图1 社交隔离未导致焦虑抑郁样行为

Fig.1 Social isolation (SI) does not cause anxiety- or depressive-like behaviors in mice. A: Schematic diagram of SI modeling in mice (n=8). B: Diagram of open field test (OFT) of the mice. C: Statistical plot of exploration time in the central areas of the mice during OFT. D: Statistical plot of total distance traveled of the mice in OFT. E: Diagram of elevated plus-maze (EPM) test of the mice. F: Statistical plot of exploration time in the open arms of the mice in EPM test. G: Statistical plot of open arm entry frequency of the mice in EPM test. H: Diagram of forced swimming test (FST) of the mice. I: Statistical plot of inactivity duration of the mice during the final 4 min in FST. J: Behavioral patterns of the mice in tail suspension test (TST). K: Statistical plot of inactivity duration of the mice during the final 4 min in TST.

图2 社交隔离未影响物体识别和工作记忆

Fig.2 SI does not affect object recognition and working memory of mice. A: Diagram of novel object recognition test (NORT) of the mice. B: Statistical chart of cognitive indices in the two groups of mice during the second phase of NORT. C: Diagram of Y-maze test of the mice. D: Statistical chart of exploration time of the mice in the novel arm in Y-maze test (n=8).

图3 社交隔离不影响社交能力和社交记忆, 但会出现社交回避

Fig.3 SI does not affect social competence or social memory, but may cause social avoidance in mice. A: Diagram of social interaction test (SIT) of the mice. B: Statistical chart of social interaction ratios of the mice in SIT. C: Statistical chart of time spent by target mice in the social interaction zone in the two groups. D: Diagram of TCST social competence stage. E: Statistical chart of exploration time of the mice toward the social mice and objects. F: Statistical chart of social preference index of the social mice in the two groups. G: Diagram of TCST social memory stage. H: Statistical chart of exploration time toward familiar mice and new mice in the two groups. I: Statistical chart of social preference index of the new mice in the two groups. **P<0.01, ***P<0.001 (n=8).

图4 社交隔离导致mPFC Glu能神经元c-Fos表达增加

Fig.4 SI leads to increased c-Fos expression in the glutamatergic neurons in the medial prefrontal cortex (mPFC) of mice. A: Flowchart of c-Fos staining experiments in mice. B: Representative immunofluorescence images of c-Fos staining in PrL and IL subregions of the mPFC (Scale bar=100 μm). C: Statistical plot of average density of c-Fos-positive neurons in the PrL, IL subregions and mPFC brain region of the mice. D: Representative immunofluorescence images of co-staining for c-Fos and CaMKIIα in the mPFC brain region of the mice (Scale bar=40 μm). White arrows indicate co-localized cells expressing c-Fos and CaMKIIα. E: Statistical plot of co-localization of c-Fos- and CaMKIIα-positive neurons in the two groups. F: Representative immunofluorescence images of co-staining for c-Fos and GABA in the mPFC brain region of the mice (Scale bar=40 μm). White arrows indicate co-localized cells expressing c-Fos and GABA. G: Statistical plot of co-localization of c-Fos- and GABA-positive neurons in the two groups. *P<0.05, **P<0.01 (n=3).

图5 SI小鼠在SIT中mPFC Glu能神经元钙活性增加

Fig.5 Increased calcium activity in mPFC glutamatergic neurons in SI mice during SIT. A: Flowchart of optical fiber recording in the two groups of mice. B: Schematic diagram of calcium indicator GCaMP6m injection and fiber embedding in mPFC neurons expressing CaMKIIα. C: Schematic diagram of viral injection sites (Scale bar=100 μm). D: Co-staining of GCaMP6m (green) and CaMKIIα (red). White arrows indicate co-localized cells expressing GCaMP6m and CaMKIIα (Scale bar=50 μm). E: Pie chart showing co-labeling rates of GCaMP6m and CaMKIIα. F: Heat map illustrating Ca2+ signal changes during optical fiber recording in the first stage of SIT in the two groups of mice. G: Curve plots of average Ca2+ fluorescence signals in the first stage in the two groups. H: Statistical plot of ΔF/F peak signals in the first stage in the two groups. I: Heat map illustrating Ca2+ signal changes during optical fiber recording in the second stage of SIT in the two groups. J: Curve plots of average Ca2+ fluorescence signals in the second stage in the two groups. K: Statistical plot of ΔF/F peak signals in the second stage in the two groups. ***P<0.001 (n=5).

图6 化学遗传学激活mPFC Glu能神经元诱导社交回避

Fig.6 Chemical genetics activation of mPFC glutamatergic neurons induces social avoidance in mice. A: Experimental flowchart of chemogenetic activation of mPFC glutamatergic neurons. B: Schematic diagram of viral injection protocol and injection sites. C: Fluorescence image showing viral expression of hM3Dq in mPFC (Scale bar=100 μm). D: Fluorescence image of co-staining with hM3Dq-mCherry (red) and CaMKIIα (green) in mPFC sections. White arrows indicate co-localized cells, and the pie chart displays co-localization rates of hM3Dq and CaMKIIα (Scale bar= 50 μm). E: Representative immunofluorescence images of c-Fos staining in mPFC regions and statistical plot of average c-Fos density in mPFC of the mice (Scale bar=200 μm; n=3). F: SIT diagram. G: Statistical plot of social interaction rates during social interaction tests in SIT in the two groups. H: Statistical plot of time spent in social interaction areas by target mice in SIT in the two groups. *P<0.05, **P<0.01 (n=8).

图7 光遗传学激活mPFC Glu能神经元诱导了社交回避

Fig.7 Optogenetic activation of mPFC glutamatergic neurons induces social avoidance in mice. A: Experimental flowchart for optogenetic activation of mPFC glutamatergic neurons. B: Schematic diagram of viral injection protocol and injection sites. C: Fluorescence image showing viral expression of hChR2 in mPFC (Scale bar=100 μm). D: Fluorescence image of co-staining hChR2-mCherry (red) and CaMKIIα (green) in mPFC sections (Scale bar=50 μm). White arrows indicate co-localized cells. The pie chart displays co-localization rates of hChR2 and CaMKIIα. E: Representative immunofluorescence images of c-Fos staining in mPFC regions and statistical plot of average c-Fos density in the mPFC of the mice (Scale bar=200 μm; n=3). F: SIT pattern diagram. G: Statistical plot of social interaction rates during social interaction tests in SIT in the two groups. H: Statistical plot of time spent in social interaction areas by target mice in SIT in the two groups. *P<0.05, **P<0.01, ***P<0.001 (n=8).

图8 化学遗传学抑制mPFC Glu能神经元缓解SI小鼠社交回避

Fig.8 Chemical genetics inhibition of mPFC glutamatergic neurons alleviates social avoidance in SI mice. A: Flowchart of the experimental protocol for chemogenetic inhibition of mPFC glutamatergic neurons. B: Schematic diagram of the viral injection protocol and injection sites. C: Fluorescence image showing the localization of hM4Di virus expression in the mPFC (Scale bar= 100 μm). D: Fluorescence image of co-staining with hM4Di-mCherry (red) and CaMKIIα (green) in mPFC sections (Scale bar= 50 μm). White arrows indicate co-localized cells. The pie chart shows the co-localization rate of hM4Di and CaMKIIα. E: Representative immunofluorescence images of c-Fos staining in the mPFC regions (Scale bar=100 μm) and statistical plot of the average c-Fos density in the mPFC in the two groups (n=3). F: SIT protocol diagram. G: Statistical plot of social interaction rates among the 3 groups during the social interaction test in SIT. H: Statistical plot of time spent by target mice in the social interaction regions in SIT. *P<0.05, **P<0.01, ***P<0.001 (n=8).

图9 光遗传学抑制mPFC Glu能神经元缓解SI小鼠社交回避

Fig.9 Optogenetic inhibition of mPFC glutamatergic neurons alleviates social avoidance in SI mice. A: Flowchart of the optogenetic inhibition of mPFC glutamatergic neurons. B: Schematic diagram of viral injection protocol and injection sites. C: Fluorescence image showing the injection site of NpHR virus in the mPFC (Scale bar=100 μm). D: Fluorescence image demonstrating co-staining of NpHR-mCherry (red) and CaMKIIα (green) in mPFC sections (Scale bar=50 μm). White arrows indicate co-localized cells. The pie chart shows the co-localization rate of NpHR and CaMKIIα. E: Representative immunofluorescence images of c-Fos staining in the mPFC regions (Scale bar=100 μm) and average c-Fos density in the mPFC in the two groups (n=3). F: SIT protocol diagram. G: Statistical plot of social interaction rates among the 3 groups during the social interaction test in the SIT. H: Statistical plot of time spent by target mice in the social interaction regions in SIT. *P<0.05, **P<0.01, ***P<0.001 (n=8).