| Objective Fragile X syndrome(FXS),a common hereditary mental retardation disease,is due to the reduced or lack expression of coding production(fragile X mental retardation protein,FMRP),which is caused by the absence or silence of Fmr1 gene.Fmr1 KO mice with fragile X mental retardation gene,is distinguished from WT mice in behavior,cognitive,and emotion.This study was to explore the effects of light signal change on sleep-wake in Fmr1 KO mice using the stereotopic localization technology and multiple polysomnography technology,and to provide theoretical basis and practical reference for the diagnosis and treatment of FXS.Methods1 Experimental animals A total of twenty-four 10-week-old male mice(SPF grade,20~25g)was used in this study.Among these animals,Fmr1 KO C57BL/6J mice(n = 11)were provided by Zhang Chen researcher from state key laboratory of biomembrane and membrane bioengineering,college of life sciences,Peking University,China.C57BL/6J mice(n =13)were provided by the laboratory animal center of Anhui Medical University,China.According to the difference in genotype,Fmr1 KO C57BL/6J mice were as the experimental group,and C57BL/6J mice were as the control group.2 Genotyping A small amount of tail tissue was obtained from the mice on two weeks postpartum.PCR amplification was performed,and the products were identified by 1% DNA agarose gel electrophoresis.The mice,whose strips were in 131 bp,were wild type,as well as those in ~400 bp were Fmr1 KO mice compared to marker after image develop.The genetic male mice needed in this study were selected for feeding.3 Animal operation The mice were intraperitoneally anesthetized using 4% pentobarbital sodium at the dose of 0.04 ml/g.After the pain reflex fell away,the mice were fixed on the mouse stereotaxic adapter,and brain electricity electrode and muscle electricity electrode were installed successively,to record the cortex electrical activity and the nuchal muscle electrical activity.The wound was treated with penicillin powder and sutured.After sobered up,the mice were housed in the shielded boxes with the light-on time at 8:00am ~ 8:00 pm.4 Recording method The electroencephalogram(EEG)and electromyography(EMG)were recorded under12 h:12 h light-dark cycle,in 9:00 am ~ 11:00 am with the light turning on and in 9:00am ~ 11:00 am with the light turning off.Sleep-wake conditions of the mice include wakefulness(W),slow wave sleep(SWS)and fast wave sleep(FWS).5 Data processing Sleep-wake data were divided into segmentation by each 4 seconds.The experiment data were analyzed using SPSS 20.0 software,as two groups’ data were analyzed by ANOVA,and the mean value of the two samples was tested by t-test.The results were presented with mean ± SD.P < 0.05 was considered as significant difference,and P <0.01 as extremely significant difference.Graphpad Prism 5 software was used for drawing.Results1 The results of the mice genotyping Before the operation,both Fmr1 KO and WT mice were identified by PCR.The size of fragment samplified with the genome DNA of Fmr1 KO and WT homozygous mice were ~400 bp and 131 bp,respectively.Fmr1 KO and WT male mice were selected.2 In normal light condition,WT and Fmr1 KO mice had a consistent circadian rhythm phenomenon in wakefulness and sleep In 12 h:12 h light-dark cycle(the light turning on at 8:00 am and the light turning off at8:00 pm),the effects of light signal change on the total sleep time(TST)and wakefulness time in Fmr1 KO(n = 4)and WT mice(n = 6)had a consistent day-night rhythm.There was no statistically significant difference between the two groups(P >0.05).3 Effects of light off on the amount of wakefulness in WT mice during9:00 am ~11:00am The quantity of wakefulness in WT mice(n = 6)was significantly changed(P < 0.01),in the 2-hour light off period(9:00 am ~ 11:00 am),compared with the corresponding time of the normal 12 h:12 h light-dark cycle(turn on the light at 8:00 am and turn off the light at 8:00 pm).Especially in the first 1 hour after turning off the light,the amount was increased remarkably(P < 0.01).Concretely,the amount of wakefulness was increased by 386.18%(P < 0.01)in the first stage(9:00 am ~ 9:20 am),the amount of wakefulness was increased by 502.64%(P<0.01)in the second stage(9:20 am ~ 9:40am)and the amount of wakefulness increased by 77.12%(P < 0.01)in the third stage(9:40 am ~10:00 am).4Effects of light off on the amount of wakefulness in Fmr1 KO mice during9:00 am~ 11:00am The relative increase of the wakefulness in Fmr1 KO mice(n = 6)was decreased significantly compared with the variation of the WT mice in the corresponding period of light-on and light-off(P < 0.01).Concretely,the amount of wakefulness was increased by 77.23%(P < 0.05)in the first stage(9:00 am ~ 9:20 am),was increased by 205%(P< 0.01)in the second stage(9:20 am ~ 9:40 am)and increased by 76.57%(P < 0.01)in the third stage(9:40 am ~10:00 am).5 Effects of light off on the change rate of TST in WT and Fmr1 KO mice during9:00 am ~ 11:00am Compared with the corresponding period in the normal 12h:12h light-dark cycle(the light turning on at 8:00 am and the light turning off at 8:00 pm),the change rate of TST in WT and Fmr1 KO mice were both decreased in the light off period(9:00 am ~ 11:00am).During the 20 min(9:00 am ~ 9:20 am)with the light off,the change rate of TST in Fmr1 KO mice was lower than that in WT mice remarkably(P < 0.01).During other periods,both WT and Fmr1 KO mice were affected by light off.However,no significant difference was obtained in the change rate of TST between WT and Fmr1 KO mice(P > 0.05).6 Effects of light off on the number of transitions in WT mice during 9:00 am ~11:00 am Compared with the normal light period,during the light off period(9:00 am ~ 10:00 am)the transitions of SWS-W,W-SWS,and FWS-W were increased from 5.14 ± 0.51 to28.86 ± 1.77(P < 0.01),6.00 ± 0.31 to 32.86 ± 1.65(P < 0.01)and 0.86 ± 0.26 to 4.00 ±0.44(P < 0.01)in WT mice,respectively.Compared with the normal light period of the corresponding time,the transition number of SWS-W,W-SWS,and FWS-W in the WT mice were significantly weakened during the period of 10:00 am ~ 11:00 am,however there was still statistically significant(P < 0.05).7 Effects of light off on the number of transitions in Fmr1 KO mice during 9:00 am~ 11:00 am Compared with the normal light period,during the light off period(9:00 am ~ 10:00 am)the transition number of SWS-W,W-SWS,and FWS-W were increased from 5.29 ±0.36 to 15.71 ± 0.57(P < 0.01),6.29 ± 0.42 to 24.71 ± 0.68(P < 0.01)and 1.00 ± 0.31 to 9.43 ± 0.48(P < 0.01),respectively.The transition numbers of SWS-W,W-SWS,and FWS-W in Fmr1 KO mice were not statistically significant during the period of 10:00 am ~ 11:00 am compared with the normal light period at the corresponding time(P >0.05).8 Effects of light off on the change rate of stage transition in WT and Fmr1 KO mice during 9:00 am ~ 11:00am Compared with the corresponding period in the normal 12h:12h light-dark cycle(the light turning on at 8:00 am and the light turning off at 8:00 pm),during the light off period(9:00 am ~ 11:00 am)the transition number of SWS-W,W-SWS and FWS-W were all increased in both WT and Fmr1 KO mice.During the light off period in 9:00am~10:00 am the change rate of SWS-W,W-SWS and FWS-W between WT and Fmr1 KO mice were all remarkably significant difference(P < 0.01).The change rate of SWS-W in Fmr1 KO mice was significantly lower than WT mice in 10:00 am ~ 11:00am(P < 0.05).However,no difference was obtained in the change rate of W-SWS and FWS-W between WT and Fmr1 KO mice(P > 0.05).Conclusions The sleep-wake of Fmr1 KO mice was not susceptible to light signal change compared with WT mice. |
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