Article Figures & Data
Figures
- Figure 1.
TAM chow causes food avoidance and results in less TAM exposure compared with TAM injection. A, Experimental model and timeline. Adult Nestin-CreERT2+/−;Rosa26-LoxP-STOP-LoxP-EYFP+/− mice were either injected with TAM (intraperitoneally) or voluntarily fed TAM chow. B, C, Body mass (in grams) of female (day: F(1.637,24.56) = 8.314, p = 0.0029; TAM: F(1,20) = 45.35, p < 0.0001; day × TAM interaction: F(3,45) = 23.42, p < 0.0001; B) and male (day: F(0.5896,9.630) = 1.338, p = 0.2412; TAM: F(1,23) = 21.96, p = 0.0001; day × TAM interaction: F(3,49) = 10.40, p < 0.0001; C) mice during and after TAM treatment. Mean ± SEM of n = 4–11 mice/time point. D, TAM chow consumption estimation (g of food not recovered to weigh) per mouse (ms) per day, averaged within cage (sex: F(1,22) = 6.139, p = 0.0214; day: F(4,22) = 8.310, p = 0.0003; interaction: F(4,22) = 1.159, p = 0.3558). Mean ± SEM of n = 2–4 cages shown. E, Estimated cumulative TAM exposure (milligram (mg) of TAM) in injected and chow-fed mice (sex: F(1,25) = 39.49, p < 0.0001; TAM: F(2,25) = 51.35, p < 0.0001; interaction: F(2,25) = 2.626, p = 0.0922). Mean ± SEM shown from n = 11–13 mice (injection) or n = 1–2 cages (chow). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Sidak’s multiple comparisons test following a mixed-effects analysis (B, C) or two-way ANOVA (D, E).
- Figure 2.
Recombination efficiency is higher following TAM injection than TAM diet. A, Representative images of EYFP+ labeling in the DG. Scale bar: 100 μm. B, Density of EYFP+ cells (cells/μm2) in the DG (ordinary one-way ANOVA, F(3,35) = 6.885, p = 0.0009). Mean ± SEM of n = 7–11 mice. C, Representative images of EYFP, SOX2, and GFAP labeling in the dentate gyrus. Scale bar: 20 μm. D, The percent of phenotypic GFAP+/SOX2+ RGL-NSCs and GFAP-/SOX2+ IPCs that were EYFP+ (RGLs: ordinary one-way ANOVA, F(3,35) = 11.43, p < 0.0001; IPCs: ordinary one-way ANOVA, F(3,35) = 2.788, p = 0.0550). Mean ± SEM of n = 7–11 mice. E, The percent of EYFP+ cells that showed RGL-NSC phenotype (ordinary one-way ANOVA, F(3,35) = 7.531, p = 0.0005). Mean ± SEM of n = 7–11 mice. F, The percent of EYFP+ cells that showed IPC phenotype (ordinary one-way ANOVA, F(3,35) = 7.799, p = 0.0004). Mean ± SEM of n = 7–11 mice. G, The percent of EYFP+ cells that showed GFAP+ stellar astrocyte phenotype (ordinary one-way ANOVA, F(3,35) = 2.465, p = 0.0784). Mean ± SEM of n = 7–11 mice. H, Representative images of EYFP and DCX labeling in the dentate gyrus. Scale bar: 20 μm. I, The percent of EYFP+ cells that were DCX+ (ordinary one-way ANOVA, F(3,35) = 34.97, p < 0.0001). Mean ± SEM of n = 7–11 mice. J, Representative images of EYFP labeling throughout the hippocampus. DG = dentate gyrus. Scale bar: 500 μm. K, Percent of hippocampal EYFP+ cells found in the CA1, CA3, and dentate gyrus (two-way repeated measures ANOVA; TAM × area: F(6,70) = 3.338, p = 0.0060; TAM: F(3,35) = 1.051, p = 0.3822; area: F(1.186,41.50) = 20 157, p < 0.0001; subject: F(35,70) = 6.492e-007, p > 0.9999). Mean ± SEM of n = 7–11 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Tukey’s multiple comparisons test.
- Figure 3.
Recombination efficiency and labeled cell types do not depend on sex. A, Density of EYFP+ cells (cell/μm2) in the dentate gyrus by sex (two-way ANOVA; interaction: F(3,31) = 2.335, p = 0.0931; sex: F(1,31) = 0.9785, p = 0.3302; TAM: F(3,31) = 8.017, p = 0.0004). Mean ± SEM of n = 3–7 mice. B, The percent of phenotypic GFAP+/SOX2+ RGL-NSCs (two-way ANOVA; interaction: F(3,31) = 2.003, p = 0.1341; sex: F(1,31) = 0.5780, p = 0.4528; TAM: F(3,31) = 11.87, p < 0.0001) and GFAP-/SOX2+ IPCs (two-way ANOVA; interaction: F(3,31) = 2.152, p = 0.1138; sex: F(1,31) = 0.3609, p = 0.5524; TAM: F(3,31) = 3.040, p = 0.0436) that were EYFP+, with sex on the horizontal axis. Mean ± SEM of n = 3–7 mice. C, The percent of EYFP+ cells that showed RGL-NSC phenotype, analyzed separately by sex (two-way ANOVA; interaction: F(3,31) = 1.147, p = 0.3457; sex: F(1,31) = 0.1478, p = 0.7032; TAM: F(3,31) = 7.335, p = 0.0007). Mean ± SEM of n = 3–7 mice. D, The percent of EYFP+ cells that showed IPC phenotype, analyzed separately by sex (two-way ANOVA; interaction: F(3,31) = 0.4952, p = 0.6883; sex: F(1,31) = 1.078, p = 0.3072; TAM: F(3,31) = 6.650, p = 0.0013). Mean ± SEM of n = 3–7 mice. E, The percent of EYFP+ cells that showed GFAP+ stellar astrocyte phenotype, analyzed separately by sex (two-way ANOVA; interaction: F(3,31) = 2.982, p = 0.0464; sex: F(1,31) = 0.1198, p = 0.7316; TAM: F(3,31) = 2.411, p = 0.0857). Mean ± SEM of n = 3–7 mice. F, The percent of EYFP+ cells that showed DCX+ immature neuron phenotype, analyzed separately by sex (two-way ANOVA; interaction: F(3,31) = 1.580, p = 0.2141; sex: F(1,31) = 0.2172, p = 0.6444; TAM: F(3,31) = 33.62, p < 0.0001). Mean ± SEM of n = 3–7 mice. G, Percent of hippocampal EYFP+ cells found in the CA1, CA3, and dentate gyrus (DG) in male (two-way repeated measures ANOVA; group × region: F(6,36) = 6.259, p = 0.0001; group: F(3,18) = 1.636, p = 0.2162; region: F(1.303,23.46) = 14,034, p < 0.0001; subject: F(18,36) = 3.413e-007, p > 0.9999) and female (two-way repeated measures ANOVA; group × region: F(6,26) = 0.2057, p = 0.9719; group: F(3,13) = 1.090, p = 0.3880; region: F(1.193,15.51) = 12,616, p < 0.0001; subject: F(13,26) = 1.595e-006, p > 0.9999) mice at d7 and d14 dpi and 10 and 14 d after TAM chow initiation. Mean ± SEM of n = 3–5 female mice and 4–7 male mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Sidak’s multiple comparisons test (A–F) or Tukey’s multiple comparisons test (G).
- Figure 4.
TAM injection suppresses progenitor cell proliferation three weeks after TAM. A, Experimental model and timeline. Adult wild-type mice were injected with TAM daily for 5 d then perfused three weeks later. Mice received three daily BrdU injections on the last 3 d of TAM and one EdU injection 2 h before perfusion. B, C, Average body mass of females (two-way repeated measures ANOVA; day × TAM: F(1,9) = 11.17, p = 0.0086; day: F(1,9) = 22.53, p = 0.0010; TAM: F(1,9) = 21.55, p = 0.0012; subject: F(9,9) = 0.9448, p = 0.5330) and males (two-way repeated measures ANOVA; day × TAM: F(1,10) = 0.3575, p = 0.5632; day: F(1,10) = 24.83, p = 0.0006; TAM: F(1,10) = 0.6152, p = 0.4510; subject: F(10,10) = 6.138, p = 0.0042) at the end of TAM treatment (d4) and three weeks later (d27). Mean ± SEM of n = 5–6 female and 6 male mice. D, Density of BrdU+ cells (cells/μm2 × 104) in the dentate gyrus (unpaired t test, t(21) = 1.613, p = 0.1217). Mean ± SEM of n = 11–12 mice. E, Density of dentate gyrus BrdU+ cells co-labeled with DCX and/or NeuN (per μm2 × 104; two-way repeated measures ANOVA; cell type × TAM: F(2,42) = 2.461, p = 0.0976; cell type: F(2,42) = 371.0, p < 0.0001; TAM: F(1,21) = 1.843, p = 0.1890; subject: F(21,42) = 1.698, p = 0.0714). Mean ± SEM of n = 11–12 mice. F, Percent of BrdU+ dentate gyrus cells co-labeled for DCX and/or NeuN (two-way repeated measures ANOVA; cell type × TAM: F(2,42) = 3.027, p = 0.0591; TAM: F(1,21) = 0.1108, p = 0.7425; cell type: F(1.606,33.73) = 440.6, p < 0.0001; subject: F(21,42) = 0.5522, p = 0.9275). Mean ± SEM of n = 11–12 mice. G, Representative images of BrdU, DCX, and NeuN labeling. Scale bar: 20 μm. H, Density of EdU+ cells (cells/μm2 × 104) in the dentate gyrus (unpaired t test, t(21) = 2.814, p = 0.0104). Mean ± SEM of n = 11–12 mice. I, Density (cells/μm2 × 104) of EdU+ phenotypic RGL-NSCs, IPCs, and other cells in the dentate gyrus (two-way ANOVA; interaction: F(2,63) = 13.82, p < 0.0001; cell type: F(2,63) = 287.1, p < 0.0001; TAM: F(1,63) = 8.275, p = 0.0055). Mean ± SEM of n = 11–12 mice. J, Density (cells/μm2 × 104) of RGL-NSCs and IPCs in the dentate gyrus (two-way repeated measures ANOVA; cell type × TAM: F(1,21) = 3.203, p = 0.0879; cell type: F(1,21) = 8.683, p = 0.0077; TAM: F(1,21) = 15.23, p = 0.0008; subject: F(21,21) = 1.029, p = 0.4741). Mean ± SEM of n = 11–12 mice. K, Representative images of EdU, SOX2, and GFAP labeling. Scale bar: 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Sidak’s multiple comparisons test.
- Figure 5.
TAM injection effects on cell survival and proliferation do not differ by sex. A, Density of BrdU+ cells (cells/μm2 × 104) in the dentate gyrus (two-way ANOVA; interaction: F(1,19) = 0.09813, p = 0.7575; sex: F(1,19) = 3.232, p = 0.0881; TAM: F(1,19) = 3.076, p = 0.0956). Mean ± SEM of n = 5–6 mice. B, Density of dentate gyrus BrdU+ cells (cells/μm2 × 104) co-labeled with DCX and/or NeuN (three-way repeated measures ANOVA; cell type: F(1.823,34.63) = 343.0, p < 0.0001; sex: F(1,19) = 0.5866, p = 0.4531; TAM: F(1,19) = 1.581, p = 0.2238; cell type × sex: F(2,38) = 0.5649, p = 0.5731; cell type × TAM: F(2,38) = 2.197, p = 0.1250; sex × TAM: F(1,19) = 0.2991, p = 0.5908; cell type × sex × TAM: F(2,38) = 0.02294, p = 0.9773). Mean ± SEM of n = 6 female mice and 5–6 male mice. C, Percent of BrdU+ dentate gyrus cells labeled for DCX and/or NeuN (three-way repeated measures ANOVA; cell type: F(1.608,30.55) = 401.7, p < 0.0001; sex: F(1,19) = 0.4138, p = 0.5277; TAM: F(1,19) = 0.08557, p = 0.7731; cell type × sex: F(2,38) = 0.1847, p = 0.8321; cell type × TAM: F(2,38) = 2.828, p = 0.0716; sex × TAM: F(1,19) = 3.418, p = 0.0801; cell type × sex × TAM: F(2,38) = 0.04,942), p = 0.9518. Mean ± SEM of n = 5–6 mice. D, Density of EdU+ cells (cells/μm2 × 104) in the dentate gyrus (two-way ANOVA; interaction: F(1,19) = 2.991, p = 0.0999; sex: F(1,19) = 1.313, p = 0.2660; TAM: F(1,19) = 7.928, p = 0.0110). Mean ± SEM of n = 5–6 mice. E, Density (cells/μm2 × 104) of dentate gyrus EdU+ phenotypic RGL-NSCs, IPCs, and other cells (three-way repeated measures ANOVA; cell type: F(2,38) = 274.4, p < 0.0001; sex: F(1,19) = 1.313, p = 0.2660; TAM: F(1,19) = 7.928, p = 0.0110; cell type × sex: F(2,38) = 0.4635, p = 0.6326; cell type × TAM: F(2,38) = 12.86, p < 0.0001; sex × TAM: F(1,19) = 2.991, p = 0.0999; cell type × sex × TAM: F(2,38) = 0.1659, p = 0.8477). Mean ± SEM of n = 5–6 female mice and 6 male mice. F, Density (cells/μm2 × 104) of RGL-NSCs and IPCs in the dentate gyrus, by sex (three-way repeated measures ANOVA; cell type: F(1,19) = 9.308, p = 0.0066; sex: F(1,19) = 0.1083, p = 0.7457; TAM: F(1,19) = 14.12, p = 0.0013; cell type × sex: F(1,19) = 3.346, p = 0.0831; cell type × TAM: F(1,19) = 3.699, p = 0.0696; sex × TAM: F(1,19) = 0.3142, p = 0.5817; cell type × sex × TAM: F(1,19) = 0.7541, p = 0.3960). Mean ± SEM of n = 5–6 female mice and 6 male mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Sidak’s multiple comparisons test.
- Figure 6.
TAM chow alters the phenotype of surviving cells 35 d after initial exposure. A, Experimental model and timeline. Mice were injected with BrdU in the last 3 d of the 14-d TAM chow period. EdU was injected 35 d after initial chow exposure. B, Grams of food consumed per mouse (ms) per day over each 3- to 4-d period of diet exposure and TAM consumed in milligrams (mg) (two-way repeated measures ANOVA; day × TAM: F(3,18) = 2.322, p = 0.1095; day: F(1.564,9.384) = 8.978, p = 0.0088; TAM: F(1,6) = 45.86, p = 0.0005; subject: F(6,18) = 0.9896, p = 0.4613). Mean ± SEM of n = 4 mice. C, In female mice, body mass in grams 14 and 35 d after diet initiation (two-way repeated measures ANOVA; day × TAM: F(1,9) = 10.62, p = 0.0099; day: F(1,9) = 42.05, p = 0.0001; TAM: F(1,9) = 0.8454, p = 0.3818; subject: F(9,9) = 0.7591, p = 0.6560). Mean ± SEM of n = 5–6 mice. D, In male mice, body mass in grams 14 and 35 d after diet initiation (two-way repeated measures ANOVA; day × TAM: F(1,10) = 18.18, p = 0.0017; day: F(1,10) = 62.45, p < 0.0001; TAM: F(1,10) = 8.540, p = 0.0152; subject: F(10,10) = 1.886, p = 0.1658). Mean ± SEM of n = 6 mice. E, Density of BrdU+ cells (cells/μm2 × 104) in the dentate gyrus (unpaired t test, t(21) = 1.675, p = 0.1087). Mean ± SEM of n = 11–12 mice. F, Density of dentate gyrus BrdU+ cells (cells/μm2 × 104) co-labeled with DCX and/or NeuN (two-way repeated measures ANOVA; cell type × TAM: F(2,42) = 8.654, p = 0.0007; cell type: F(1.207,25.35) = 83.41, p < 0.0001; TAM: F(1,21) = 8.675, p = 0.0077; subject: F(21,42) = 1.516, p = 0.1239). Mean ± SEM of n = 11–12 mice. G, Percent of BrdU+ dentate gyrus cells labeled for DCX and/or NeuN (two-way repeated measures ANOVA; TAM × cell type: F(2,42) = 6.501, p = 0.0035; TAM: F(1,21) = 16.13, p = 0.0006; cell type: F(1.545,32.44) = 151.5, p < 0.0001; subject: F(21,42) = 0.3789, p = 0.9903). Mean ± SEM of n = 11–12 mice. H, Representative images of BrdU, DCX, and NeuN labeling. Scale bar: 20 μm. I, Density of EdU+ cells (cells/μm2 × 104) in the dentate gyrus (unpaired t test, t(21) = 1.043, p = 0.3090). Mean ± SEM of n = 11–12 mice. J, Density (cells/μm2 × 104) of dentate gyrus EdU+ phenotypic RGL-NSCs, IPCs, and other cells (two-way repeated measures ANOVA; cell type × TAM: F(2,42) = 1.157, p = 0.3241; cell type: F(1.066,22.38) = 210.3, p < 0.0001; TAM: F(1,21) = 1.087, p = 0.3090; subject: F(21,42) = 0.9645, p = 0.5209). Mean ± SEM of n = 11–12 mice. K, Density (cells/μm2 × 104) of RGL-NSCs and IPCs in the dentate gyrus (two-way repeated measures ANOVA; cell type × TAM: F(1,21) = 1.042, p = 0.3191; cell type: F(1,21) = 3.794, p = 0.0649; TAM: F(1,21) = 2.404, p = 0.1360; subject: F(21,21) = 1.138, p = 0.3846). Mean ± SEM of n = 11–12 mice. L, Representative images of EdU, SOX2, and GFAP labeling. Scale bar: 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Sidak’s multiple comparisons test.
- Figure 7.
TAM diet effects on cell survival and proliferation do not differ by sex. A, Grams of TAM and vehicle chow consumed by male (n = 2 cages/sex) and female (n = 2 cages/sex) mice per mouse (ms) per day over each 3- to 4-d period of diet exposure. B, Cumulative TAM consumed in milligrams (mg) by sex (n = 2 cages/sex). C, Density of BrdU+ cells (cells/μm2 × 104) in the dentate gyrus, by sex (two-way ANOVA; interaction: F(1,19) = 0.6818, p = 0.4192; sex: F(1,19) = 0.5413, p = 0.4709; TAM: F(1,19) = 2.921, p = 0.1037). Mean ± SEM of n = 5–6 mice. D, Density of dentate gyrus BrdU+ cells (cells/μm2 × 104) co-labeled with DCX and/or NeuN in females and males (three-way repeated measures ANOVA: cell type: F(1.213,23.05) = 78.09, p < 0.0001; sex: F(1,19) = 0.2586, p = 0.6169; TAM: F(1,19) = 8.323, p = 0.0095; cell type × sex: F(2,38) = 0.02142, p = 0.9788; cell type × TAM: F(2,38) = 8.256, p = 0.0011; sex × TAM: F(1,19) = 0.3225, p = 0.5767; cell type × sex × TAM: F(2,38) = 0.5548, p = 0.5787). Mean ± SEM of n = 5–6 female mice and 6 male mice. E, Percent of BrdU+ dentate gyrus cells labeled for DCX and/or NeuN in male and female mice (three-way repeated measures ANOVA: cell type: F(1.557,29.58) = 140.5, p < 0.0001; sex: F(1,19) = 0.3592, p = 0.5561; TAM: F(1,19) = 14.62, p = 0.0011; cell type × sex: F(2,38) = 0.6323, p = 0.5369; cell type × TAM: F(2,38) = 5.877, p = 0.0060; sex × TAM: F(1,19) = 0.3233, p = 0.5763; cell type × sex × TAM: F(2,38) = 0.06,331, p = 0.9387). Mean ± SEM of n = 5–6 mice. F, Density of EdU+ cells (cells/μm2 × 104) in the dentate gyrus in male and female mice (two-way ANOVA; interaction: F(1,19) = 0.2581, p = 0.6173; sex: F(1,19) = 0.1190, p = 0.7339; TAM: F(1,19) = 0.9220, p = 0.3490). Mean ± SEM of n = 5–6 mice. G, Density (cells/μm2 × 104) of dentate gyrus EdU+ phenotypic RGL-NSCs, IPCs, and other cells in male and female mice (three-way ANOVA: cell type: F(1.058,20.10) = 190.3, p < 0.0001; sex: F(1,19) = 0.1190, p = 0.7339; TAM: F(1,19) = 0.9220, p = 0.3490; cell type × sex: F(2,38) = 0.05511, p = 0.9465; cell type × TAM: F(2,38) = 1.094, p = 0.3451; sex × TAM: F(1,19) = 0.2581, p = 0.6173; cell type × sex × TAM: F(2,38) = 0.08267, p = 0.9208). Mean ± SEM of n = 5–6 female mice and 6 male mice. H, Density (cells/μm2 × 104) of RGL-NSCs and IPCs in the dentate gyrus of male and female mice (three-way ANOVA: cell type: F(1,19) = 3.943, p = 0.0617; sex: F(1,19) = 2.181, p = 0.1561; TAM: F(1,19) = 2.703, p = 0.1166; cell type × sex: F(1,19) = 4.370, p = 0.0503; cell type × TAM: F(1,19) = 1.338, p = 0.2618; sex × TAM: F(1,19) = 0.1703, p = 0.6845; cell type × sex × TAM: F(1,19) = 0.1708, p = 0.6840). Mean ± SEM of n = 5–6 female mice and 6 male mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Sidak’s multiple comparisons test.
Tables
Primary
antibodyVendor/product
numberRRID Dilution Secondary Vendor/product
numberDilution RRID Rabbit anti-GFP ThermoFisher AB_221569 1:1000 Donkey Alexa Fluor
488 anti-rabbitFisher 1:500 AB_2535792 A11122 A21206 Mouse anti-GFAP EMD Millipore AB_11212597 1:1000 Donkey Alexa Fluor
647 anti-mouseFisher 1:500 AB_162542 MAB360 A31571 Rat anti-SOX2 Affymetrix eBioscience AB_11219471 1:1000 Donkey Alexa Fluor
594 anti-ratFisher 1:500 AB_2535795 14–9811 A21209 Rabbit anti-DCX Cell Signaling AB_561007 1:500 Donkey Alexa Fluor
555 anti-rabbitFisher 1:500 AB_162543 4604 A31572 Mouse anti-NeuN EMD Millipore AB_2298772 1:1000 Donkey Alexa Fluor
647 anti-mouseFisher 1:500 AB_162542 MAB377 A31571 Rat anti-BrdU Bio-Rad AB_609568 1:500 Donkey Alexa Fluor
488 anti-mouseFisher 1:500 AB_141607 OBT0030 A21202 List of primary and secondary antibodies.
List of other major reagents.
Figure 2-1
Raw data supporting Figure 2. Download Figure 2-1, XLSX file.
Figure 4-1
Raw data supporting Figure 4. Download Figure 4-1, XLSX file.
Figure 6-1
Raw data supporting Figure 6. Download Figure 6-1, XLSX file.
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