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. 2020 Nov 16;14:564106. doi: 10.3389/fncel.2020.564106

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Copyright © 2020 Morella, Hallum and Brambilla.

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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(A,C) Pre-treatment with glutamate antagonists CNQX 10 μM and AP5 10 μM increases BDNF-induced ERK activation (two-way ANOVA, effect of BDNF F_(1,28) = 341.384, p < 0.0001, effect of AP5/CNQX F_(1,28) = 19.969, p < 0.0001, effect of interaction F_(1,28) = 13.553, p = 0.001. Bonferroni’s post hoc, not stimulated vs. BDNF: ****p < 0.0001, BDNF vs. BDNF + AP5 + CNQX: ****p < 0.0001). Data are shown as mean ± SEM, n = 8 per group. The values of mean, SEM and fold change relative to not stimulated control are shown in panel (C). (B) Representative pictures of anti-phospho-ERK1/2 immunohistochemistry. Scale bar: 50 μm. (D,F) Pre-treatment with glutamate antagonists CNQX 10 μM and AP5 10 μM has no effect on BDNF-induced H3 activation (two-way ANOVA, effect of BDNF F_(1,23) = 50.80, p < 0.0001, effect of AP5/CNQX F_(1,23) = 1.272, p = 0.2710, effect of interaction F_(1,23) = 1.279, p = 0.2697. Bonferroni’s post hoc, not stimulated vs. BDNF ***p = 0.0007, AP5 + CNQX vs. BDNF + AP5 + CNQX ****p < 0.0001, BDNF vs. BDNF + AP5 + CNQX p = 0.2082). Data are shown as mean ± SEM. Not stimulated and AP5 + CNQX: n = 6 per group, BDNF: n = 7, BDNF + AP5 + CNQX: n = 8. The values of mean, SEM, and fold change relative to not stimulated vehicles are shown in panel (F); n.s.: not significant. (E) Representative pictures of phospho-H3 immunofluorescence, where the red channel (NeuN) is merged with the green channel (phospho-H3). Scale bar: 50 μm. (G,I) Pre-treatment with D1-antagonist SCH23390 upregulates BDNF-induced ERK phosphorylation (two-way ANOVA, effect of BDNF F_(1,28) = 331.98, p < 0.0001, effect of SCH23390 F_(1,28) = 31.82, p < 0.0001, effect of interaction F_(1,28) = 15.51, p = 0.0005. Bonferroni’s post hoc, not stimulated vs. BDNF ***p < 0.001, BDNF vs. BDNF + SCH23390 ***p < 0.001). Data are shown as mean ± SEM, n = 8 per group. The values of mean, SEM, and fold change relative to not stimulated control are shown in panel (I). (H) Representative pictures of anti-phospho-ERK1/2 immunohistochemistry. Scale bar: 50 μm. (J,L) Pre-treatment with D1-antagonist SCH23390 does not affect BDNF-induced H3 phosphorylation (two-way ANOVA, effect of BDNF F_(1,28) = 76.97, p < 0.0001, effect of SCH23390 F_(1,28) = 0.8778, p = 0.3568, effect of interaction F_(1,28) = 0.01749, p = 0.8957. Bonferroni’s post hoc, not stimulated vs. BDNF ****p < 0.0001, SCH23390 vs. BDNF + SCH23390 ****p < 0.0001, BDNF vs. BDNF + SCH23390 p > 0.9999). Data are shown as mean ± SEM, n = 8 per group. The values of mean, SEM, and fold change relative to not stimulated vehicles are shown in panel (L); n.s.: not significant. (K) Representative pictures of phospho-H3 immunofluorescence, where the red channel (NeuN) is merged with the green channel (phospho-H3). Scale bar: 50 μm.