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北京大学学报（医学版） ›› 2022, Vol. 54 ›› Issue (2): 209-216. doi: 10.19723/j.issn.1671-167X.2022.02.002

• 论著 • 上一篇    下一篇

CACNA1H基因敲除对小鼠孤独症样行为及海马神经元形态学的影响

焦翠1,王俭妹1,况海霞1,武志红1,△(),柳涛1,2,△()   

  1. 1.南昌大学第一附属医院 儿科,南昌 330006
    2.南昌大学第一附属医院 医学科研中心,南昌 330006
  • 收稿日期:2020-02-18 出版日期:2022-04-18 发布日期:2022-04-13
  • 通讯作者: 武志红,柳涛 E-mail:1390959916@qq.com;liutao1241@ncu.edu.cn
  • 基金资助:
    国家自然科学基金(31660289);江西省杰出青年人才资助计划(20171BCB23091);南昌大学研究生创新专项基金(CX2018166);江西省自然科学基金(20202BABL206049)

Effects of CACNA1H gene knockout on autistic-like behaviors and the morphology of hippocampal neurons in mice

JIAO Cui1,WANG Jian-mei1,KUANG Hai-xia1,WU Zhi-hong1,△(),LIU Tao1,2,△()   

  1. 1. Department of Pediatrics, the First Affiliated Hospital of Nanchang University, Nanchang 330006, China
    2. Center for Experimental Medicine, the First Affiliated Hospital of Nanchang University, Nanchang 330006, China
  • Received:2020-02-18 Online:2022-04-18 Published:2022-04-13
  • Contact: Zhi-hong WU,Tao LIU E-mail:1390959916@qq.com;liutao1241@ncu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31660289);Excellent Young Scientist Foundation of Jiangxi(20171BCB23091);Graduate Innovation Foundation of Nanchang University(CX2018166);Natural Science Foundation of Jiangxi Province(20202BABL206049)

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摘要：

目的: 研究CACNA1H基因敲除(knockout, KO)对小鼠孤独症样行为及海马神经元形态学的影响。方法: 25只3~4周龄C57BL/6背景的CACNA1H KO小鼠作为实验组,26只同年龄同背景的野生型(wild type,WT)小鼠作为对照组。通过三箱实验和旷场实验观察小鼠社交、焦虑和重复刻板行为后测量其脑质量与脑体积,用尼氏染色法(Nissl staining)观察海马神经元数目。将CACNA1H杂合子小鼠与Thy1-GFP-O小鼠杂交,构建CACNA1H-/--Thy1+(KO-GFP)及CACNA1H+/+-Thy1+(WT-GFP)小鼠,观察海马神经元树突棘密度及成熟度。结果: 三箱实验中,社交测试阶段,KO小鼠在陌生鼠箱中的时间比空箱更长(F1,14=95.086,P<0.05;Post-Hoc:P<0.05),探索的偏好指数与对照组相比差异无统计学意义(t=1.044,P>0.05);新社交对象识别测试阶段,KO小鼠在新陌生鼠箱与陌生鼠箱中的时间差异无统计学意义(F1,14=18.062,P<0.05;Post-Hoc:P>0.05),探索的偏好指数低于对照组(t=2.390,P<0.05)。旷场实验中,KO小鼠在旷场中心活动时间明显少于对照组(t=2.503,P<0.05),自梳理时间明显多于对照组(t=-2.299,P<0.05)。形态学结果显示,KO小鼠脑质量/体质量和脑体积与对照组相比差异均无统计学意义(t=0.356,P>0.05;t=-0.660,P>0.05),但其海马齿状回区神经元数目较对照组减少(t=2.323,P<0.05),且KO-GFP小鼠海马齿状回区树突棘密度较对照组增加(t=-2.374,P<0.05),而成熟度差异无统计学意义(t=-1.935,P>0.05)。结论: CACNA1H KO小鼠具有孤独症样行为,可能与海马齿状回区神经元数目减少及树突棘密度增高有关。

关键词: CACNA1H, 小鼠, 基因敲除, 孤独症谱系障碍, 行为测试

Abstract:

Objective: To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice. Methods: In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H -/--Thy1+(KO-GFP) and CACNA1H +/+-Thy1+ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed. Results: In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F1,14=95.086, P<0.05; Post-Hoc: P<0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F1,14=18.062, P<0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P<0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P<0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P<0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P<0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P<0.05), without any significant difference in spine maturity (t=-1.935, P>0.05). Conclusion: CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.

Key words: CACNA1H, Mice, knockout, Autism spectrum disorder, Behavior test

中图分类号: 

  • R749.94

图1

CACNA1H基因敲除对小鼠社交行为的影响"

表1

WT与KO小鼠的三箱实验结果"

Genotype Sociability Social novelty recognition
O/s S1/s PI S1/s S2/s PI
WT 158.05±19.93 353.36±25.35* 0.62±0.07 153.24±27.87 346.03±38.77# 0.52±0.08
KO 155.33±15.55 349.44±19.24* 0.50±0.10 226.18±32.21 254.84±33.45 0.15±0.15△

图2

CACNA1H基因敲除对小鼠焦虑及自梳理行为的影响 A, representative track and heat maps of mice movements in the open field test; B, the time spend in the center was decreased in KO mice (n=18) compared to that of the WT mice (n=17); C, the grooming time was significantly increased in KO mice (n=12) compared to that of the WT mice (n=15). Abbreviations as in Figure 1. * P<0.05."

表2

WT与KO小鼠的旷场实验结果"

Parameter Genotype Time/s
Anxiety (time in center) WT 53.87±6.01
KO 36.30±3.63*
Repetitive behavior (self-grooming) WT 69.86±13.53
KO 131.66±24.92*

图3

CACNA1H基因敲除对小鼠大脑形态及海马神经元数目的影响"

表3

WT与KO小鼠体质量、脑质量/体质量和脑体积测量"

Genotype Body weight/g Brain weight/body weight Brain size/mL
WT 7.05±0.97 0.06±0.00 0.39±0.00
KO 7.43±0.99 0.06±0.01 0.40±0.02

表4

WT与KO小鼠海马区尼氏染色神经元相对数"

Genotype Hippocampus
CA1 CA2 CA3 CA4 DG
WT 1.00±0.04 1.00±0.08 1.00±0.07 1.00±0.06 1.00±0.03
KO 0.95±0.08 0.85±0.06 0.86±0.11 0.86±0.06 0.86±0.05*

图4

CACNA1H基因敲除对小鼠树突棘的影响"

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