Social isolation induces social avoidance in mice: the mediating role of the medial prefrontal cortex glutamatergic neurons

doi:10.12122/j.issn.1673-4254.2026.09.01

Abstract

Abstract:

Objective To investigate social isolation (SI)-induced abnormal behaviors and changes in neuronal activity in the medial prefrontal cortex (mPFC) of mice. Methods Male C57BL/6J mice kept under normal conditions or SI mice housed in individual cages for 2 weeks were subjected to intracerebral microinjection of a control virus, a chemogenetic activation virus, or an optogenetic activation virus (n=8). The mice were assessed for behavioral changes and examined for neuronal changes in mPFC using c-fos immunofluorescence staining and co-staining with specific antibodies against CaMKIIα (for excitatory neurons) and GABA (for inhibitory neurons). In vivo optical fiber recording was used to detect real-time neuronal changes in the mice during behavioral assessment. The effects of chemogenetics and optogenetics for modulating activities of glutamatergic neurons in the mPFC were observed on social behaviors of both the control and SI mice. Results Behavioral tests showed that two weeks of SI did not cause obvious anxiety or depression-like behaviors or impair object recognition memory, working memory, social ability or social memory, but induced social avoidance behaviors in the mice. Immunofluorescence staining revealed significantly increased c-fos-positive neurons in the mPFC of SI mice, consisting mostly of excitatory glutamatergic neurons. In vivo optical fiber recording showed that during the second stage of social interaction test (SIT), the activity of mPFC glutamatergic neurons increased significantly in SI mice. Chemogenetic and optogenetic activation of mPFC glutamatergic neurons induced obvious social avoidance in the control mice, and inhibition of these neurons significantly alleviated social avoidance in SI mice. Conclusion Two weeks of SI can induce social avoidance behaviors in mice and enhances the activity of mPFC glutamatergic neurons.

Key words: social isolation, medial prefrontal cortex, glutamatergic neurons

Quan YUAN, Dongdong ZHANG, Jingtao WANG, Hanjun SONG. Social isolation induces social avoidance in mice: the mediating role of the medial prefrontal cortex glutamatergic neurons[J]. Journal of Southern Medical University, 2026, 46(9): 1993-2005.

Figures/Tables 9

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.

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).

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).

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).

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).

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).

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).

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).

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).

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