neurons treated with vehicle (Ctrl), BDNF (20 ng/mL) or BDNF plus the pan-Trk inhibitor, K252a (200 nmol/L) for 5 min before surface biotinylation. A Representative blots of surface (biotinylated, S) and total (T) ASIC1a, with GAPDH as a cytoplasmic protein control. B Quantification of surface ASIC1a levels by S/T ratio (normalized to Ctrl). Data are presented as the mean ± SEM. [BDNF/Ctrl, 1.48 ± 0.02; BDNF/(K252a + BDNF), 1.47 ± 0.01; (K252a + BDNF)/Ctrl, 1.00 ± 0.02, **P < 0.01, n = 4 experiments, no significant difference between Ctrl and BDNF + K252a, one-way ANOVA multiple comparison]. C Quantification of total ASIC1a levels by ASIC1a/GAPDH ratio (normalized to Ctrl). Data are presented as the mean ± SEM. [BDNF/Ctrl, 0.94 ± 0.01; BDNF/(K252a + BDNF), 0.96 ± 0.04; (K252a + BDNF)/Ctrl, 0.98 ± 0.02, n = 4 experiments, no significant difference between any two groups, one-way ANOVA multiple comparison. D, E Asic1a^−/− neurons co-transfected with GFP-hASIC1a and mCherry were treated with vehicle, BDNF, or BDNF plus K252a as above before surface staining. D Representative images of surface hASIC1a stained by ASC06-IgG1, as well as that of total hASIC1a by GFP and neuron morphology by mCherry (scale bar, 20 μm). E Quantification of surface hASIC1a level based on ASC06-IgG1/GFP intensity ratio. Data are presented as the mean ± SEM. [Vehicle, 0.69 ± 0.05 (n = 11 neurons); BDNF, 0.88 ± 0.05 (n = 10 neurons); BDNF + K252a, 0.63 ± 0.06 (n = 8 neurons), *P < 0.05, **P < 0.01, no significant difference between Ctrl and BDNF + K252a, one-way ANOVA multiple comparison. F, G FRAP assays of dendritic spines of Asic1a^−/− neurons co-transfected with mCherry and hASIC1a-²⁹⁸pHluorin²⁹⁹. After 5 min of baseline recording, neurons were perfused with ECS containing vehicle, BDNF, or BDNF plus K252a for another 5 min. Then a selected mushroom spine head was bleached with the 488 nm laser for 1 min, followed by another 20 min of recording. F Representative time-lapse images (scale bars, 1 μm). G Quantification of FRAP. After 20 min of recovery, the pHluorin fluorescence returns to 40.20 ± 1.66% (n = 122 spines) of baseline for Ctrl, 49.98 ± 1.48% (n = 100 spines) for BDNF, and 32.22 ± 1.05% (n = 134 spines) for K252a + BDNF. Data points represent the mean ± SEM, **P < 0.01, ****P < 0.0001, no significant difference between Ctrl and BDNF + K252a, two-way ANOVA multiple comparison. H, I Effect of BDNF on lateral mobility of surface hASIC1a in dendritic shafts and spines. H Representative images and reconstructed trajectories of surface hASIC1a SPT (scale bars, 1 μm). I MSD versus time plot for the mobility of surface hASIC1a puncta on dendritic shafts and spines with or without BDNF treatment. Data points represent the mean ± SEM, n = 19 cells for Ctrl and n = 21 cells for BDNF-treated. Paired t test on area under the curve showed no significant difference between shafts and spines in either the Ctrl or BDNF treatment group, but **P < 0.01 for Ctrl versus BDNF-treated at either shafts or spines.