Skip to main content
. 2018 Mar 1;11:62. doi: 10.3389/fnmol.2018.00062

Figure 2.

Figure 2

BDNF upregulates ATF4 expression in processes, cell bodies and nuclei of cultured hippocampal neurons. (A) DIV 21 hippocampal neurons were treated with 25 ng/ml of BDNF or vehicle for 15 mins or 4 h, then immunofluorescently labeled for ATF4 (green) and beta-3-tubulin (red) and for Hoescht 33342 staining (blue). The second row shows a longer exposure to reveal processes. Representative images are shown. Scale bar = 10 μm. (B) Quantification of the ATF4 immunostaining fluorescence intensity of cell bodies and processes for the cultured neurons described in (A). Details for the separate quantification in cell bodies and processes are given in Experimental Procedures. Data were used to compute the intensity ratios for ATF4 vs. beta-3-tubulin at each time with the ratios at time 0 set to 100%. n = 28–42 randomly chosen cell bodies and 7–9 randomly chosen neurite fields per group. Values are means ± SEM. **p = 0.0001, *p = 0.008 compared with controls. p values were calculated vs. contol using the Kruskal-Wallis test (overall p = 0.002) with the post hoc Conover method adjusted by the FDR procedure of Benjamini-Hochberg. (C) BDNF changes the expression of ATF4 in a subpopulation of hippocampal neurons. The values (in arbitary units, A.U.) for ATF4/beta-3-tubulin immunofluorescence ratios in individual cell bodies were determined as in (B) and grouped into bins (from 1 A.U to 11 A.U. in the three upper panels and as indicated in the lower panel). Graphs show the distributions of percentages of cell bodies in each bin at different times of BDNF treatment. n = 28–42 randomly chosen cells per time point. (D) BDNF treatment (4 h, 25 ng/ml) increases ATF4 protein in both nuclear-enriched and synaptosomal-enriched cell fractions. DIV 21 hippocampal neurons treated with BDNF or vehicle were separated into nuclear-enriched (P1, black bars) and synaptosomal-enriched (P2, white bars) fractions as described in “Materials and Methods” section and ATF4 protein levels were determined by Western immunoblotting. Upper panel shows a representative immunoblot, lower panel quantification with values for control set at 100. Values are means ± SEM, n = 3 independent experiments, each with duplicate cell lysates. *p < 0.029, **p < 0.014 vs. controls (no BDNF), t-test.