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. 2025 Mar 8;16(1):2333.
doi: 10.1038/s41467-025-57810-w.

Maternal behavior promotes resilience to adolescent stress in mice through a microglia-neuron axis

Hongyu Chen #  1   2 , Ruifeng Xu #  1   2   3 , Jianhao Wang #  1   2 , Feng Gao #  1   2 , Yida Lv  1   2 , Xiang Li  1   2 , Fang Li  1   2 , Junqin Zhao  1   2 , Xi Zhang  1   2 , Jiabei Wang  1   2 , Ruicheng Du  1   2 , Yuke Shi  1   2 , Hang Yu  1   2 , Shuai Ding  1   2 , Wenxin Li  1   2 , Jing Xiong  1   2 , Jie Zheng  4   5 , Liang Zhao  3 , Xin-Ya Gao  6   7 , Zhi-Hao Wang  8   9
Affiliations

Maternal behavior promotes resilience to adolescent stress in mice through a microglia-neuron axis

Hongyu Chen et al. Nat Commun. .

Abstract

Early life experience modulates resilience to stress in later life. Previous research implicated maternal care as a key mediator of behavioral responses to the adversity in adolescence, but details of molecular mechanisms remain elusive. Here, we show social stress activates transcription factor C/EBPβ in mPFC neurons of adolescent mice, which transcriptionally upregulates Dnm1l and promotes mitochondrial dysfunction, thereby conferring stress susceptibility in adolescent mice. Moreover, different maternal separation differentially regulates adolescent stress susceptibility. Mechanistically, this differential effect depends on maternal behavior-stimulated IGF-1, which inhibits neuronal C/EBPβ through mTORC1-induced C/EBPβ-LIP translation. Furthermore, we identify maternal behavior-stimulated IGF-1 is mainly released from mPFC microglia. Notably, increased maternal care under an environmental enrichment condition or maternal behavior impairment induced by repeated MPOAEsr1+ cells inhibition in dams prevents or promotes stress susceptibility via microglial-to-neuronal IGF-1-C/EBPβ-DRP1 signaling. In this work, these findings have unveiled molecular mechanisms by which maternal behavior promotes stress resilience in adolescents.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Social defeat stress induces mitochondrial dysfunction in the mPFC of the adolescent susceptible mice.
a Schematic representation of the AcSD experimental course and behavioral test process for C57BL/6J male mice. SPT, sucrose preference test; AcSD, accelerated social stress defeat; SIT, social interaction test. b Graphic representation of AcSD stress paradigm for adolescent mice. c Scatter plot depicting the distribution of social interaction ratio for control (without experiencing AcSD), susceptible (ratio < 100), and resilient (ratio ≥ 100) after the AcSD paradigm. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. d Measurement of the time in interaction zone of the above mice without or with target. e The percentages of susceptible (red) and resilient (blue) mice exposed to AcSD. Measurement of the total liquid intake (f) and the percentage of sucrose consumption (g) in SPT. Data in (c–g) are presented as the mean ± SEM (n = 30 mice for the control group, n = 37 mice for the susceptible group, n = 38 mice for the resilient group; one-way ANOVA and Bonferroni’s multiple comparison test). h Graphic representation of mitochondrial membrane potential (MMP) and ATP levels in the mPFC and the hippocampus of adolescent mice. Measurement of mitochondrial MMP (i) and ATP levels (j) in the mPFC and the hippocampus from WT male mice exposed to AcSD or control. Data in (i, j) are presented as the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). k Schematic representation of the experimental procedure for AcSD paradigm and behavioral test process for C57BL/6J male mice with administration of Mdivi1, GW0742 or ACSF (Artificial Cerebrospinal Fluid). l–n Behavioral tests of WT male mice exposed to AcSD paradigm with administration of Mdivi1, GW0742 or ACSF. Measurement of social interaction ratio (l), time in interaction zone without or with target (m), and the percentage of sucrose preference (n) in WT male mice with indicative treatments. Data in (l–n) are presented as the mean ± SEM (n = 12 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). See also Supplementary Figs. 1, 2. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Neuronal DRP1 in the mPFC contributes to stress susceptibility in adolescent mice.
a Western blotting showing the levels of p-DRP1 and DRP1 expression in the mPFC of adolescent mice after AcSD. Data are representative of three independent experiments. b Quantification of p-DRP1/DRP1, DRP1/Tubulin protein levels in the mPFC of adolescent mice after AcSD. c Measurement of Dnm1l mRNA levels in the mPFC of the above mice; Correlation analysis between social interaction ratio and relative Dnm1l mRNA levels in the mPFC of the above mice. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r2 and p value are shown. Data in (b, c) are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). d Immunofluorescence co-staining of NeuN (green) and DRP1 (Red) on the brain sections of the mPFC from indicative mice exposed to AcSD. Scale bars: 20 μm. e Quantification of DRP1 intensity in NeuN+ cells in the mPFC from indicative mice exposed to AcSD; Correlation analysis between social interaction ratio and DRP1 intensity in NeuN+ cells in the above mice. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r2 and p value are shown. Data are presented the mean ± SEM (n = 8 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). f Immunofluorescence co-staining of Iba1 (green) and DRP1 (Red) on the brain sections of the mPFC from indicative mice exposed to AcSD. Scale bars: 20 μm. g Quantification of DRP1 intensity in Iba1+ cells in the mPFC from indicative mice exposed to AcSD. h Immunofluorescence co-staining of GFAP (green) and DRP1 (Red) on the brain sections of the mPFC from indicative mice exposed to AcSD. Scale bars: 20 μm. i Quantification of DRP1 intensity in GFAP+ cells in the mPFC from indicative mice exposed to AcSD. Data in (g, i) are presented the mean ± SEM (n = 8 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). j Schematic representation of the experimental procedure for AcSD paradigm and behavioral test process for WT mice, with virus injection of AAV-hSyn-shControl-EGFP or AAV-hSyn-shDnm1l-EGFP. k The location of the cannula tips and fluorescence microscopy image of representative field showed GFP expression in the mPFC injected with AAV-hSyn-shDnm1l-EGFP. Scale bar: 500 μm. n = 3 mice for each group. l Representative immunoblots of p-DRP1 and DRP1 protein levels in the mPFC neurons of WT mice exposed to Control or AcSD paradigm with virus injection of AAV-hSyn-shControl-EGFP or AAV-hSyn-shDnm1l-EGFP. Data are representative of three independent experiments. m Measurement of mitochondrial MMP and ATP levels in the mPFC of WT mice with indicative treatments. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). n Measurement of social interaction ratio and the percentage of sucrose preference in WT mice with indicative treatments. Data are presented the mean ± SEM (n = 10 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). o Schematic representation of the experimental procedure for SSD paradigm and behavioral test process for WT mice, with virus injection of AAV-hSyn-EGFP or AAV-hSyn-Dnm1l-EGFP. p The location of the cannula tips and fluorescence microscopy image of representative field showed GFP expression in the mPFC injected with AAV-hSyn-Dnm1l-EGFP. Scale bar: 500 μm. n = 3 mice for each group. q Representative immunoblots of p-DRP1 and DRP1 protein levels in the mPFC neurons of WT mice exposed to Control or SSD paradigm with virus injection of AAV-hSyn-EGFP or AAV-hSyn-Dnm1l- EGFP. Data are representative of three independent experiments. r Measurement of mitochondrial MMP and ATP levels in the mPFC of WT mice with indicative treatments. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). s Measurement of social interaction ratio and the percentage of sucrose preference in WT mice with indicative treatments. Data are presented the mean ± SEM (n = 9 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). See also Supplementary Figs. 3–5. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Neuronal C/EBPβ acts as a transcription activator for Dnm1l gene.
a Binding score of transcription factors bound to the Dnm1l promoter region determined by ChIP assays (ChIP-Atlas MACS2) in HeLa S3 cells obtained from the Signaling Pathway Project. b Measurement of mRNA levels of Gabpa, Ctcf, Rest, Cebpb in the control and susceptible mice. Data are presented the mean ± SEM (n = 6 for each group; two-sided unpaired t-test with Welch’s correction). c Correlation analysis between social interaction ratio and relative Cebpb mRNA levels in WT mice exposed to AcSD or control. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r2 and two-sided p value are shown (n = 6 for each group). d Representative immunoblots of p-C/EBPβ and C/EBPβ protein levels in the mPFC of WT mice exposed to AcSD or control. Data are representative of three independent experiments. e The immunostaining of p-C/EBPβ (Red) with Dnm1l mRNA (Green) in situ hybridization on the mPFC sections of WT mice exposed to AcSD or control. Scale bar: 10 μm. Correlation analysis between relative p-C/EBPβ intensity and social interaction ratio (f), or relative Dnm1l mRNA intensity (g) in WT mice exposed to AcSD or control. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r2 and two-sided p value are shown (n = 8 for each group). h Immunofluorescence co-staining of C/EBPβ (green) and DRP1 (Red) on the brain sections of the mPFC in WT mice exposed to AcSD or control. Scale bars: 20 μm. i Quantification of C/EBPβ intensity in the mPFC from WT mice exposed to AcSD or control. Data are presented the mean ± SEM (n = 8 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). j Correlation analysis between relative DRP1 intensity and relative C/EBPβ intensity in WT mice exposed to AcSD or control. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r2 and two-sided p value are shown (n = 8 for each group). k Snapshots of C/EBPβ ChIP-Seq at locus of Dnm1l gene in HeLa cells, HepG2 cells, HCT116 cells and MSCs cells. l Luciferase assays with different truncates of Dnm1l promoter in HEK293 cells co-transfected with GST or GST-C/EBPβ. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). m Representative immunoblots of C/EBPβ protein expressions in HEK293 cells co-transfected with variants lengths of Dnm1l promoter and GST-C/EBPβ. Data are representative of three independent experiments. n Luciferase assays with different truncates of Dnm1l promoter and representative immunoblotting bands of C/EBPβ protein expressions in HEK293 cells co-transfected with different truncates of Dnm1l promoter and si-control or si-C/EBPβ. Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). o The consensus C/EBPβ-binding motif and sequence comparison of human and mouse Dnm1l promoters. p EMSA assay demonstrates that C/EBPβ binds the Dnm1l promoter. Nuclear extract proteins (NE) are isolated from HEK293 cells transfected with GST-C/EBPβ for 48 h. EMSA assay is recruited to detect the C/EBPβ binding ability on site –1369 to –1060 of Dnm1l promoter with probe (GTGGCGCAATC), mutation probe (GTGTATCAATC). Data are representative of three independent experiments. Probe bands indicate probe without binding of C/EBPβ protein. Shift bands indicate the C/EBPβ protein—Probe complex. Super-shift band indicates the C/EBPβ antibody—C/EBPβ protein—Probe complex. q ChIP-PCR assays demonstrate C/EBPβ specifically binds to Dnm1l promoter binding motif of genomic DNA. The C/EBPβ—DNA crosslinking ChIP samples are obtained from HEK293 cells and immunoprecipitated with anti-C/EBPβ or IgG antibodies. After reversing crosslinks, PCR are performed by using primer pairs at –1404 to –1195 of Dnm1l promoter. PCR assay also include each input sample. The positive control is demonstrated with anti-Histone H3 antibody coupling with GAPDH primers. Data are representative of three independent experiments. r, s Representative immunoblots and quantification of human and mouse C/EBPβ and DRP1 protein expressions in primary mouse neurons. Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). t Measurement of Dnm1l mRNA levels in primary mouse neurons. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). u Immunofluorescence co-staining of C/EBPβ (green) and DRP1 (Red) in primary mouse neurons. Scale bars: 10 μm. Quantification of C/EBPβ intensity (v) and DRP1 intensity (w) in primary mouse neurons. Data in (v) are presented the mean ± SEM (n = 10 for each group; ND not detected; two-sided unpaired t-test with Welch’s correction). Data in (w) are presented the mean ± SEM (n = 10 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). See also Supplementary Fig. 6. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Neuronal C/EBPβ acts as a transcription activator for Dnm1l and regulates stress susceptibility in adolescent mice.
a Immunofluorescence co-staining of NeuN (green) and C/EBPβ (Red) on the brain sections of the mPFC in WT mice exposed to AcSD or control. Scale bars: 20 μm. b Quantification of C/EBPβ intensity in NeuN+ cells on the brain sections of the mPFC in WT mice exposed to AcSD or control. c Correlation analysis between social interaction ratio and C/EBPβ intensity in NeuN+ cells in WT mice exposed to AcSD or control. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r2 and p value are shown. Data in (b, c) are presented the mean ± SEM (n = 8 for control group, n = 9 for susceptible group, n = 9 for resilient group; one-way ANOVA and Bonferroni’s multiple comparison test). d Schematic representation of the experimental procedure for AcSD paradigm and behavioral test process for WT or C/EBPβ+/- mice, with virus injection of AAV-hSyn-EGFP or AAV-hSyn-Dnm1l-EGFP. e The location of the cannula tips and fluorescence microscopy image of representative field showed GFP expression in the mPFC injected with AAV-hSyn-Dnm1l-EGFP. Scale bar: 500 μm. n = 3 mice for each group. f, g Representative immunoblots and quantification of C/EBPβ and DRP1 protein expressions in the mPFC neurons of WT and C/EBPβ+/- mice with indicative treatments. Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). Measurement of mitochondrial MMP (h) and ATP levels (i) in the mPFC of WT and C/EBPβ+/- mice with indicative treatments. Data in (h, i) are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). j, k Behavioral tests of WT and C/EBPβ+/- mice exposed to AcSD paradigm with virus injection of AAV-hSyn-EGFP or AAV-hSyn-shDnm1l-EGFP. Measurement of social interaction ratio, time in interaction zone without or with target (j), and the percentage of sucrose preference (k) in WT and C/EBPβ+/- mice with indicative treatments. Data in (j, k) are presented as the mean ± SEM (n = 10 mice for WT + GFP + AcSD group; n = 9 mice for C/EBPβ+/- + GFP + AcSD group; n = 9 mice for C/EBPβ+/- + Dnm1l + AcSD group; one-way ANOVA and Bonferroni’s multiple comparison test). l The location of the cannula tips and fluorescence microscopy image of representative field showed GFP expression in the mPFC injected with AAV-hSyn-shDnm1l-EGFP. Scale bar: 500 μm. n = 3 mice for each group. m Schematic representation of the experimental procedure for SSD paradigm and behavioral test process for WT or LAP Tg mice, with virus injection of AAV-hSyn-EGFP or AAV-hSyn-shDnm1l-EGFP. n, o Representative immunoblots and quantification of human and mouse C/EBPβ and DRP1 protein expressions in the mPFC neurons of WT and LAP Tg mice with indicative treatments. Data are representative of three independent experiments. Data are presented the mean ± SEM (n  = 5 for each group; ND, not detected; one-way ANOVA and Bonferroni’s multiple comparison test). Measurement of mitochondrial MMP (p) and ATP levels (q) in the mPFC of WT and LAP Tg mice with indicative treatments. Data in (p, q) are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). r, s Behavioral tests of WT and LAP Tg mice exposed to SSD paradigm with virus injection of AAV-hSyn-shControl-EGFP or AAV-hSyn-shDnm1l-EGFP. Measurement of social interaction ratio, time in interaction zone without or with target (r), and the percentage of sucrose preference (s) in WT and LAP Tg mice with indicative treatments. Data in (r, s) are presented as the mean ± SEM (n = 11 mice for WT + shControl + Control group, n = 11 mice for WT + shControl + SSD group, n = 11 mice for LAP Tg + shControl + Control group, n = 10 mice for LAP Tg + shControl + SSD group, n = 11 mice for LAP Tg + shDnm1l + Control group, n = 10 mice for LAP Tg + shDnm1l + SSD group; one-way ANOVA and Bonferroni’s multiple comparison test). See also Supplementary Fig. 7. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Different patterns of maternal separation trigger different outcomes in stress susceptibility via IGF-1-mediated C/EBPβ activity.
a Schematic representation of the upstream signaling pathway of early life experience affecting C/EBPβ/DRP1 axis, contributing to adolescent depression. b Schematic representation of the experimental procedure for maternal separation on different durations, and AcSD paradigm. c Measurement of C/EBPβ activity in the mPFC neurons of WT mice exposed to different maternal separations and AcSD. d Measurement of Cebpb mRNA levels in the mPFC neurons of WT mice exposed to different maternal separations and AcSD. Data in (c, d) are presented as the mean ± SEM (n = 4 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). e, f. Representative immunoblots and quantification of LAP/LIP ratio in the mPFC neurons of WT mice from (b). Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 4 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). g Schematic representation of molecular pathway hypothesis that IGF-1 induces inhibition of neuronal C/EBPβ activity. h Measurement of the IGF-1 levels in the blood, the mPFC and the hippocampus of WT mice from (b). Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). i Representative immunoblots of LAP/LIP ratio, p-IGF-1R/IGF-1R, p-Akt/Akt and p-S6K1/S6K1 in the primary mouse cortex neurons with vehicle or IGF-1. Data are representative of three independent experiments. j Schematic representation of the experimental procedure for maternal separation for 10 min × 2, and AcSD paradigm and behavioral tests with the mPFC infusion of anti-IgG or anti-IGF-1. k Representative immunoblots of LAP/LIP ratio and DRP1 expressions in the mPFC neurons of WT mice exposed to maternal separation for 10 min × 2, and AcSD paradigm with the mPFC infusion of anti-IgG or anti-IGF-1. Data are representative of three independent experiments. l, m Behavioral tests of WT mice exposed to maternal separation for 10 min × 2, and AcSD paradigm with the mPFC infusion of anti-IgG or anti-IGF-1. Measurement of social interaction ratio, time in interaction zone with target (l), and the percentage of sucrose preference (m) in WT mice with indicative treatments. Data in (l, m) are presented as the mean ± SEM (n = 8 mice for anti-IgG group, n = 10 mice for anti-IGF-1 group; unpaired t-test with Welch’s correction). n Schematic representation of the experimental procedure for AcSD paradigm and behavioral tests with the mPFC infusion of vehicle or IGF-1. o Representative immunoblots and quantification of LAP/LIP ratio, and p-DRP1 and DRP1 expressions in the mPFC neurons of WT susceptible mice exposed to AcSD paradigm with the mPFC infusion of vehicle or IGF-1. Data are representative of three independent experiments. Data in (o) are presented as the mean ± SEM (n = 6 for each group; two-sided unpaired t-test with Welch’s correction). p Scatter plot depicting the distribution of social interaction ratio for susceptible (ratio < 1), and resilient (ratio ≥ 1) after the AcSD paradigm. The red, and blue dots represent the susceptible, and resilient groups, respectively. Data in (p) are presented as the mean ± SEM (n = 22 for susceptible group; n = 26 for resilient group; two-sided unpaired t-test with Welch’s correction). q, r Behavioral tests of WT susceptible mice exposed to AcSD paradigm with the mPFC infusion of vehicle or IGF-1. Measurement of social interaction ratio, time in interaction zone with target (q), and the percentage of sucrose preference (r) in WT susceptible mice with indicative treatments. Data in (q, r) are presented as the mean ± SEM (n = 9 mice for each group; two-sided unpaired t-test with Welch’s correction). See also Supplementary Figs. 8–10. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Maternal behavior protects against stress vulnerability via upregulating mPFC IGF-1 levels in adolescent mice.
a Graphic representation of maternal behavior in mice; Measurement of maternal behavior sample points in WT mice exposed to maternal separation for 0, 10 min × 2, and 180 min × 1. Data are presented the mean ± SEM (n = 11 mice for no maternal separation group, n = 8 mice for maternal separation on 10 min × 2 group, n = 8 mice for maternal separation on 180 min × 1 group; one-way ANOVA and Bonferroni’s multiple comparison test). b Correlation analysis between social interaction ratio and maternal behavior sample points in WT mice experiencing maternal separation. The white, red, and blue dots represent 0, 10 min × 2, and 180 min × 1 groups, respectively. The Spearman correlation coefficient r2 and two-sided p value are shown (n = 8 mice for no maternal separation group, n = 8 mice for 10 min × 2 group, n = 10 mice for 180 min × 1 group). Correlation analysis between mPFC IGF-1 levels and maternal behavior sample points (c), or social interaction ratio (d) in WT mice experiencing maternal separation. The white, red, and blue dots represent 0, 10 min × 2, and 180 min × 1 groups, respectively. The Spearman correlation coefficient r2 and two-sided p value are shown (n = 6 mice for each group). e Graphic representation of L and LnL treatment; Schematic representation of the experimental procedure for maternal separation on 180 min × 1 or not, and AcSD paradigm and behavioral tests with L or LnL dams. f Measurement of maternal behavior sample points in WT mice exposed to maternal separation for 180 min × 1 or 0, with L or LnL dams. Data are presented the mean ± SEM (n = 6 mice for 180 × 1 + L group, n = 6 mice for 180 × 1 + LnL group, n = 6 mice for 0 + L group; n = 7 mice for 0 + LnL group; one-way ANOVA and Bonferroni’s multiple comparison test). g Measurement of IGF-1 levels in the mPFC, in the hippocampus, and in the blood in WT mice exposed to maternal separation for 180 min × 1 or 0, with L or LnL dams. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). h Representative immunoblots of LAP/LIP ratio and DRP1 expressions in the mPFC neurons of WT mice exposed to maternal separation for 180 min × 1 or 0, with L or LnL dams. Data are representative of three independent experiments. i, j Behavioral tests of WT mice exposed to maternal separation for 180 min × 1 or 0, with L or LnL dams. Measurement of social interaction ratio (i), and the percentage of sucrose preference (j) in WT mice with indicative treatments. Data in (i, j) are presented as the mean ± SEM (n = 13 mice for 180 × 1 groups, n = 15 mice for 0 groups; one-way ANOVA and Bonferroni’s multiple comparison test). k Schematic representation of the experimental procedure for maternal separation on 10 min × 2 or not, and AcSD paradigm and behavioral tests with saline- or CNO-treated dams. l The location of the cannula tips in Esr-2A-Cre female mice; Fluorescence microscopy image of representative field showed mCherry expression in the MPOA injected with AAV-DIO-hM4Di-mCherry. Scale bar: 100 μm. n = 4 dams for each group. m Measurement of maternal behavior sample points in WT mice exposed to maternal separation for 10 min × 2 or 0, with saline- or CNO-treated dams. Data are presented the mean ± SEM (n = 6 mice for 0 + Saline group, n = 6 mice for 10 × 2 + Saline group, n = 5 mice for 0 + CNO group; n = 6 mice for 10 × 2 + CNO group; one-way ANOVA and Bonferroni’s multiple comparison test). n Measurement of IGF-1 levels in the mPFC, in the hippocampus and in the blood in WT mice exposed to maternal separation for 10 min × 2 or 0, with saline- or CNO-treated dams. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). o Representative immunoblots of LAP/LIP ratio and DRP1 expressions in mPFC neurons of WT mice exposed to maternal separation for 10 min × 2 or 0, with saline- or CNO-treated dams. Data are representative of three independent experiments. p, q Behavioral tests of WT mice exposed to maternal separation for 10 min × 2 or 0, with saline- or CNO-treated dams. Measurement of social interaction ratio (p), and the percentage of sucrose preference (q) in WT mice with indicative treatments. Data in (p, q) are presented as the mean ± SEM (n = 14 mice for 0 + Saline group, n = 14 mice for 10 × 2 + Saline group, n = 13 mice for 0 + CNO group; n = 14 mice for 10 × 2 + CNO group; one-way ANOVA and Bonferroni’s multiple comparison test). r. Schematic representation of molecular pathway that various maternal behavior levels mediate stress susceptibility via the “two-hit” hypothesis. See also Supplementary Figs. 11–16. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Microglia are primary source of maternal behavior-mediated IGF-1 levels in the mPFC of adolescent mice.
a Schematic representation of the experimental procedure for WT mice with L or LnL dams in group 1, and saline- or CNO-treated dams in group 2. b The location of the cannula tips in Esr-2A-Cre female mice; Fluorescence microscopy image of representative field showed mCherry expression in the MPOA injected with AAV-DIO-hM4Di-mCherry. Scale bar: 100 μm. n = 3 dams for each group. c Measurement of maternal behavior sample points in WT mice with L or LnL dams, and saline- or CNO-treated dams. Data are presented the mean ± SEM (n = 3 mice for each group; two-sided unpaired t-test with Welch’s correction). d Measurement of IGF-1 levels in the mPFC of WT mice with L or LnL dams, and saline- or CNO-treated dams. Data are presented the mean ± SEM (n = 8 for L group; n = 8 for LnL group; n = 8 for Saline group; n = 7 for CNO group; two-sided unpaired t-test with Welch’s correction). e Measurement of Igf1 mRNA levels in the mPFC of WT mice with L or LnL dams, and saline- or CNO-treated dams. Data are presented the mean ± SEM (n = 8 for L or LnL group, n = 9 mice for Saline or CNO group; two-sided unpaired t-test with Welch’s correction). The immunostaining of Iba1 (Green) with Igf1 mRNA (Red) in situ hybridization on the mPFC sections of WT mice with L or LnL dams (f), and saline- or CNO-treated dams (g). Scale bar: 10 μm (f), 10 μm (g); Quantification of Igf1 mRNA intensity in Iba1+ cells, and Iba1—cells. Data in (f, g) are presented the mean ± SEM (n = 10 for L or LnL group, n = 12 for Saline or CNO group; two-sided unpaired t-test with Welch’s correction). h Schematic representation of the experimental procedure of PLX3397 treatment, and AcSD paradigm and behavioral tests for WT mice with L or LnL dams. i The image of cannula placements for injection of PLX3397. White arrow indicates cannula target region and dotted line regions correspond to the position of the cannula. Bregma: −0.22 mm, Scale bar: 500 μm. n = 3 mice for each group. j Measurement of maternal behavior sample points in WT mice exposed to L or LnL dams, and AcSD paradigm with PLX3397 treatment. Data are presented the mean ± SEM (n = 5 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). k Measurement of IGF-1 levels in the mPFC from WT mice with indicative treatments. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). l Measurement of C/EBPβ activity in the mPFC neurons from WT mice with indicative treatments. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). m Measurement of Dnm1l mRNA levels in the mPFC neurons from WT mice with indicative treatments. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). n Measurement of mitochondrial MMP and ATP levels in the mPFC of WT mice with indicative treatments. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). o, p Behavioral tests of WT mice exposed to L or LnL dams, and AcSD paradigm with PLX3397 treatment. Measurement of social interaction ratio and time in interaction zone without or with target (o), and the percentage of sucrose preference (p) in WT mice with indicative treatments. Data in (o, p) are presented as the mean ± SEM (n = 10 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). See also Supplementary Figs. 17, 18. Source data are provided as a Source Data file.
Fig. 8
Fig. 8. Microglial-derived IGF-1 inhibits neuronal C/EBPβ-DRP1 axis to prevent stress susceptibility in adolescent mice.
a Schematic representation of the experimental procedure of virus injection, and AcSD paradigm and behavioral tests for Cx3cr1-Cre mice with LnL dams. b The location of the cannula tips and fluorescence microscopy image of representative field showed mCherry expression in the mPFC injected with AAV-hSyn-mCherry; Fluorescence microscopy image of representative field showed GFP expression and Iba1 (Purple) in the mPFC injected with AAV-MG1.2-DIO-GFP in Cx3cr1-Cre mice. Scale bar: 100 μm (left), 50 μm (right). n = 3 mice for each group. c, d Representative immunoblots and quantification of LAP/LIP ratio, and IGF-1 and DRP1 expressions in Cx3cr1-Cre mice exposed to AcSD paradigm and virus injection of AAV-MG1.2-DIO-shControl or -shIGF-1 and AAV-hSyn-mCherry or -LIP, with LnL dams. Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 4 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). e Measurement of maternal behavior sample points in Cx3cr1-Cre mice with indicative treatments. Data are presented the mean ± SEM (n = 5 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). f Measurement of Dnm1l mRNA levels in the mPFC neurons of Cx3cr1-Cre mice with indicative treatments. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). g Measurement of mitochondrial MMP and ATP levels in the mPFC of Cx3cr1-Cre mice with indicative treatments. Data are presented the mean ± SEM (n = 8 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). h, i Behavioral tests of Cx3cr1-Cre mice exposed to AcSD paradigm and virus injection of AAV-MG1.2-DIO-shControl or -shIGF-1 and AAV-hSyn-mCherry or -LIP, with LnL dams. Measurement of social interaction ratio (h), and the percentage of sucrose preference (i) in Cx3cr1-Cre mice with indicative treatments. Data in (h, i) are presented as the mean ± SEM (n = 12 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). j Schematic representation of the experimental procedure of virus injection, and AcSD paradigm and behavioral tests for Cx3cr1-Cre mice with CNO-treated dams. k The location of the cannula tips and fluorescence microscopy image of representative field showed mCherry expression in the mPFC injected with AAV-hSyn-mCherry; Fluorescence microscopy image of representative field showed GFP expression and Iba1 (Purple) in the mPFC injected with AAV-MG1.2-DIO-GFP in Cx3cr1-Cre mice. Scale bar: 100 μm (left), 50 μm (right). n = 3 mice for each group. l, m Representative immunoblots and quantification of LAP/LIP ratio, and IGF-1 and DRP1 expressions in Cx3cr1-Cre mice exposed to AcSD paradigm and virus injection of AAV-MG1.2-DIO-GFP or -IGF-1 and AAV-hSyn-mCherry or -LAP, with CNO-treated dams. Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 4 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). n Measurement of maternal behavior sample points in Cx3cr1-Cre mice with indicative treatments. Data are presented the mean ± SEM (n = 4 mice for each group; one-way ANOVA and Bonferroni’s multiple comparison test). o Measurement of Dnm1l mRNA levels in the mPFC neurons of Cx3cr1-Cre mice with indicative treatments. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). p Measurement of mitochondrial MMP and ATP levels in the mPFC of Cx3cr1-Cre mice with indicative treatments. Data are presented the mean ± SEM (n  = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). q, r Behavioral tests of Cx3cr1-Cre mice exposed to AcSD paradigm and virus injection of AAV-MG1.2-DIO-GFP or -IGF-1 and AAV-hSyn-mCherry or -LAP, with CNO-treated dams. Measurement of social interaction ratio (q), and the percentage of sucrose preference (r) in Cx3cr1-Cre mice with indicative treatments. Data in (q, r) are presented as the mean ± SEM (n = 10 mice for GFP + mCherry group; n = 9 mice for IGF-1 + mCherry group; n = 9 mice for IGF-1 + LAP group; one-way ANOVA and Bonferroni’s multiple comparison test). See also Supplementary Fig. 19. Source data are provided as a Source Data file.
Fig. 9
Fig. 9. Maternal behavior in the early period can mediate later-life stress resilience by a microglial-to-neuronal IGF-1-C/EBPβ-DRP1 signaling in the mPFC of adolescent mice.
In the mPFC of adolescent mice with low maternal care, social stress activates transcription factor C/EBPβ in mPFC neurons, upregulating Dnm1l expression and promoting mitochondrial dysfunction, which confers to stress susceptible in adolescent mice. However, high maternal care stimulates microglia to relieve IGF-1, which inhibit neuronal C/EBPβ activity through mTOR-induced C/EBPβ-LIP translation via binding to IGF-1R on the neurons in the mPFC of offspring, which protects stress resilience in adolescence. Source data are provided as a Source Data file.
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