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丘脑室旁核谷氨酸能神经元可能参与调控Shank3基因敲除小鼠异常睡眠行为

陈昌锋¹, 王烈成¹, 刘勇², 陈磊^2,*

¹安徽医科大学基础医学院，合肥 23003；²安徽中医药大学第一附属医院药学部，合肥 230031

摘要

本研究旨在观察Shank3基因敲除(Shank3-KO)小鼠焦虑样行为、节律和睡眠行为的变化，同时阐明Shank3-KO小鼠异常睡眠行为的可能机制。用高架十字迷宫、旷场实验和悬尾实验检测小鼠焦虑样行为，用跑轮装置检测小鼠节律行为，并用多导睡眠记录仪实时同步记录小鼠脑电图和肌电图，用免疫荧光染色法检测SHANK3 在野生型小鼠前扣带皮层(anteriorcingulate cortex, ACC)、丘脑室旁核(paraventricular thalamus, PVT)、伏隔核(nucleus accumbens, NAc)、基底外侧杏仁核(basolateral amygdala, BLA)和海马CA2 区等脑区的表达分布情况，并检测光照期PVT、ACC和NAc脑区的c-Fos 表达情况，用免疫荧光双标实验检测PVT区c-Fos 与囊泡谷氨酸转运体2 (vesicular glutamate transporter 2，Vglut2，谷氨酸能神经元标志物)共定位情况。高架十字迷宫实验结果显示，与野生型小鼠相比，Shank3-KO小鼠的开臂停留时间减少，进入开臂的次数减少；旷场实验结果显示，Shank3-KO 小鼠进入中心区域的次数减少，在中心区域的移动时间减少；悬尾实验结果显示，Shank3-KO小鼠不动时间增加。跑轮节律实验结果显示，Shank3-KO小鼠在持续黑暗期的相移时间增加。脑电图/肌电图结果显示，与野生型小鼠相比，光照期Shank3-KO组小鼠觉醒时间增加，非快速眼动(non-rapid eye movement, NREM)睡眠时间减少，觉醒次数增加，NREM睡眠次数减少，NREM睡眠向觉醒的转换增加，觉醒向NREM睡眠的转换减少。SHANK3在野生型小鼠的ACC、PVT和NAc等脑区均有表达分布；进入光照期2 h 时，Shank3-KO小鼠PVT区c-Fos 表达上调，且大多与Vglut2 共定位。以上结果提示，Shank3-KO小鼠焦虑水平提高，内在节律的调控水平下降，光照期觉醒次数增加，PVT区谷氨酸能神经元可能参与Shank3-KO小鼠光照期异常睡眠行为的调控。

关键词： Shank3; 焦虑; 节律; 睡眠

Glutamatergic neurons in thalamic paraventricular nucleus may be involved in the regulation of abnormal sleep behavior of Shank3 gene knockout mice

CHEN Chang-Feng¹, WANG Lie-Cheng¹, LIU Yong², CHEN Lei^2,*

¹School of Basic Medicine Sciences, Anhui Medical University, Hefei 230032, China；²Department of Pharmacy, First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230031, China

Abstract

The purpose of this study was to investigate the anxiety-like behaviors, circadian rhythms and sleep, and to elucidate the possible underlying mechanisms of the abnormal sleep behavior in Shank3 gene knockout (Shank3-KO) mice. The anxiety-like behaviors were detected by elevated plus-maze (EPM) test, open field test (OFT) and tail suspension test (TST). The circadian rhythms were detected by running wheel test. The electroencephalogram (EEG)/electromyogram (EMG) recordings were performed synchronically by polysomnograph. The distribution of SHANK3 in anterior cingulate cortex (ACC), paraventricular thalamus (PVT), nucleus accumbens (NAc), basolateral amygdala (BLA) and hippocampal CA2 region in wild type (WT) mice was detected by immunofluorescence assay. The protein expression of c-Fos in PVT, ACC and NAc was also detected by immunofluorescence assay during light cycle. The colocalization of c-Fos and vesicular glutamate transporter 2 (Vglut2, a marker for glutamatergic neurons) in the PVT was detected by immunofluorescence double labeling experiment. The results of EPM test showed that, compared with the WT mice, the Shank3-KO mice showed less time in open arms and less number of open arm entries. The results of OFT showed that the Shank3- KO mice showed less time in central area and less number of central area entries. The immobility time of Shank3-KO mice was increased in the TST. The results of running wheel rhythm test showed that the phase shift time of Shank3-KO mice in the continuous dark period was increased. The results of EEG/EMG recording showed that, compared with the WT mice, the duration of wakefulness in Shank3-KO mice was increased and the duration of non-rapid eye movement (NREM) sleep was decreased during light phase; The bout number of wakefulness was increased, the bout number of NREM sleep was decreased, NREM-wake transitions were increased, and wake-NREM transitions were decreased during light phase. SHANK3 was expressed in ACC, PVT, NAc and BLA in the WT mice. The expression of c-Fos in the PVT of Shank3-KO mice was up-regulated 2 h after entering the light phase, and majority of c-Fos was co-localized with Vglut2. These results suggest that the anxiety level of Shank3-KO mice is increased, the regulation of the internal rhythms is decreased, and the bout number of wakefulness is increased during light phase. The glutamatergic neurons in PVT may be involved in the regulation of abnormal sleep behavior in Shank3-KO mice during the light phase.

Key words: Shank3; anxiety; circadian rhythms; sleep

收稿日期：　　录用日期：

通讯作者：陈磊　　E-mail:

DOI: 10.13294/j.aps.2025.0041

引用本文：

陈昌锋, 王烈成, 刘勇, 陈磊. 丘脑室旁核谷氨酸能神经元可能参与调控Shank3基因敲除小鼠异常睡眠行为[J]. 生理学报 2025; 77 (5): 792-800.

CHEN Chang-Feng, WANG Lie-Cheng, LIU Yong, CHEN Lei. Glutamatergic neurons in thalamic paraventricular nucleus may be involved in the regulation of abnormal sleep behavior of Shank3 gene knockout mice. Acta Physiol Sin 2025; 77 (5): 792-800 (in Chinese with English abstract).