敲除MT3R/Nqo2基因对大鼠睡眠/觉醒行为和EEG能谱的影响

Effects of knockout of MT3R/Nqo2 gene on sleep/wake behaviors and EEG power spectrum in rats

目的利用CRISPR/Cas9基因编辑工具构建褪黑素3型受体(MT3R/Nqo2)基因敲除的SD大鼠,观察基因敲除后大鼠的睡眠/觉醒行为变化和EEG能谱改变,以探究褪黑素调控睡眠/觉醒的受体机制。方法利用非同源重组修复引入突变的方式,造成MT3R/Nqo2基因蛋白读码框移码突变,使该基因功能缺失,建立MT3R/Nqo2基因敲除的SD大鼠,并通过PCR、RT-PCR和FISH进行基因水平、转录水平和形态学的敲除验证,然后对敲除组与对照组大鼠进行睡眠/觉醒行为分析和睡眠/觉醒各时相脑电能谱分析。结果MT3R/Nqo2基因敲除后,与对照组大鼠相比,敲除组大鼠在黑暗期(活跃期)开始后的前3 h(ZT12~15)出现觉醒时长减少、NREM和REM睡眠时长增加(P<0.05)。另外,在该时期,敲除MT3R/Nqo2基因后大鼠还出现睡眠/觉醒转化次数显著增加,觉醒片段长度缩短,觉醒片段化现象(P<0.05)。脑电能谱分析显示,与对照组大鼠相比,敲除组大鼠NREM睡眠期θ波能量升高(P<0.05)。结论MT3R/Nqo2基因敲除使大鼠NREM睡眠期EEG高频能量异常升高,NREM睡眠期稳态恢复过程受损,导致大鼠在黑暗期(活跃期)开始后的前3 h(ZT12-15)觉醒减少,睡眠增加,觉醒片段化。

Objective To observe the sleep/wake behaviors and EEG power spectrum in melatonin type 3 receptor(MT3R/Nqo2)knockout rats with CRISPR/Cas9 gene editing tool in order to explore the receptor mechanism of melatonin in regulation of sleep/wake behaviors.Methods SD rats with MT3R/Nqo2 knockout were created by introducing a mutation in the MT3R/Nqo2 gene frame shift by nonhomologous recombination repair causing the gene dysfunction,and the knockout was confirmed at the genetically,transcriptionally,and morphologically by PCR,RT-PCR,and FISH,respectively.Then the changes in sleep/wake behaviors and in EEG power spectrum were observed in the knockout rats and control rats.Results After MT3R/Nqo2 knockout,the knockout rats showed decreased arousal time and increased NREM and REM sleep time during the first 3 h(ZT12~15)after the onset of the dark phase(active phase)when compared with the control rats(P<0.05).In addition,a significant increase in the number of sleep/wake transitions,a reduction in the duration of wakefulness,and appearance of wake fragmentation were also observed after MT3R/Nqo2 knockout during this period(P<0.05).EEG power spectrum analysis revealed that NREM sleepθband energy was elevated in the knockout rats than the control rats(P<0.05).Conclusion MT3R/Nqo2 knockout results in abnormal power spectrum of EEG especially high-frequency energy during NREM sleep,disrupts sleep homeostasis,leads to decreased arousal,increased sleep,and fragmented arousals in the first 3 h(ZT12~15)after the onset of the dark phase(active phase)in rats.