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. 2017 Jul 10;11:186. doi: 10.3389/fncel.2017.00186

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Copyright © 2017 Micheli, Ceccarelli, Gioia, D’Andrea, Farioli-Vecchioli, Costanzi, Saraulli, Cestari and Tirone.

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) or licensor 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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Treatment with fluoxetine significantly reduces the length of the S phase in Tis21 knockout 2-month-old dentate gyrus progenitor cells. (A) Representative confocal images of cycling cells (40× magnification), with 3D reconstruction from Z-stack and orthogonal projections of triple- and double-positive cells. Cycling cells are shown: (i) cells in S-phase during the 3 h interval after the IdU pulse and before CldU (IdU⁺/CldU⁻ cells, green, arrow; or NeuroD1⁺/IdU⁺/CldU⁻, blue-green, arrows with dotted line); (ii) cells in S-phase during the CldU pulse (IdU⁻/CldU⁺cells, red, arrowhead); (iii) or cells that were in S-phase during both pulses and continued throughout the cell cycle (IdU⁺/CldU⁺, yellow-green, asterisk; or NeuroD1⁺/IdU⁺/CldU⁺, white, at lines intersection). Scale bar, 50 μm. (B) Treatment timeline: 2-month-old wild-type and Tis21 knockout mice, after fluoxetine treatment for 21 days, were injected with IdU and CldU 3 h and 45 min, respectively, before analysis. (B′) Equation to calculate the S-phase length. The ratio of cells that have left the S phase during the interval of 3 h between injections (IdU⁺/CldU⁻ cells) to the total number of IdU⁺ cells is equal to 3 h/Ts. (C) Graphs showing the measurement of the length of the S phase (Ts) for the total population of progenitor cells and (D) for the NeuroD1⁺ progenitor cells population in the different experimental conditions. (C) A shortening of Ts is evident only in the total population of progenitor cells, in the Tis21^KO + FLX mice with respect to the Tis21^WT and Tis21^KO mice. Analysis of simple effects: *p < 0.05, PLSD ANOVA test. (D) NeuroD1⁺ progenitor cell population, analysis of simple effects: NS p > 0.05, PLSD ANOVA test. Data are mean ± SEM of the analysis of five (C) or 3–4 (D) animals per group (see Supplementary Table S1). (E) Treatment timeline and (F) analysis of S phase length (Ts) for the total proliferating stem/progenitor cells or (G) for the NeuroD1⁺ progenitor cells in 2-week-old wild-type and Tis21 knockout mice. *p < 0.05, or ***p < 0.001 Student’s t test. Data are mean ± SEM of the analysis of three animals per group.