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. 2018 Oct 30;12:391. doi: 10.3389/fncel.2018.00391

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Copyright © 2018 Hiester, Becker, Bowen, Schwartz and Kennedy.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Blocking postsynaptic exocytosis prevents stabilization of AMPA receptors initially recruited by activity in dendritic spines. (A) TeNT light chain blocks activity-triggered RE fusion and accumulation of surface TfR-SEP. Representative images of dissociated hippocampal neurons expressing TfR-SEP with mCherry (mCh, top panels) or mCh and TeNT (mCh-2A-TeNT, bottom panels). Images were taken pre- (left) and 15 min post (right) cLTP stimulation. The plot to the right shows TfR-SEP signal plotted as a function of time following cLTP (black bar). N = 8–10 neurons per condition. (B) Quantification of the normalized total dendritic SEP-GluA1 signal before and after cLTP stimulation for control (n = 7 neurons) and TeNT expressing (n = 9 neurons) neurons. ^**p < 0.001, ^*p < 0.05 (Paired two-tailed Student's t-test). (C) Representative examples of SEP-GluA1 spine accumulation from control (left) and TeNT expressing (right) neurons before (0 min), 20 and 50 min following cLTP stimulation. The top row shows the SEP-GluA1 signal, the middle row shows the mCh signal and the bottom row shows the merge of the two channels. The dotted line represents an outline of the cell morphology based on the mCh signal. Scale bar, 1 μm. (D) Traces showing the spine SEP-GluA1 and mCh signals in control (solid lines) and TeNT-expressing (dashed lines) neurons as a function of time for the spines shown in (C). The gray box indicates the duration of the cLTP stimulus. (E) Cumulative probability of maximum SEP-GluA1 accumulation within randomly selected spines following cLTP stimulation for control neurons (red line, n = 119 spines from 6 neurons) and neurons expressing TeNT (black dashed line, n = 138 spines from 9 neurons). p = 0.66, Kolmogorov-Smirnov test. (F) Quantification of the SEP-GluA1 signal retention over time in selected dendritic spines for control (filled circles, n = 52 spines from 7 neurons) and TeNT expressing (open circles, n = 87 spines from 9 neurons) neurons. Only spines that acquired SEP signal >25% over baseline were selected for this analysis. The gray box indicates the duration of the cLTP stimulus. ^***p < 0.001 (Two-way ANOVA, Bonferroni multiple comparisons test). (G) Shown is a cumulative probability plot of the decrease in spine SEP-GluA1 signal 50 min following cLTP stimulation in control neurons (red line) and neurons expressing TeNT (black dashed line). p = 0.0082, Kolmogorov-Smirnov test.