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Journal of Peking University (Health Sciences) ›› 2022, Vol. 54 ›› Issue (2): 209-216. doi: 10.19723/j.issn.1671-167X.2022.02.002

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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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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

CLC Number: 

  • R749.94

Figure 1

Effects of CACNA1H gene knockout on social behavior in mice A and D, representative heat maps of mice movements in the sociability test session and the social novelty recognition test session in the three-chamber test, respectively; B and E, averaged time (s) spend in each chamber of WT (n=9) and KO (n=7) mice; C and F, averaged preference index from exploration time of WT (n=9) and KO (n=7) mice. KO, knockout; WT, wild type; O, object; S1, stranger 1; S2, stranger 2; M, middle. * P<0.05."

Table 1

Results of three-chamber test of WT and KO mice"

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△

Figure 2

Effects of CACNA1H gene knockout on anxiety and self-grooming behavior in mice"

Table 2

Results of open field test of WT and KO mice"

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*

Figure 3

Effects of CACNA1H gene knockout on brain morphology and the number of neurons of the hippocampus in mice A, representative brain morphology of WT and KO mice; B, the body weight of WT (n=9) and KO (n=7) mice; C, the ratio between body weight and brain weight of WT (n=9) and KO (n=7) mice; D, the brain size of WT (n=9) and KO (n=7) mice; E, representative Nissl staining figures of hippocampal sections from WT and KO mice (the white squares represent the areas for analysis); F, relative number of neurons in each subregion of the hippocampus in WT (n=8) and KO (n=6) mice, the number in the histogram represents the number of slices. DG, dentate gyrus; other abbreviations as in Figure 1. * P<0.05."

Table 3

Body weight, brain weight/body weight, and brain size of WT and KO mice"

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

Table 4

Relative number of neurons in the hippocampus of WT and KO mice counted by Nissl staining"

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*

Figure 4

Effects of CACNA1H gene knockout on dendritic spines in mice A, classification of dendritic spines; B, photomicrographs showing representation of dendritic spines in hippocampal dentate gyrus neurons of WT-GFP and KO-GFP mice; C, CACNA1H gene knockout significantly increased the density of dendritic spines in KO mice (WT-GFP n=5, KO-GFP n=5); D, percentage of mature spines in WT-GFP (n=5) and KO-GFP (n=5) mice. The number in the histogram represents the number of segments of dendrite. GFP, green fluorescent protein; other abbreviations as in Figure 1. * P<0.05."

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