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. 2020 Jun 3;10:9028. doi: 10.1038/s41598-020-65531-x

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© The Author(s) 2020

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of TrkB mutants of on dendritic spine maturation. (A). To determine the effects of TrkB mutants on dendritic spines size, rat hippocampal neurons - transiently transfected with Clover fluorescent protein-actin and WT/mutant TrkB - were analysed for spine area before (−) and after (+) recombinant BDNF on DIV11. Representative images are shown in Figs. 3C and 4C (images used for quantification). Cumulative frequency plots for control + /− BDNF and mutants + BDNF are shown. In control, stimulation with BDNF (grey vs black), significantly increases the size of spines. p < 2.2*10⁻¹⁶, Kolmogorov–Smirnov (K-S) test. Expression of mutant forms of Trkb stunts BDNF’s ability to promote spine growth (colours vs grey). p < 0.001, K-S test. (B). To determine the effect of TrkB mutants on post synaptic density sizes, rat hippocampal neurons were transiently transfected with ClFP-Actin and WT/mutant TrkBs (DIV5) and stimulated with vehicle or BDNF (DIV7-11), followed by staining for PSD-95. Representative images are shown (Fig. 4C). Cumulative frequency plots for control + /− BDNF and mutants + BDNF are shown. In control, stimulation with BDNF (grey vs black), significantly increases the area if PSD-95 staining. p < 3*10⁻¹⁵, K-S test. Expression of mutant Trkb diminishes BDNF’s stimulatory effect on postsynaptic density size (colours vs grey). p < 2.2*10⁻¹⁶, K-S test. (C). PSD-95 staining of dendritic spines (mushroom and stubby) is shown in the left-hand panel and proportion of spines positive for PSD-95 staining following expression of WT/mutants is shown in the right-hand panel. PSD-95 staining is reduced for all mutants in the stimulated and unstimulated state. *p < 0.01, student’s t-test ± SEM. (D). To determine the effects of TrkB mutants on the surface expression of GluR1-containing AMPA receptors in dendritic spines, rat hippocampal neurons were transiently transfected with ClFP-actin and WT/mutant TrkBs, followed by live staining for GluR1 in the presence or absence of recombinant BDNF (DIV7-11). Representative images are shown (left-hand panel) and the percentage of dendritic spines (mushroom and stubby) positive for surface GluR1 staining is shown in the right-hand panel. In control, stimulation with BDNF significantly (*p < 0.01, student’s t-test ± SEM) increases GluR1 staining in spines, but shows the reverse effect (i.e. decrease) in 3 of the 4 mutants tested (^#p < 0.05, student’s t-test ± SEM).