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Early growth response 2 in the mPFC regulates mouse social and cooperative behaviors

Lab Animal volume 52, pages 37–50 (2023)Cite this article

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Abstract

Adolescent social neglect impairs social performance, but the underlying molecular mechanisms remain unclear. Here we report that isolation rearing of juvenile mice caused cooperation defects that were rescued by immediate social reintroduction. We also identified the transcription factor early growth response 2 (Egr2) in the medial prefrontal cortex (mPFC) as a major target of social isolation and resocialization. Isolation rearing increased corticosteroid production, which reduced the expression of Egr2 in the mPFC, including in oligodendrocytes. Overexpressing Egr2 ubiquitously in the mPFC, but not specifically in neurons nor in oligodendroglia, protected mice from the isolation rearing-induced cooperation defect. In addition to synapse integrity, Egr2 also regulated the development of oligodendroglia, specifically the transition from undifferentiated oligodendrocyte precursor cells to premyelinating oligodendrocytes. In conclusion, this study reveals the importance of mPFC Egr2 in the cooperative behavior that is modulated by social experience, and its unexpected role in oligodendrocyte development.

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Fig. 1: The effect of isolation rearing on social interaction and cooperation ability of mice.
Fig. 2: Identification of potential genes regulating cooperative behavior.
Fig. 3: Knocking down Egr2 impairs mice’s sociability, cooperation behaviors and myelination.
Fig. 4: Overexpressing Egr2 in the mPFC of SI mice mitigates sociability and cooperation defects and restores synaptic and myelination impairments.
Fig. 5: Specifically overexpressing Egr2 in oligodendrocytes in the mPFC of SI mice restored the social interaction but not cooperation performance.
Fig. 6: Elevated corticosterone level in SI mice potentially downregulates Egr2 expression.
Fig. 7: Egr2 is expressed in early- and late-stage oligodendrocytes and Egr2 knockdown dampens oligodendrocyte differentiation.

Data availability

The authors declare that all data generated or analyzed during this study are included in this published article and Supplementary Information, or available from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank L. Zhu (Tongji Medical College of HUST) and Q. Zhou (Nanjing Medical University) for their insightful comments. We also thank Z. Xia for technical assistance with the EM and Y. Zhang for technical assistance with the oligodendrocyte culture. This work was supported by the grants from the National Natural Science Foundation of China (81801378 and 81871117).

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Author notes

  1. These authors contributed equally: Yanli Zhang, Weixi Feng.

Authors and Affiliations

  1. Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, China

    Yanli Zhang, Weixi Feng, Ze Wang, Yingting Pang, Yuxi Jin, Sijia Chen, Shixin Ding, Tianqi Wang, Ying Zou, Peng Sun, Yan Chen, Hu Feng, Chengyu Sheng & Ming Xiao

  2. Brain Institute, Nanjing Brain Hospital, Nanjing Medical University, Nanjing, China

    Yanli Zhang, Ze Wang, Yuxi Jin, Sijia Chen, Shixin Ding, Yan Chen & Ming Xiao

  3. Center for Global Haealth, Nanjing Medical University, Nanjing, China

    Weixi Feng, Yingting Pang, Tianqi Wang, Ying Zou, Peng Sun, Hu Feng & Ming Xiao

  4. Department of Neurology, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China

    Huang Huang

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Contributions

M.X. and C.S. designed the experiments. Y. Zhang and W.F. performed most of the experiments and analysis. Y. Zhang, W.F., Z.W., Y.P., Y.J., S.C., S.D., T.W., Y. Zou, P.S., Y.C., H.H. and H.F. performed the behavioral experiments. The manuscript was written by M.X., C.S., Y. Zhang and W.F. All authors discussed the results, made the figures and edited the manuscript. M.X., C.S. and H.H. directed and supervised all aspects of the study.

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Correspondence to Chengyu Sheng or Ming Xiao.

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Extended data

Extended Data Fig. 1 The effect of SI on mouse locomotion, exploration, spatial working memory, anxiety-like behaviors, and fear memory.

Postnatal day 21 mice were housed singly or in groups for 3 weeks before the behavioral tests. a, Locomotion, and exploration behavior of GH and SI mice at P56 in the open field. b, The spatial working memory performance of P57 GH and SI mice in the Y-maze testing. c, The anxiety-like behavior of P58 GH and SI mice examined by the elevated plus-maze test. d, Fear memory performance of GH and SI mice at P58-59 in the fear conditioning test. Data are represented as medians and IQR (n = 18 in each group). Data were analyzed by two-tailed Student’s t-tests. GH: group housing; SI: social isolation.

Source data

Extended Data Fig. 2 The sociability, cooperation, locomotion, exploration, spatial working memory, anxiety-like behaviors, and fear memory defects of SI mice are rescued by resocialization.

a, Diagram showing the timeline of the experiment, including weanling, social isolation, resocialization, behavioral tests (shown in b-i), and then qPCR (shown in Fig. 2j), Western blot (shown in Fig. 2k, l and Extended Data Fig. 3g,h), EM (shown in Extended Data Fig. 3a-d), immunohistochemistry (shown in Extended Data Fig. 3e,f), immunofluorescence (shown in Fig. 6i,j and Supplementary Fig. 3a, b), or ELISA analysis (shown in Fig. 6e). b, c, The social behavior performance and social memory of mice in social test. d, e, Graphs showing the time before the first drinking (drinking latency), the times of drinking (drinking number), and the total time spent on drinking (drinking time) each day in the training (d) and testing (e) periods. f, Locomotion, and exploration behavior in the open field. g, The anxiety-like behavior examined by the elevated plus-maze test. h, The spatial working memory performance in the Y-maze testing. i, Fear memory performance in the fear conditioning test. (GH: n = 15 mice; GH-SI: n = 15 mice; SI: n = 14 mice); Data are represented as medians and IQR in c, f-i; and mean + SEM in d and e (n = 7 pairs of mice per group). Data in c and f-i were analyzed by One-way ANOVA followed by Tukey’s post hoc test; and data in d and e were analyzed by Repeated Measures ANOVA with post hoc Student-Newman-Keuls test. GH: group housing; SI: social isolation; GH-SI: resocialization of SI mice with GH mice.

Source data

Extended Data Fig. 3 The mPFC impairment of SI mice is rescued by resocialization.

a, Representative EM images showing myelin (the top panel), oligodendrocyte nuclear heterochromatin (the middle panel), and synaptic morphology (the bottom panel) in the mPFC, respectively. Scale bar, 500 nm. b, The scatter plots of g ratios with linear least squares fitting (g ratio vs. axon caliber) in GH (106 axons), GH-SI (107axons), and SI (110 axons) mice (n = 4 per group). c, Graph showing the area percentage of nuclear heterochromatin in GH (17 nuclei), GH-SI (19 nuclei), and SI (15) mice (n = 4 per group). d, Postsynaptic density thickness, synaptic cleft width, presynaptic active zones length, and synaptic curvature in GH (70 synapses), GH-SI (68 synapses) and SI (63 synapses) mice (n = 4 per group). e-h Representative immunostaining images (e) and Western blot bands (g) showing MBP and PSD-95 expression in the mPFC of GH, GH-SI, and SI mice; corresponding quantification analyses are shown in f for e (n = 4 per group) and h for g (n = 8); scale bar, 50 µm. Data are represented as mean ± SEM in c and d; and as medians and IQR in f and h. Data in b-h were analyzed by One-way ANOVA followed by Tukey’s post hoc test. GH: group housing (P87); SI: social isolation (P87); GH-SI: resocialization of SI mice with GH mice (P87).

Source data

Extended Data Fig. 4 Knocking down Egr2 in the mPFC of GH mice does not affect exploring activities, spatial working memory, anxiety-like behavior, and fear memory.

a, Graphs showing the time before the first drinking (drinking latency), the times of drinking (drinking number), and the total time spent on drinking (drinking time) each day in different groups at P46-57 in the 7 consecutive training days. b, Locomotion, and exploration behavior in different groups at P58 in the open field. c, The anxiety-like extent examined in different groups at P60 by the elevated plus-maze test. d, The spatial working memory performance of mice at P59 in the Y-maze testing. e, Fear memory performance of mice at P60-61 in fear conditioning test. Data are represented as mean + SEM in a; and as medians and IQR in b-e (n = 20 per group). Data in a were analyzed by repeated-measures ANOVA with post hoc Student-Newman-Keuls test; and in b–e by Two-way ANOVA followed by Tukey’s post hoc test. GH-GFP: GH mice with AAV-GFP virus injection; GH-Egr2-RNAi: GH mice with AAV-Egr2-RNAi virus injection; SI-GFP: SI mice with AAV-GFP virus injection; SI-Egr2-RNAi: SI mice with AAV-Egr2-RNAi virus injection.

Source data

Extended Data Fig. 5 Overexpression of Egr2 in the mPFC of SI mice mitigates abnormal anxiety-like behavior, but not exploring activities, spatial working memory and fear memory.

a, Graphs showing the time before the first drinking (drinking latency), the times of drinking (drinking number), and the total time spent on drinking (drinking time) each day in different groups at P46-57 in the 7 consecutive training days. b, Locomotion and exploration behavior of P58 mice were tested in the open field. c, The anxiety-like behavior of P60 mice was examined in the elevated plus-maze test. d, The spatial working memory performance of P59 mice was tested in the Y-maze test. e, Fear memory performance in different groups at P60-61 in fear conditioning test. Data are represented as mean + SEM in a; and as medians and IQR in b-e (data from 18 mice in GH-GFP group, 22 mice in GH-Egr2 group, 30 mice in SI-GFP group and 28 mice in SI-Egr2 group, respectively). Data in a were analyzed by repeated-measures ANOVA with post hoc Student-Newman-Keuls test; and data in b-e were analyzed by Two-way ANOVA followed by Tukey’s post hoc test. GH-GFP: GH mice with AAV-GFP virus injection; GH-Egr2: GH mice with AAV-Egr2 virus injection; SI-GFP: SI mice with AAV-GFP virus injection; SI-Egr2: SI mice with AAV-Egr2 virus injection.

Source data

Extended Data Fig. 6 Specifically overexpressing Egr2 in oligodendrocytes in the mPFC of SI mice restores myelin damage, but has no effect on synaptic damage.

a, b, Representative immunostaining images and quantification analysis of Egr2 expression in the mPFC of GH-CNP-GFP, GH-CNP-Egr2, SI-CNP-GFP and SI-CNP-Egr2 mice (n = 6). Scale bar, 50 µm. c, d, Western blot and quantification analysis showing Egr2 protein levels in the mPFC of GH-CNP-GFP, GH-CNP-Egr2, SI-CNP-GFP and SI-CNP-Egr2 mice (n = 5-6). e, f, Immunostaining images and quantification analysis of MBP and PSD-95 expression in the mPFC (n = 6 mice per group). Scale bar, 50 µm. g-i, Western blot and quantification analysis of MBP and PSD-95 expression in the mPFC of GH-CNP-GFP, GH-CNP-Egr2, SI-CNP-GFP, and SI-CNP-Egr2 mice (n = 5-6). Data are represented as medians and IQR, and analyzed by Two-way ANOVA followed by Tukey’s post hoc test. GH-CNP-GFP: GH mice with AAV-CNP-GFP virus injection (P61); GH-CNP-Egr2: GH mice with AAV-CNP-Egr2 virus injection (P61); SI-CNP-GFP: SI mice with AAV-CNP-GFP virus injection (P61); SI-CNP-Egr2: SI mice with AAV-CNP-Egr2 virus injection (P61).

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Extended Data Fig. 7 Overexpression of Egr2 in oligodendrocytes in the mPFC of SI mice mitigates abnormal anxiety-like behavior, but has no effect on fear memory, exploring activities and spatial working memory.

a, Graphs showing the time before the first drinking (drinking latency), the times of drinking (drinking number), and the total time spent on drinking (drinking time) each day in different groups at P46-57 in the 7 consecutive training days. b, Locomotion and exploration behavior of P58 mice were tested in the open field. c, The anxiety-like behavior was examined in P60 mice using the elevated plus-maze test. d, The spatial working memory performance in each group of P59 mice was tested in the Y-maze test. e, Fear memory performance of P60-61 mice was tested using the fear conditioning test. Data are represented as mean + SEM in a; and as medians and IQR in b-e (data from 20 mice in GH-CNP-GFP group, 16 mice in GH-CNP-Egr2 group, 16 mice in SI-CNP-GFP group and 16 mice in SI-CNP-Egr2 group, respectively). Data in a were analyzed by repeated-measures ANOVA with post hoc Student-Newman-Keuls test; and data in b-e were analyzed by Two-way ANOVA followed by Tukey’s post hoc test. GH-CNP-GFP: GH mice with AAV-CNP-GFP virus injection, GH-CNP-Egr2: GH mice with AAV-CNP-Egr2 virus injection, SI-CNP-GFP: SI mice with AAV-CNP-GFP virus injection, SI-CNP-Egr2: SI mice with AAV-CNP-Egr2 virus injection.

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Extended Data Fig. 8 Specifically overexpressing Egr2 in neurons in the mPFC of SI mice has no effect on sociability or cooperation performance.

a, Diagram showing the timeline of the experiment, including weanling, AAV virus injection, group housing or social isolation, and then behavioral tests (shown in Extended Data Fig. 8b–f and Extended Data Fig. 9a-d), and immunofluorescence, EM, immunohistochemistry or Western blot analysis (shown in Extended Data Fig. 8a and Extended Data Fig. 10a–h). b, Representative immunofluorescence images showing GFP expression in some Tuj1- or Neun-labeled neurons, but not in NG2- or PDGFRα-labeled oligodendrocyte of P61 mice in the mPFC. Scale bar, 50 µm. c, d, Heat maps and graphs showing the sociability performance and social memory in the three-chamber test. e, Graphs showing the time before the first drinking (drinking latency), the times of drinking (drinking number), and the total time spent on drinking (drinking time) each day in different groups in the 7 consecutive training days. f, hSyn-Egr2 in the mPFC of SI mice did not reverse their cooperation performance, as assessed by drinking latency, drinking number, and drinking time. Data are represented as medians and IQR in c and d (16 mice per group); and mean + SEM in e and f (8 pairs of mice per group). Data in c and d were analyzed by two-way ANOVA followed by Tukey’s post hoc test; and data in e and f were analyzed by repeated-measures ANOVA with post hoc Student-Newman-Keuls test. GH-hSyn-GFP: GH mice with AAV-hSyn-GFP virus injection; GH-hSyn-Egr2: GH mice with AAV-hSyn-Egr2 virus injection; SI-hSyn-GFP: SI mice with AAV-hSyn-GFP virus injection; SI-hSyn-Egr2: SI mice with AAV-hSyn-Egr2 virus injection.

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Extended Data Fig. 9 Specifically overexpressing Egr2 in neurons in the mPFC of SI mice mitigates abnormal anxiety-like behavior, but has no effect on fear memory, exploring activities and spatial working memory.

a, Locomotion, and exploration behavior were tested at P58 for each group of mice in the open field. b, The anxiety-like behavior was examined in P60 mice by the elevated plus-maze test. c, The spatial working memory performance was tested at P59 for each group mice in the Y-maze testing. d, Fear memory performance of P60-61 mice in the fear conditioning test. Data are represented as medians and IQR from 16 mice in every group, and analyzed by two-way ANOVA followed by Tukey’s post hoc test. GH-hSyn-GFP: GH mice with AAV-hSyn-GFP virus injection; GH-hSyn-Egr2: GH mice with AAV-hSyn-Egr2 virus injection; SI-hSyn-GFP: SI mice with AAV-hSyn-GFP virus injection; SI-hSyn-Egr2: SI mice with AAV-hSyn-Egr2 virus injection.

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Extended Data Fig. 10 Specifically overexpressing Egr2 in neurons ameliorates impairments of synapses but not myelination in the mPFC of SI mice.

a, Representative EM images showing myelin (in the top panel), oligodendrocyte nuclear heterochromatin (in the middle panel) and synaptic morphology (in the bottom panel) in the mPFC, respectively. Scale bar, 500 nm. b, Scatter plot of g ratios with linear least squares fitting in GH-hSyn-GFP (175 axons), GH-hSyn-Egr2 (171 axons), SI-hSyn-GFP (193 axons), and SI-hSyn-Egr2 (157 axons) mice (n = 5 per group). c, Graphs showing altered percentage of oligodendrocyte nuclear heterochromatin in GH-hSyn-GFP (27 nuclei), GH-hSyn-Egr2 (30 nuclei), SI-hSyn-GFP (25 nuclei), and SI-hSyn-Egr2 (29 nuclei) mice (n = 5 per group). d, Graphs showing quantified analysis of postsynaptic dense, synaptic cleft width, length of the active zones and synaptic curvature in GH-hSyn-GFP (102 synapses), GH-hSyn-Egr2 (106 synapses), SI-hSyn-GFP (138 synapses), and SI-hSyn-Egr2 (125 synapses) mice (n = 5 per group). e-h, Immunohistology (e, f) and Western blot (g, h) showing MBP and PSD-95 expression in the mPFC (n = 6 for each group). Scale bar, 50 µm. Data are represented as mean ± SEM in c and d; and medians and IQR in f-h. Data in b-d and f-h were analyzed by two-way ANOVA followed by Tukey’s post hoc test. GH-hSyn-GFP: GH mice with AAV-hSyn-GFP virus injection (P61); GH-hSyn-Egr2: GH mice with AAV-hSyn-Egr2 virus injection (P61); SI-hSyn-GFP: SI mice with AAV-hSyn-GFP virus injection (P61); SI-hSyn-Egr2: SI mice with AAV-hSyn-Egr2 virus injection mice (P61).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–19.

Reporting Summary

Supplementary Video 1

Social cooperation training.

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Social cooperation behavior.

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Social uncooperation behavior.

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Zhang, Y., Feng, W., Wang, Z. et al. Early growth response 2 in the mPFC regulates mouse social and cooperative behaviors. Lab Anim 52, 37–50 (2023). https://doi.org/10.1038/s41684-022-01090-0

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