收稿日期: 2020-02-18
网络出版日期: 2022-04-13
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
Received date: 2020-02-18
Online published: 2022-04-13
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)
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。
焦翠 , 王俭妹 , 况海霞 , 武志红 , 柳涛 . 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[J]. 北京大学学报（医学版）, 2022 , 54(2) : 209 -216 . DOI: 10.19723/j.issn.1671-167X.2022.02.002
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
| [1] | Baio J, Wiggins L, Christensen DL, et al. Prevalence of autism spectrum disorder among children aged 8 years: Autism and Deve-lopmental Disabilities Monitoring Network, 11 Sites, United States, 2014[J]. MMWR Surveill Summ, 2018, 67(6):1-23. |
| [2] | Bai D, Yip BHK, Windham GC, et al. Association of genetic and environmental factors with autism in a 5-country cohort[J]. JAMA Psychiatry, 2019, 76(10):1035-1043. |
| [3] | Iakoucheva LM, Muotri AR, Sebat J. Getting to the cores of autism[J]. Cell, 2019, 178(6):1287-1298. |
| [4] | Andrade A, Brennecke A, Mallat S, et al. Genetic associations between voltage-gated calcium channels and psychiatric disorders[J]. Int J Mol Sci, 2019, 20(14):3537. |
| [5] | Rebellato P, Kaczynska D, Kanatani S, et al. The T-type Ca2+ channel Cav3.2 regulates differentiation of neural progenitor cells during cortical development via caspase-3[J]. Neuroscience, 2019, 402:78-89. |
| [6] | Souza IA, Gandini MA, Zhang FX, et al. Pathogenic Cav3.2 channel mutation in a child with primary generalized epilepsy[J]. Mol Brain, 2019, 12(1):86. |
| [7] | Splawski I, Yoo DS, Stotz SC, et al. CACNA1H mutations in autism spectrum disorders[J]. J Biol Chem, 2006, 281(31):22085-22091. |
| [8] | Chourasia N, Osso-Rivera H, Ghosh A, et al. Expanding the phenotypic spectrum of CACNA1H mutations[J]. Pediatr Neurol, 2019, 93:50-55. |
| [9] | Feng XJ, Ma LX, Jiao C, et al. Nerve injury elevates functional Cav3.2 channels in superficial spinal dorsal horn[J]. Mol Pain, 2019, 15:1744806919836569. |
| [10] | Takumi T, Tamada K, Hatanaka F, et al. Behavioral neuroscience of autism[J]. Neurosci Biobehav Rev, 2020, 110:60-76. |
| [11] | Kaidanovich-Beilin O, Lipina T, Vukobradovic I, et al. Assessment of social interaction behaviors[J]. J Vis Exp, 2011(48):e2473. |
| [12] | Seibenhener ML, Wooten MC. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice[J]. J Vis Exp, 2015(96):e52434. |
| [13] | Harris KM, Jensen FE, Tsao B. Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: Implications for the maturation of synaptic physiology and long-term potentiation[J]. J Neurosci, 1992, 12(7):2685-2705. |
| [14] | Bader PL, Faizi M, Kim LH, et al. Mouse model of Timothy syndrome recapitulates triad of autistic traits[J]. Proc Natl Acad Sci USA, 2011, 108(37):15432-15437. |
| [15] | Iossifov I, O’roak BJ, Sanders SJ, et al. The contribution of de novo coding mutations to autism spectrum disorder[J]. Nature, 2014, 515(7526):216-221. |
| [16] | D’gama AM, Pochareddy S, Li M, et al. Targeted DNA sequencing from autism spectrum disorder brains implicates multiple genetic mechanisms[J]. Neuron, 2015, 88(5):910-917. |
| [17] | Lee YH, Yamrom B, Wigler M, et al. Low load for disruptive mutations in autism genes and their biased transmission[J]. Proc Natl Acad Sci USA, 2015, 112(41):E5600-E5607. |
| [18] | Takata A, Miyake N, Tsurusaki Y, et al. Integrative analyses of de novo mutations provide deeper biological insights into autism spectrum disorder[J]. Cell Rep, 2018, 22(3):734-747. |
| [19] | Gangarossa G, Laffray S, Bourinet E, et al. T-type calcium channel Cav3.2 deficient mice show elevated anxiety, impaired memory and reduced sensitivity to psychostimulants[J]. Front Behav Neurosci, 2014, 8:92. |
| [20] | Tao J, Hildebrand ME, Liao P, et al. Activation of corticotropin-releasing factor receptor 1 selectively inhibits Cav3.2 T-type calcium channels[J]. Mol Pharmacol, 2008, 73(6):1596-1609. |
| [21] | Henbid MT, Marks WN, Collins MJ, et al. Sociability impairments in genetic absence epilepsy rats from Strasbourg: Reversal by the T-type calcium channel antagonist Z944[J]. Exp Neurol, 2017, 296:16-22. |
| [22] | Chen CC, Shen JW, Chung NC, et al. Retrieval of context-asso-ciated memory is dependent on the Ca(v)3.2 T-type calcium channel[J]. PLoS One, 2012, 7(1):e29384. |
| [23] | Bauman M, Kemper TL. Histoanatomic observations of the brain in early infantile autism[J]. Neurology, 1985, 35(6):866-874. |
| [24] | Courchesne E, Mouton PR, Calhoun ME, et al. Neuron number and size in prefrontal cortex of children with autism[J]. Jama, 2011, 306(18):2001-2010. |
| [25] | Chemin J, Nargeot J, Lory P. Neuronal T-type alpha 1H calcium channels induce neuritogenesis and expression of high-voltage-activated calcium channels in the NG108-15 cell line[J]. J Neurosci, 2002, 22(16):6856-6862. |
| [26] | Cai Y, Tang X, Chen X, et al. Liver X receptor beta regulates the development of the dentate gyrus and autistic-like behavior in the mouse[J]. Proc Natl Acad Sci USA, 2018, 115(12):E2725-E2733. |
| [27] | Ito H, Morishita R, Nagata KI. Autism spectrum disorder-asso-ciated genes and the development of dentate granule cells[J]. Med Mol Morphol, 2017, 50(3):123-129. |
| [28] | Bernal Sierra YA, Haseleu J, Kozlenkov A, et al. Genetic tracing of Cav3.2 T-type calcium channel expression in the peripheral nervous system[J]. Front Mol Neurosci, 2017, 10:70. |
| [29] | Martínez-Cerdeño V. Dendrite and spine modifications in autism and related neurodevelopmental disorders in patients and animal models[J]. Dev Neurobiol, 2017, 77(4):393-404. |
| [30] | Katsarou AM, Galanopoulou AS, Moshe SL. Epileptogenesis in neonatal brain[J]. Semin Fetal Neonatal Med, 2018, 23(3):159-167. |
| [31] | Aguado C, Garcia-Madrona S, Gil-Minguez M, et al. Ontogenic changes and differential localization of T-type Ca(2+) channel subunits Cav3.1 and Cav3.2 in mouse hippocampus and cerebellum[J]. Front Neuroanat, 2016, 10:83. |
| [32] | Huang IY, Hsu YL, Chen CC, et al. Excavatolide-B enhances contextual memory retrieval via repressing the delayed rectifier potassium current in the hippocampus[J]. Mar Drugs, 2018, 16(11):405. |
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