A Preliminary Study On The Relationship Between Cacna1h Gene And Autism Spectrum Disorder

Objective:Autism spectrum disorder(ASD)is a common neurodevelopmental disorder.The etiology of ASD is complicated and the pathogenesis is not clear.Up to now,there is no specific drug to treat ASD,which is mainly focused on behavioral and educational interventions.Cacna1h encoding T-type calcium channel 3.2 subtype(Ca_v3.2).Clinical studies found CACNA1H mutation in some autistic patients,but lack of biological evidence and validation of animal models.Environmental enrichment(EE)has been confirmed to be able to stimulate the generation of brain synapses through physical and social environments,thereby promoting increased brain activity and improving social and emotional abnormalities in autistic patients.To further clarify the relationship between Cacna1h and ASD,we used the Cacna1h gene knockout(KO)mice to explore their ASD-like behavior and the underlying mechanisms.The effects and mechanisms of EE intervention were further explored.Methods:1.Fifty-six wild type(WT)and 53 KO juvenile mice,62 WT and 52 KO adult mice,5 juvenile and adult WT(Thy1~+)and KO(Thy1~+)mice were randomly selected.With WT as the control group and KO as the experimental group,the following experiments were conducted.(1)Behavioral observations:(1)The three-chamber test was used to assess the sociability and the social novelty recognition of mice;(2)The Y-maze test was used to evaluate the spatial working memory ability of mice;(3)The open field test was used to analyze the anxiety,restricted and repetitive behavior and spontaneous locomotor activity of mice;(4)The forced swimming test was used to evaluate the depression-like behavior of mice.(2)After the behavioral experiment,the body weight,brain weight and brain size of the partial mice in the same litter were measured,and the number of brain neurons were observed by Nissl staining.(3)The dendritic spines of WT(Thy1~+)and KO(Thy1~+)mice were observed.2.Eighteen WT and 24 KO mice,5 WT(Thy1~+)and 5 KO(Thy1~+)mice were recruited at 21 days after birth and were put into EE cages until 56 days after birth.The effects of EE intervention on mice were evaluated by standard cage(STD)feeding as the control group and EE feeding as the experimental group.Results:1.(1)The three-chamber test:compared with WT mice,no significant difference was observed in the exploration preference of juvenile(0.62±0.07 vs 0.50±0.10)and adult KO(0.46±0.07 vs 0.47±0.05)mice to strangers in the sociability test session;but the exploration preferences of juvenile(0.52±0.05 vs 0.15±0.15)and adult(0.37±0.07 vs 0.17±0.07)KO mice to new strangers were significantly reduced in the social novelty recognition test session.(2)The Y-maze test:compared with WT mice,no significant difference was observed in the spontaneous alternations of juvenile(0.57±0.02 vs 0.51±0.04)and adult KO(0.56±0.02 vs 0.59±0.03)mice,nor was any significant difference observed in the number of arm entries of juvenile KO mice(26±2 times for WT vs 25±3 times for KO).However,the number of arm entries was significantly increased in adult KO(41±3 times)mice compared to that of the WT mice(28±2 times).(3)The open field test:compared with WT mice,juvenile and adult KO mice spent less time in the center of the open field(juvenile:53.87±6.01 s vs 36.30±3.63 s;adult 67.51±6.92 s vs 46.93±4.12s),but the self-grooming time(juvenile:69.86±13.53 s vs 131.66±24.92 s;adult17.52±5.18 s vs 46.06±6.02 s)and the total distance of motion(juvenile:12 837.82±687.70 cm vs 15 304.49±769.02 cm;adult 15 779.61±721.08 cm vs 19 020.03±1449.29 cm)were significantly increased.(4)The forced swimming test:compared with WT mice,there was no significant difference in the immobility time of juvenile(0.19±0.19 s vs 0.67±0.45 s)and adult KO(32.06±12.87 s vs 29.83±16.70 s)mice in the swimming tank.2.There were no significant differences among the body weight(juvenile:7.05±0.97 g vs 7.43±0.99 g;adult:16.64±0.66 g vs 18.56±0.93 g),brain weight(juvenile:0.06±0.00 vs 0.06±0.01;adult:0.03±0.00 vs 0.02±0.00)and brain size(juvenile:0.39±0.00 mL vs 0.40±0.01 mL;adult:0.43±0.02 mL vs 0.43±0.03mL)in juvenile and adult KO mice compared with that of the WT mice with the same age.Nissl staining:the number of neurons in the medial prefrontal cortex(mPFC)(theⅡ/Ⅲlayers:1.00±0.03 vs 0.87±0.03;theⅤlayers:0.98±0.04 vs 0.89±0.05;theⅥlayers:1.00±0.05 vs 0.84±0.05),the dentate gyrus(DG)(1.00±0.03 vs 0.86±0.05)and the basolateral amygdala(BLA)(1.00±0.06 vs 0.82±0.04)of juvenile KO mice were lower than that of the WT mice with the same age,without any significant difference between adult KO and WT mice(theⅡ/Ⅲlayers of mPFC:1.00±0.12 vs 0.98±0.06;theⅤlayers of mPFC:1.15±0.10 vs 1.09±0.09;theⅥlayers of mPFC:1.00±0.06 vs 0.91±0.06;DG:1.00±0.03 vs 0.99±0.05;BLA:1.00±0.08 vs 0.98±0.05).3.The density of dendritic spine:the spine density in the DG of juvenile KO mice(8.32±0.20 vs 10.03±0.69),and in the mPFC(9.88±0.24 vs 12.76±0.43),the DG(9.83±0.24 vs 11.50±0.71)and the BLA(10.65±0.32 vs 12.52±0.68)of adult KO mice were higher than that of WT mice with the same age.Maturity of dendritic spines:no significant difference was observed between the WT and KO juvenile(mPFC:37.80±2.48 vs 40.40±2.50;DG:34.40±1.03 vs 39.60±2.48;BLA:36.20±1.62 vs 39.00±2.70)and adult(mPFC:57.20±3.81 vs 68.80±4.07;DG:53.40±3.50 vs 62.50±5.39;BLA:59.00±3.61 vs 64.00±3.99)mice.4.Behavioral test after EE intervention:in the three-chamber test,EE significantly increased the exploration preference of KO mice to both stranger mice and new stranger mice(0.47±0.05 vs 0.64±0.06;0.36±0.08 vs 0.40±0.07).In the open field test,after EE intervention,no significant change was found in the center time of the open field of KO mice(46.93±4.12 s vs 43.76±8.52 s),which was significantly decreased(67.51±6.92 s vs 44.82±8.72 s)in WT mice.In addition,there were no significant changes in the self-grooming time(46.06±6.02 s vs 43.67±12.11 s)and the total distance of motion(19 020.03±1449.29 cm vs 18 404.39±1494.10 cm)in KO mice compared with that of the WT mice.5.Dendritic spine density after EE intervention:no significant changes were observed in the mPFC(12.76±0.43 vs 12.14±1.07)and the BLA(12.52±0.68 vs13.60±0.50)of KO mice.However,EE significantly increased the spine density of WT mice(mPFC:9.88±0.24 vs 11.30±0.36);BLA:10.65±0.32 vs 13.04±0.63).Moreover,no significant change was observed in the DG of the two genotypes of mice(WT:9.83±0.24 vs 10.88±1.10;KO:11.50±0.71 vs 11.38±0.60);Dendritic spine maturity:it was increased in the mPFC of KO mice by EE(68.80±4.07 vs70.00±3.51),without any significant changes in the DG(62.50±5.39 vs 64.00±2.98)and the BLA(64.00±3.99 vs 62.20±5.18).Conclusions:Our study confirmed that the deletion of Cacna1h gene induced autistic-like behavior in mice,which may be related to the decrease of the number of neurons and the increase of dendritic spine density in the mPFC,the DG and the BLA.EE intervention can improve partial autistic-like behavior of KO mice,which may be due to the increased maturity of dendritic spine in mPFC.These data may promote the understanding of the genetic mechanisms of ASD and provide a theoretical basis for drug development targeting the Cacna1h gene.