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Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2026 Sep 20;46(9):1993–2005. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2026.09.01

社交隔离诱导小鼠社交回避：mPFC谷氨酸能神经元的介导作用

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

YUAN Quan 1,2, ZHANG Dongdong 1, WANG Jingtao 1, SONG Hanjun 1,✉
Editor: 余 诗诗
PMCID: PMC13624992  PMID: 42812047

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.

Keywords: social isolation, medial prefrontal cortex, glutamatergic neurons

社交隔离是当今社会普遍存在的一个问题，在这种情况下会对我们的身体健康、心理健康和行为方面产生影响［1， 2］。在现代社会中，社交缺失和人口老龄化导致社交隔离越来越普遍［3］，处于社交孤独和被孤立状态的人群逐渐增大。由于新型冠状病毒肺炎疫情的爆发，被动的社交隔离使得人们越来越关注心理社会因素对于健康的影响。长期的社交隔离有许多危害，会导致焦虑、抑郁、社交障碍、认知功能障碍等行为的出现；还会导致睡眠质量下降、血压升高、心血管功能和免疫系统异常等［4-8］。可以说，社交隔离在某种程度上全部或部分剥夺了人的社会化过程，阻碍了人的正常形成与发展过程，参与了各种精神障碍的发生过程［9］。研究表明可以利用像素跟踪系统评估社交隔离对小鼠身体活动的影响，监测同笼饲养伴侣小鼠与隔离小鼠的社交运动行为，与有伴侣的小鼠相比，社交隔离的小鼠的社交活动水平较低［10］。除此之外，有研究表明，2周的社交隔离会导致SI小鼠在社交过程中与新型小鼠互动的时间较短［11］。

前额叶皮层位于大脑皮层额叶的前端，在进化过程中，前额叶区域相对于大脑皮层其余部分的尺寸不断增大，占皮层总面积的30%［12］。内侧前额叶皮层（mPFC）是前额叶皮层的一个关键子区，它位于中央沟的前方和扣带沟的内侧，是大脑中负责高级认知和情绪调节的重要区域，也是自然条件下处理复杂信息的重要核团［13］。mPFC主要由兴奋性锥体神经元和抑制性GABA能中间神经元组成，两种类型的神经元共同参与皮层信息调控，起着至关重要的作用［14］。mPFC是调节社交行为的关键脑区。在人类童年和青春期，mPFC神经机制的成熟发育对社会功能快速发展发挥关键作用。在脑发育关键时期，经历社交隔离会导致mPFC和神经发育不成熟，进而导致社交功能障碍［15］。

目前关于成年期社交隔离对小鼠行为学影响的研究结论有所不同，有研究表明成年期社交隔离会产生焦虑、抑郁样行为等异常行为［9］。为进一步探讨成年期社交隔离对啮齿类动物行为学的影响，本研究选用6周雄性小鼠构建成年期社交隔离小鼠模型，系统评估成年期社交隔离对小鼠焦虑、抑郁、认知及社会行为的影响，并依据行为学检测、c-Fos免疫荧光染色、在体光纤记录以及化学遗传学和光遗传学的结果，探讨mPFC Glu能神经元介导社交隔离小鼠社交回避行为中的变化。

1. 材料和方法

1.1. 实验材料

1.1.1. 实验动物

实验小鼠为野生型成年C57BL/6J雄性（6周），体质量18~24 g，购买于珠海百试通生物科技有限公司。小鼠饲养在实验动物中心SPF级标准屏障系统，饲养笼具类型为独立通气笼盒 （IVC），笼盒规格为380 mm×204 mm×162 mm，遵循12 h光照和12 h黑暗循环（光照时间为每天8∶00~20∶00），室温维持温度（20~25 ℃）和湿度（40%~60%）。小鼠可随意获得水和粮食。所有动物实验程序均获得佳木斯大学基础医学院生物与医学伦理委员会批准（伦理批号：JDJCYXY20250035）。

1.1.2. 实验仪器及试剂

光纤记录系统、有线智能光遗传学系统（南京奥冠生物科技有限公司）；TopScanLite行为学采集系统（CleverSysInc.，Reston，VirginiaTopScanliteliteVersion2.00）；脑立体定位仪（瑞沃德生命科技有限公司）；摇床（海门市其林贝尔仪器制造有限公司）；PBS磷酸盐缓冲剂（鼎国昌盛生物技术有限责任公司）；4%多聚甲醛固定液（四和生物科技股份有限公司）；Bas试剂盒（艾克发生物技术有限公司）；氯氮平-N-氧化物CNO（Target Mol）；一抗Rabbit anti-Fos（CST）；Mouse anti-CaMKIIα（Abcam）；Rabbit anti-GABA（Sigma）；二抗Alexa FLuor555-conjugated Donkey anti-Mouse、Alexa FLuor488-conjugated Donkey anti-Rabbit（Invitrogen）；AAV2/9-mCaMKIIα-GCaMP6m-WPRE-pA、AAV2/9-CaMKIIα-mCherry-WPRE-pA、AAV2/9-mCaMKIIα-hM3Dq-mCherry-WPRE-pA、AAV2/9-mCaMKIIα-hChR2（H134R）-mCherry-WPRE-pA（上海泰儿图生物科技有限公司）。

1.2. 方法

1.2.1. 社交隔离模型

选用 6周鼠龄的 C57BL/6J小鼠，单独饲养，垫料减半，笼子周围用黑色塑料袋包裹住，隔离时间为 2周。在开始造模之前需要将小鼠放入标准条件下饲养1周，以适应环境。在造模期间，对照组小鼠处于标准条件下饲养。

1.2.2. 行为学测试

所有行为学测试都在9∶00~18∶00进行，每次测试前将测试小鼠提前40 min~1 h放入行为学室以适应环境。

1.2.2.1. 社交互动测试（SIT）

社交互动测试包括2个阶段，每个阶段时间为150 s。该实验所用到的仪器设备是一个开放矿场（长40 cm×宽40 cm×高40 cm）和一个空的、带孔的透明笼子（长10 cm×宽6.5 cm×高40 cm）。最后计算实验小鼠的社交互动比率，社交互动比率=实验小鼠在透明笼里面有目标小鼠时在相互作用区花费的时间/在透明笼里面没有目标小鼠时实验小鼠在相互作用区中花费的时间。

1.2.2.2. 三箱社交测试（TCST）

三箱社交测试包括适应阶段、社交阶段、社交记忆阶段，每个阶段时间为10 min。该实验所用到的仪器设备是由3个连通的室组成的，整体是由透明的墙壁和蓝色哑光底部构成的，每个室的尺寸为长40 cm×宽20 cm×高20 cm。通过记录的时间计算社交偏好指数。Stranger1社交偏好指数=Stranger1的社交时间/Stranger1和Object社交的总时间×100%。Stranger2社交偏好指数=Stranger2的社交时间/Stranger1和Stranger2社交的总时间×100%。

1.2.2.3. 开放矿场测试（OFT）

OFT所用到的实验设备是一个深蓝色不透明的箱子（长40 cm×宽40 cm×高40 cm）。实验开始时，将小鼠放入箱底部中心区域，并允许小鼠自由探索10 min。记录小鼠在中心区域花费的时间和运动总距离。

1.2.2.4. 高架十字迷宫测试（EPM）

EPM所用到的实验设备是一个距离地面高50 cm的十字迷宫，由2个开放臂（长35 cm×宽5 cm×高15 cm）和两个闭合臂（长35 cm×宽5 cm×高15 cm）以及连接4个臂的中心区域（长5 cm×宽5 cm）组成。实验开始时，将小鼠放入其中一开放臂，并允许小鼠探索10 min。记录小鼠在开放臂停留的时间以及进入开放臂的次数。

1.2.2.5. 强迫游泳测试（FST）

将小鼠放入装有20 cm水深的圆柱形玻璃水箱（直径16 cm×高28 cm）中，记录小鼠6 min内不动的时间，分析最后4 min内小鼠的不动时间。

1.2.2.6. 尾部悬挂实验（TST）

用胶带将小鼠悬挂于20 cm×20 cm×30 cm的箱子中（距离小鼠尾部末端1 cm处粘上胶带），使其头部距离地面约5 cm，记录小鼠6 min内的不动时间，分析后4 min内小鼠的不动时间。

1.2.2.7. 新物体识别测试（NORT）

NORT包括适应阶段、训练阶段和测试阶段，每个测试阶段均为10 min。NORT所用到的实验设备是一个不透明的蓝色箱体（长40 cm×宽40 cm×高40 cm），以及放在蓝色箱体里的A、B、C 3种物体（其中A、B为同种物体）。识别指数=对C的探索时间/（对C的探索时间+对A的探索时间）× 100%。

1.2.2.8. Y迷宫

Y迷宫包括训练阶段和测试阶段，训练阶段为10 min，测试阶段为5 min。Y迷宫用到的实验设备是一个蓝色不透明的Y型迷宫，该迷宫由3个相同的臂（长35 cm×宽5 cm×高10 cm）以及中心区域（边长为5 cm的三角形）组成的，两臂之间的夹角为120°。记录测试阶段小鼠探索新奇臂的时间。

1.2.3. 心脏灌注以及制备脑片

小鼠麻醉后固定在手术板上，用0.9%的生理盐水进行灌注，后用4%的多聚甲醛固定，然后取脑。取脑后将脑组织放到4%的多聚甲醛里固定过夜，用梯度蔗糖脱水后，进行冰冻切片。

1.2.4. 免疫组织荧光

PBS清洗3次后进行抗原修复，自然晾至室温后再次用PBS清洗，进行封闭，封闭结束后于4 ℃冰箱一抗孵育（兔抗c-Fos，1∶300；鼠抗CaMKIIα，1∶300；兔抗GABA，1∶500），48 h后PBS冲洗，进行二抗孵育（1∶800），2 h后再次冲洗，然后贴片、封片和显影。

1.2.5. 脑立体定位注射

制作微量注射针。麻醉小鼠，进行固定、调平、打孔以及注射病毒，最后根据实验目的选择直接缝合伤口或植入光纤陶瓷插芯针。（mPFC：ML：±0.33 mm；AP：+2.1 mm；DV：-2.1 mm。）

1.2.6. 在体光纤记录

将0.2 μL的AAV2/9-mCaMKIIα-GCaMP6m-WPRE-pA单侧立体定位注射到mPFC并植入光纤。病毒表达到高峰时，在行为中进行钙信号检测，实时记录GCaMP6m荧光信号的变化。ΔF/F=（F-F0）/F0，有效荧光信号值（F）为470 nm与410 nm信号差值；基线荧光值（F0）为行为学测试前稳定记录的平均荧光强度。

1.2.7. 化学遗传学

将0.2 μL的AAV2/9-CaMKIIα-hM3D（Gq）-mCherry或AAV2/9-CaMKIIα-hM4D（Gi）-mCherry或对照病毒AAV2/9-CaMKIIα-mCherry立体定位注射到双侧mPFC。病毒表达达到高峰期时，进行行为学检测。在行为学检测前45 min，对注射实验病毒和对照病毒的小鼠进行腹腔注射CNO（0.33 mg/kg）。

1.2.8. 光遗传学

将0.2 μL的AAV2/9-mCaMKIIα-hChR2（H134R）‑mCherry或AAV2/9-mCaMKIIα-eNpHR3.0-mCherry或对照病毒AAV2/9-CaMKIIα-mCherry立体定位注射到双侧mPFC，同时在mPFC植入光纤。病毒表达达到高峰期时，进行行为学检测。光激活给予465 nm，30 Hz，3~5 mW功率的蓝色激光刺激。光抑制给予589 nm，3~5 mW功率的黄色激光刺激。

1.3. 统计学分析

本研究中所有实验数据均通过GraphPad Prism10.1.2进行统计分析。所有实验数据以均数±标准差表示，并使用Shapiro-Wilk进行正态性检验。两组间比较若符合正态分布则采用Student t检验，反之则对数据进行对数变换以实现正态性，再进行 Student t检验。3组及以上的数据比较使用单因素方差分析。P<0.05表示差异有统计学意义。

2. 结果

2.1. 成年期社交隔离对小鼠行为的影响

结果显示，在OFT中，SI组与对照组小鼠在中心区域停留时间以及运动总距离的差异无统计学意义（P>0.05，图1A~D）。在EPM中，SI组与对照组小鼠探索开放臂的时间以及进入开放臂次数的差异无统计学意义（P>0.05，图1E~G）。在强迫游泳测试（FST）与尾部悬挂实验（TST）中，两组小鼠在后 4 min 内不动时间的差异无统计学意义（P>0.05，图1H~K）。

图1.

图1

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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.

在NORT中，SI组与对照组小鼠对新物体的识别指数的差异无统计学意义（P>0.05，图2A、B）。在Y 迷宫中，两组小鼠探索新奇臂时间的差异无统计学意义（P>0.05，图2C、D）。

图2.

图2

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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).

SIT结果显示，SI小鼠表现出社交回避行为（图3A~C）。与对照组相比，SI组的社交互动比率显著降低（P<0.001）；在第2阶段中，SI组小鼠对空笼子与社交小鼠的探索时间的差异无统计学意义（P>0.05）。在TCST第2阶段中，相比于探索物体，两组小鼠探索社交小鼠的时间均显著升高（P<0.01），社交偏好指数的差异无统计学意义（P>0.05，图3D~F）；第3阶段，相比于探索熟悉小鼠，两组小鼠探索新小鼠的时间均显著升高（P<0.01），且社交偏好指数的差异无统计学意义（P>0.05，图3G~I）。

图3.

图3

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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).

2.2. 社交隔离对mPFC Glu能神经元活性的影响

与对照组相比，SI组小鼠mPFC脑区c-Fos数量显著增加（P<0.01），且两个亚区PrL和IL的c-Fos数量也显著增加（P<0.01，图4A~C）；SI组小鼠mPFC谷氨酸能神经元的活性显著升高（P<0.05），而抑制性神经元的活性的差异无统计学意义（P>0.05，图4D~G）。

图4.

图4

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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).

采用在体光纤记录对小鼠mPFC中谷氨酸能神经元进行了钙信号检测（图5A~C）。染色结果显示，有82.2%的GCaMP6m可以特异性的表达在兴奋性神经元上（图5D、E）。钙成像记录结果显示，在SIT第1阶段中，Ctrl组小鼠和SI组小鼠在探索空笼子时，mPFC Glu能神经元钙活性的差异无统计学意义（P>0.05，图5F~H）；在SIT第二阶段中，与对照组相比，SI组小鼠在探索目标小鼠时，mPFC Glu能神经元钙活性明显增加（P<0.001，图5I~K）。

图5.

图5

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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).

2.3. 激活mPFC Glu能神经元诱导社交回避

2.3.1. 化学遗传学激活mPFC Glu能神经元诱导社交回避

特异性结果显示，有76%表达hM3Dq的神经元可以与代表兴奋性神经元抗体CaMKIIα共标（图6D）；有效性统计结果显示，与mCherry组小鼠相比，hM3Dq组小鼠mPFC中的c-Fos阳性细胞平均密度显著增多（P<0.01，图6E）。行为学测试结果显示，在SIT中，与Ctrl-mCherry组小鼠相比，Ctrl-hM3Dq组小鼠的社交互动比率显著下降（P<0.01），在第2阶段探索社交互动区域的时间与第1阶段的差异无统计学意义（P>0.05，图6F~H）。

图6.

图6

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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).

2.3.2 光遗传学激活mPFC Glu能神经元诱导社交回避 特异性结果显示，有80%表达hChR2的神经元可以与代表兴奋性神经元抗体CaMKIIα共标（图7D）；有效性统计结果显示，与mCherry组小鼠相比，hChR2组小鼠mPFC中的c-Fos阳性细胞平均密度显著增多（P<0.01）。行为学测试结果显示，在SIT中，与Ctrl-mCherry组小鼠相比，Ctrl-hChR2组小鼠的社交互动比率显著下降（P< 0.001），在第2阶段探索社交互动区域的时间与第一阶段的差异无统计学意义（P>0.05，图7F~H）。

图7.

图7

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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).

2.4. 抑制mPFC Glu能神经元可缓解社交回避

2.4.1. 化学遗传学抑制mPFC Glu能神经元可缓解社交回避

特异性结果显示，有75%表达hM4Di的神经元可以与代表兴奋性神经元抗体CaMKIIα共标（图8D）；有效性统计结果显示，与mCherry组小鼠相比，hM4Di组小鼠mPFC中的c-Fos阳性细胞平均密度显著减少（P<0.01）（图8E）。行为学测试结果显示，在SIT中，SI-mCherry组小鼠的社交互动比率显著低于Ctrl-mCherry组小鼠（P<0.001），SI-hM4Di小鼠的社交互动比率显著高于SI-mCherry小鼠（P<0.05），SI-mCherry组小鼠在第2阶段探索社交互动区域的时间与第1阶段的差异无统计学意义（P>0.05，图8F~H）。

图8.

图8

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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).

2.4.2. 光遗传学抑制mPFC Glu能神经元可缓解社交回避

特异性结果显示，有78.95%表达NpHR的神经元可以与代表兴奋性神经元抗体CaMKIIα共标（图9D）；有效性统计结果显示，与mCherry组小鼠相比，NpHR组小鼠mPFC中的c-Fos阳性细胞平均密度显著减少（P<0.05，图9E）。行为学测试结果显示，在SIT中，SI-mCherry组小鼠的社交互动比率显著低于Ctrl-mCherry组小鼠（P<0.01），SI-NpHR小鼠的社交互动比率显著高于SI-mCherry小鼠（P<0.001），SI-mCherry组小鼠在第2阶段探索社交互动区域的时间与第1阶段的差异无统计学意义（P>0.05，图9F~H）。

图9.

图9

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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).

3. 讨论

社交在人类生活中发挥着至关重要的作用，是个体生存和发展的基础。缺乏社交会导致焦虑、抑郁等精神障碍疾病发病率的增加［16-22］。在啮齿类动物中，社交隔离也会引起焦虑及抑郁样行为、社交和认知功能变化［23-30］。近年来，多种因素的影响，社交隔离的人群覆盖面不断扩大，成为全球性的公共卫生问题与社会热点议题。

mPFC是大脑调控情绪、认知、决策等功能的高级脑区，主要由兴奋性锥体神经元和抑制性GABA能中间神经元组成。其中兴奋性锥体神经元占mPFC神经元中的80%~90%，与脑内的许多神经核团发生联系［31， 32］。谷氨酸能神经元是mPFC中的主要神经元类型，负责处理大量的信息输入。研究表明mPFC谷氨酸能神经元在精神疾病中扮演着重要角色。有研究系统的阐述了多种不同社交行为的神经环路调控机制及其在疾病中的异常表现以及mPFC在调控社交行为中的核心地位，为理解社交行为的神经调控机制和开发精神疾病干预策略提供了重要理论依据［33］。

本研究选取6周雄性C57BL/6J小鼠建立社交隔离模型，通过一系列行为学实验对小鼠进行测试。在本研究中，SIT的结果表明2周的社交隔离会导致小鼠出现社交回避行为；OFT和EPM的结果表明，SI小鼠没有出现焦虑样行为。有研究表明，2周的社交隔离会导致小鼠出现焦虑样行为，这可能是由于本研究所用的实验小鼠品系以及年龄与其并不相同［11］；FST和TST的结果表明，SI小鼠没有出现抑郁样行为，先前的研究结果表明，8周龄雌性C57BL/6小鼠进行8周社交隔离后，发现隔离小鼠会产生抑郁样行为［9］，本研究与其结果存在差异可能是由于小鼠的性别以及隔离时间不同；NORT以及Y maze的结果表明，SI小鼠物体识别记忆和空间工作记忆未受影响，但有研究表明，经历10周社交隔离的大鼠会出现认知障碍［34］，这可能是由于隔离时间与选择实验动物种类不同而导致结果存在差异；TCST的结果表明，SI小鼠社交能力和社交记忆没有受损。这对于社会行为是一个有趣的分离现象。武科大田波团队联合华科大张培团队揭示了BLA印迹细胞通过BLA-ACC-dHPC和 BLA-ACC-vHPC分别调控社交偏好与社交回避，说明这两种行为在环路水平具有可分离性［35］。在mPFC层面，先前的研究表明，mPFC IL区投射至 NAcSh的神经元选择性调控社交记忆，而mPFC PL区投射至BLA的环路则通过编码负性情绪状态驱动社交回避［36， 37］。结合这些发现，本研究中社交隔离选择性诱导社交回避而不影响社交能力与社交记忆，可能原因是社交隔离对mPFC不同亚区向不同下游脑区（如BLA、vHPC）的投射产生了差异化影响。

免疫荧光染色以及在体光纤记录结果发现，SI小鼠mPFC脑区大部分都是兴奋性谷氨酸能神经元，且mPFC Glu能神经元活性增加，而抑制性神经元无显著变化。已有综述指出，多种社交缺陷模型涉及mPFC E/I平衡的偏移。通过影响mPFC神经元的发育和局部环路的E/I平衡来调节成年后的社交行为。梅林团队发现，mPFC中表达ErbB4的GABA能抑制性中间神经元（B4INs）在逃避行为发生时活动显著增强，且其活性受ErbB4激酶调控；当ErbB4活性升高时，B4INs对周围兴奋性神经元的抑制作用加强，从而诱发逃避行为［38］。此外，小清蛋白神经元和生长抑素（SST）神经元作为mPFC中两类主要的GABA能中间神经元亚型在社交行为调控中的分离作用日益受到重视［39］。徐晗教授团队在条件性社交恐惧模型中揭示了一条关键的微环路：他们证实前额叶皮层SST神经元活动的升高抑制了PV神经元的活动，使兴奋性神经元的活性增强，进而导致了社交恐惧行为的发生［40］。综上所述，可推测社交隔离导致E/I平衡向兴奋性偏移。

基于以上实验结果，我们猜测激活mPFC Glu能神经元可以诱导小鼠社交回避。为了进一步确定激活mPFC Glu能神经元能诱导小鼠出现社交回避，采用化学遗传和光遗传学调控mPFC Glu能神经元的活性，行为学结果显示，激活mPFC Glu能神经元活性可以诱导正常小鼠出现社交回避行为，而抑制mPFC Glu能神经元活性可以缓解SI小鼠的社交回避行为。以上结果进一步支持mPFC Glu能神经元在社交隔离中的关键作用。

本研究发现，社交隔离导致小鼠出现社交回避，mPFC Glu能神经元活性升高，通过化学遗传学和光遗传学技术激活mPFC Glu能神经元诱导小鼠出现社交回避，而抑制mPFC Glu能神经元可缓解社交回避这一现象，然而本研究尚未阐明具体的神经环路机制，未来仍需探索mPFC Glu能神经元在社会行为中具体神经环路机制及其作用，尽可能为社会行为的早期诊断和治疗提供更多的思路。

基金资助

国家自然科学基金培育项目佳木斯大学基础研究类（自然类）（JMSUGPZR2023-015）

Supported by National Natural Science Foundation Cultivation Project of Jiamusi University Basic Research Program (Natural Sciences)(JMSUGPZR2023-015).

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