食欲素与睡眠的实验研究
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摘要
1998年，两个实验室独立发现了食欲素A和B(orexin A和B，又称为hypocretin 1和2)。近年来的研究已经证实食欲素主要参与睡眠的调节。尤其与觉醒有明显的关系。首先发现食欲素受体—2突变的狗表现为类似人的发作性睡病，其次，剔除食欲素基因的小鼠也表现出发作性睡病。合成食欲素的神经元胞体位于外侧下丘脑。Orexing A和B均来自前食欲素原(prepro-orexin)。对于外侧下丘脑区的食欲素原是否有生理节律的变化，以及睡眠剥夺对其的影响目前有不同的报道。Fos基因作为觉醒的一个标记已经广泛用于神经生物学的研究。我们通过免疫组化的方法，通过观察orexin神经元的fos表达来进一步了解orexin与觉醒的关系。
我们用RT-PCR检测了大鼠在正常睡眠觉醒情况下以及4、8、12和24小时睡眠剥夺后，下丘脑和大脑皮层前食欲素原mRNA的表达。在正常睡眠觉醒情况下，下丘脑前食欲素原mRNA表达量，在光亮期(安静期)为：0:00，O.835±0.045；4:00，0.349±0.009；8:00，0.332±0.011；12:00，0.357±0.004；光暗期(活动期)为：16:00，0.406±0.012；20:00，0.694±0.027：24:00，0.835±0.045。而大脑皮层前食欲素原mRNA表达量，在光亮期(安静期)为：0:00，0.019±0.001；4:00，0.014±0.000：8:00，
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0.025士0.012;12:00，0.021士0.003;光暗期(活动期)为:
16:00，0 .0 16士0.007;20:00，0.031士0.004;24:00，
0.019士0.001。下丘脑前食欲素原mRNA表达量明显高于大脑皮层
并呈现出昼夜节律的变化，在光暗期(活动期)下丘脑前食欲素
原mRNA表达量要明显高于光亮期(安静期)。而大脑皮层没有类
似的变化。睡眠剥夺4小时后下丘脑和大脑皮层前食欲素原mRNA
表达量没有明显变化，睡眠剥夺前与4h睡眠剥夺后比较表达量
为:下丘脑0.349士0.009比0.398士0.034;大脑皮层
0.014士0.000比0.022士0.009。随着睡眠剥夺时间的延长，表达
逐渐增加。剥夺睡眠8h:下丘脑0.518士0.043，大脑皮层
0.197士0.027;剥夺睡眠12h:下丘脑0.636士0.047，大脑皮层
0.184士0.033;剥夺睡眠24h:下丘脑0.827士0.042，大脑皮层
0.366士0.045。与睡眠剥夺前相比较P<0.001。睡眠剥夺以后，
下丘脑和大脑皮层的前食欲素原mRNA表达量均有明显的增高。
另外，我们用免疫组化的方法观察了正常睡眠觉醒情况下和
24小时全面睡眠剥夺后大鼠下丘脑食欲素能神经元表达FoS的变
化。光亮期和光暗期大鼠下丘脑区表达FoS的食欲素能神经元数
目有明显差异，光亮期为13士1.9，而光暗期为172.7士19.6。全
面睡眠剥夺48小时后大鼠下丘脑区表达FoS的食欲素能神经元的
数目为215.8士n.0，与光亮期和光暗期相比有明显差异
(P<0.001)。在光暗期，orx+FoS+神经元占总的食欲素能神经元
的69%，在光亮期占23%，而在全面睡眠剥夺48小时的大鼠要
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高达77%。在光暗期(活动期)，大鼠下丘脑区表达FoS的食欲
素能神经元明显多于光亮期(安静期)，而24小时全面睡眠剥夺
后大鼠下丘脑表达FoS的神经元要明显多于光暗期和光亮期。结
果提示下丘脑的食欲素能神经元对觉醒有促进作用;在睡眠剥夺
期间为仍然维持长时间的觉醒状态，需要更多的食欲素能神经元
的活动。表现为FoS表达的神经元明显增多。
通过以上的实验结果可以证实，下丘脑是睡眠调节的中枢部
位，下丘脑食欲素的分泌控制着正常的睡眠一觉醒周期，并发现
了大脑皮层没有明显的节律变化。食欲素的作用是维持觉醒状态，
因此，在剥夺睡眠时为了保持长时间的觉醒状态，有更多的食欲
素能神经元活动;需要机体增加食欲素分泌。
通过本组实验的研究为进一步开展发作性睡病的研究打下基
础。
In 1998, two independent group discovered the hypocretin-1 and -2 (also referred to as orexin-A and -B). Several findings from past year have suggested that orexin might play a role in sleep regulation: first, a mutation of the orexin recaptor-2 gene has been found in narcoleptic dogs. In addition, orexin knockout mice (lacking orexin peptides) exhibit a phenotype that is strikingly similar to human narcolepsy patients. Orexins are synthesized by neurons whose somas are restricted to the lateral hypothalamus.
The expression of prepro-orexin mRNA was exzamined by RT-PCR in the hypothalamus and cerebral cortex of rat during the sleep-waking cycle and after 4, 8, 12 and 24 hours sleep deprivation. During the sleep-waking cycle, the expression of prepro-orexin mRNA in hypothalamus is higher than that in cerebral cortex, and there was a diurnal fluctuation. After 4 hours of sleep deprivation, there was no change of the expression of prepro-orexin mRNA in hypothalamus and cerebral cortex. As the prolong of time in sleep deprivation, the expression of prepro-orexin mRNA elevated gradually in
hypothalamus and cerebral cortex.
In addition, hypothalamic neurons that contain hypocreti(orexin) express c-fos were observed by immunohistochemical technique duringactive wakefulness and 48h total sleep deprivation. The majority of hypocretinergic neuron expressed c-fos during darkness. Only a small number of these cells expressed c-fos during lightness. The maximum of these cells expressed c-fos during 48h total sleep deprivation. The results shows hyporexinergic neurons in hypthalamus facilitate the wakefulness. In order to maintenance wakefulness, more hypocretinergic neuron activated. This is one of mechanisms of orexin in sleep regulation.
It is concluded the hypothalamus is a central of the regulation of sleep and wakefulness. Orexin neurotransmitter system controls the sleep-waking clcle. There was no diurnal fluctuation of orexin in cerebral cortex. The role of orexin is maintenance of wakefulness. Therefore, the secretion of orexin increased to maintain wakefulness.
我们用RT-PCR检测了大鼠在正常睡眠觉醒情况下以及4、8、12和24小时睡眠剥夺后，下丘脑和大脑皮层前食欲素原mRNA的表达。在正常睡眠觉醒情况下，下丘脑前食欲素原mRNA表达量，在光亮期(安静期)为：0:00，O.835±0.045；4:00，0.349±0.009；8:00，0.332±0.011；12:00，0.357±0.004；光暗期(活动期)为：16:00，0.406±0.012；20:00，0.694±0.027：24:00，0.835±0.045。而大脑皮层前食欲素原mRNA表达量，在光亮期(安静期)为：0:00，0.019±0.001；4:00，0.014±0.000：8:00，
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0.025士0.012;12:00，0.021士0.003;光暗期(活动期)为:
16:00，0 .0 16士0.007;20:00，0.031士0.004;24:00，
0.019士0.001。下丘脑前食欲素原mRNA表达量明显高于大脑皮层
并呈现出昼夜节律的变化，在光暗期(活动期)下丘脑前食欲素
原mRNA表达量要明显高于光亮期(安静期)。而大脑皮层没有类
似的变化。睡眠剥夺4小时后下丘脑和大脑皮层前食欲素原mRNA
表达量没有明显变化，睡眠剥夺前与4h睡眠剥夺后比较表达量
为:下丘脑0.349士0.009比0.398士0.034;大脑皮层
0.014士0.000比0.022士0.009。随着睡眠剥夺时间的延长，表达
逐渐增加。剥夺睡眠8h:下丘脑0.518士0.043，大脑皮层
0.197士0.027;剥夺睡眠12h:下丘脑0.636士0.047，大脑皮层
0.184士0.033;剥夺睡眠24h:下丘脑0.827士0.042，大脑皮层
0.366士0.045。与睡眠剥夺前相比较P<0.001。睡眠剥夺以后，
下丘脑和大脑皮层的前食欲素原mRNA表达量均有明显的增高。
另外，我们用免疫组化的方法观察了正常睡眠觉醒情况下和
24小时全面睡眠剥夺后大鼠下丘脑食欲素能神经元表达FoS的变
化。光亮期和光暗期大鼠下丘脑区表达FoS的食欲素能神经元数
目有明显差异，光亮期为13士1.9，而光暗期为172.7士19.6。全
面睡眠剥夺48小时后大鼠下丘脑区表达FoS的食欲素能神经元的
数目为215.8士n.0，与光亮期和光暗期相比有明显差异
(P<0.001)。在光暗期，orx+FoS+神经元占总的食欲素能神经元
的69%，在光亮期占23%，而在全面睡眠剥夺48小时的大鼠要
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高达77%。在光暗期(活动期)，大鼠下丘脑区表达FoS的食欲
素能神经元明显多于光亮期(安静期)，而24小时全面睡眠剥夺
后大鼠下丘脑表达FoS的神经元要明显多于光暗期和光亮期。结
果提示下丘脑的食欲素能神经元对觉醒有促进作用;在睡眠剥夺
期间为仍然维持长时间的觉醒状态，需要更多的食欲素能神经元
的活动。表现为FoS表达的神经元明显增多。
通过以上的实验结果可以证实，下丘脑是睡眠调节的中枢部
位，下丘脑食欲素的分泌控制着正常的睡眠一觉醒周期，并发现
了大脑皮层没有明显的节律变化。食欲素的作用是维持觉醒状态，
因此，在剥夺睡眠时为了保持长时间的觉醒状态，有更多的食欲
素能神经元活动;需要机体增加食欲素分泌。
通过本组实验的研究为进一步开展发作性睡病的研究打下基
础。
In 1998, two independent group discovered the hypocretin-1 and -2 (also referred to as orexin-A and -B). Several findings from past year have suggested that orexin might play a role in sleep regulation: first, a mutation of the orexin recaptor-2 gene has been found in narcoleptic dogs. In addition, orexin knockout mice (lacking orexin peptides) exhibit a phenotype that is strikingly similar to human narcolepsy patients. Orexins are synthesized by neurons whose somas are restricted to the lateral hypothalamus.
The expression of prepro-orexin mRNA was exzamined by RT-PCR in the hypothalamus and cerebral cortex of rat during the sleep-waking cycle and after 4, 8, 12 and 24 hours sleep deprivation. During the sleep-waking cycle, the expression of prepro-orexin mRNA in hypothalamus is higher than that in cerebral cortex, and there was a diurnal fluctuation. After 4 hours of sleep deprivation, there was no change of the expression of prepro-orexin mRNA in hypothalamus and cerebral cortex. As the prolong of time in sleep deprivation, the expression of prepro-orexin mRNA elevated gradually in
hypothalamus and cerebral cortex.
In addition, hypothalamic neurons that contain hypocreti(orexin) express c-fos were observed by immunohistochemical technique duringactive wakefulness and 48h total sleep deprivation. The majority of hypocretinergic neuron expressed c-fos during darkness. Only a small number of these cells expressed c-fos during lightness. The maximum of these cells expressed c-fos during 48h total sleep deprivation. The results shows hyporexinergic neurons in hypthalamus facilitate the wakefulness. In order to maintenance wakefulness, more hypocretinergic neuron activated. This is one of mechanisms of orexin in sleep regulation.
It is concluded the hypothalamus is a central of the regulation of sleep and wakefulness. Orexin neurotransmitter system controls the sleep-waking clcle. There was no diurnal fluctuation of orexin in cerebral cortex. The role of orexin is maintenance of wakefulness. Therefore, the secretion of orexin increased to maintain wakefulness.
引文
1. Sakurai T, Amamiya A, Ishii M, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulating feeding behavior. Cell, 1998, 92: 573-585
2. Wijker M, Wszolek ZK, Wolter ECH, et al. Localization of the gene for rapidly progressive autosomal dominant parkinsonism and dementia with pallidoponto-nigral degeneration to chromosome 17q21. Hum Mol Genet, 1996, 5: 151-154
3. Taheri S, Sunter D, Dakin C, et al. Diurnal variation in orexin A immunoreactivity and prepro-orexin mRNA in the rat central nervous system. Neurosci Lett 2000: 279: 109-12
4. Yoshida Y, Fujiki N, Nakajima T, et al. Fluctuation of extracellular hypocretin-1 (orexin A) levels in the rat in relation to the light-dark cycle and sleep-wake activities. Eur J Neurosci 2001: 14: 1075-81
5. Lin L, Wisor J, Shiba T, et al. Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background. Peptides 2002: 23: 2203-11
6. Piper DC, Upton N, Smith MI, et al. The novel brain neuropeptide, orexin-A, modulates the sleep-wake cycle of rats. Eur J Neurosci 2000; 12: 726-30
7. Novak CM, Albers HE. Localization of hypocretin-like immunoreactivity in the brain of the diurnal rodent, Arvicanthis niloticus. J them Neuroanat 2002; 23: 49-58
8. Espana RA, Baldo BA, Kelley AE, et al. Wake-promoting and sleep-suppressing actions of hypocretin (orexin): basal forebrain sites of action. Neuroscience 2001; 106: 699-715
9. Terao A, Peyron C, Ding J, et al Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice. Sleep 2000; 23: 867-74
10. Estabrooke IV, McCarthy MT, Ko E, et al. Fos expression in orexin neurons varies with behavioral state. J Neurosci 2001; 21: 1656-62
11. Taheri S, Sunter D, Dakin C, et al Diurnal variation in orexin A immunoreactivity and prepro-orexin mRNA in the rat central nervous system. Neurosci Lett 2000 Jan 28; 279(2): 109-12
12. Yoshida Y, Fujiki N, Nakajima T, et al Fluctuation of extracellular hypocretin-1 (orexin A) levels in the rat in relation to the light-dark cycle and sleep-wake activities. Eur J Neurosci 2001 Oct; 14(7): 1075-81
13. Lin L, Wisor J, Shiba T, et al. Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background. Peptides 2002 Dec; 23(12): 2203-11
2. Wijker M, Wszolek ZK, Wolter ECH, et al. Localization of the gene for rapidly progressive autosomal dominant parkinsonism and dementia with pallidoponto-nigral degeneration to chromosome 17q21. Hum Mol Genet, 1996, 5: 151-154
3. Taheri S, Sunter D, Dakin C, et al. Diurnal variation in orexin A immunoreactivity and prepro-orexin mRNA in the rat central nervous system. Neurosci Lett 2000: 279: 109-12
4. Yoshida Y, Fujiki N, Nakajima T, et al. Fluctuation of extracellular hypocretin-1 (orexin A) levels in the rat in relation to the light-dark cycle and sleep-wake activities. Eur J Neurosci 2001: 14: 1075-81
5. Lin L, Wisor J, Shiba T, et al. Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background. Peptides 2002: 23: 2203-11
6. Piper DC, Upton N, Smith MI, et al. The novel brain neuropeptide, orexin-A, modulates the sleep-wake cycle of rats. Eur J Neurosci 2000; 12: 726-30
7. Novak CM, Albers HE. Localization of hypocretin-like immunoreactivity in the brain of the diurnal rodent, Arvicanthis niloticus. J them Neuroanat 2002; 23: 49-58
8. Espana RA, Baldo BA, Kelley AE, et al. Wake-promoting and sleep-suppressing actions of hypocretin (orexin): basal forebrain sites of action. Neuroscience 2001; 106: 699-715
9. Terao A, Peyron C, Ding J, et al Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice. Sleep 2000; 23: 867-74
10. Estabrooke IV, McCarthy MT, Ko E, et al. Fos expression in orexin neurons varies with behavioral state. J Neurosci 2001; 21: 1656-62
11. Taheri S, Sunter D, Dakin C, et al Diurnal variation in orexin A immunoreactivity and prepro-orexin mRNA in the rat central nervous system. Neurosci Lett 2000 Jan 28; 279(2): 109-12
12. Yoshida Y, Fujiki N, Nakajima T, et al Fluctuation of extracellular hypocretin-1 (orexin A) levels in the rat in relation to the light-dark cycle and sleep-wake activities. Eur J Neurosci 2001 Oct; 14(7): 1075-81
13. Lin L, Wisor J, Shiba T, et al. Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background. Peptides 2002 Dec; 23(12): 2203-11