GABA受体激动剂消除NO合成酶抑制剂对大鼠睡/醒周期的影响
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摘要
20世纪80年代末一氧化氮(NO)生物学作用的发现告诉我们一个全新的事实：一个细胞产生的气体信号可透过细胞膜调节另一个细胞的功能。此后，NO的生物学作用受到人们越来越多的关注。体内的NO是由NO合成酶(NOS)催化L-精氨酸而产生的，是一种结构简单、易扩散的高脂溶性气体物质。它在局部产生后，可扩散到邻近细胞而发挥细胞间信使的作用。NOS在中枢神经系统中分布广泛，与睡眠有关的部位，如前脑基底部、脑桥背盖、中缝背核、嗅球和海马等均有较高密度的NOS，说明这些部位可以产生NO。由此推测内源性NO可能与睡/醒周期调控有关。在这一方面，Kapás和Krueger等人做了许多工作。他们发现，应用NOS抑制剂减少脑内NO的合成，能够引起大鼠和家兔的睡眠抑制效应；应用NO供体则延长动物的慢波睡眠。这提示NO具有促进睡眠的作用。另有研究发现，大鼠皮下注射NOS抑制剂L-NAME(N~G-nitro-L-arginine methylester)，引起觉醒增加，慢波睡眠和快眼动睡眠减少，并随剂量的增加其作用愈明显。同时发现，经侧脑室注射较小剂量就能发挥作用，而经外周用药，则需较大剂量(20倍于前者)才能显示作用，推测可能外周大剂量注射L-NAME后，其通过血脑屏障的量相对较多，L-NAME在中枢抑制NOS而发挥作用，说明L-NAME抑制睡眠的作用是中枢性的，并且呈剂量相关关系。Dzoljic在实验中应用NOS的另一抑制剂7-nitro indazole(7-NI)，发现7-NI也有睡眠抑制效应，这些都支持了NO促进睡眠的观点。
郑州大学2003届硕士毕业论文
GABA受体激动剂消除NO合成酶抑制剂对大鼠睡/醒周期的影响
睡/醒周期的形成是一个复杂的网络调控的结果，体内许多因子都参与了这一
调控网络，这些因子如白介素一1(IL一1)、肿瘤坏死因子(TNF)、生长激素释放激
素(GHRH)、血管活性肠肤(VIP)以及经典的神经递质如5一轻色氨(5一HT)、
乙酞胆碱(ACh)、去甲肾上腺素(NE)、多巴胺(DA)和卜氨基丁酸(GABA)
等，它们在睡眠的发生和调节中也发挥着重要作用。近年来的研究表明，NO在
中枢发挥信使作用，可通过调节神经递质的释放而实现。组织学研究也发现，大
鼠部分脑区内NOS与经典神经递质如5~HT、ACh、GABA等共存，提示NO与
这些递质之间具有广泛的相互调节作用。在体或离体的一些研究表明，应用NO
供体后，大鼠脑内GABA的基础释放量增加，而应用NOS抑制剂后则发生相反
的变化。这提示改变脑内NO的含量可引起脑内GABA释放量的改变。由此推测
系统应用或侧脑室局部应用NOS抑制剂引起的睡眠抑制效应可能与脑内GABA
的释放量的减少有关。这方面的研究少有报道。本研究采用多导睡眠描记技术和
皮下给药方式，观察应用GABA受体激动剂后，对NOS抑制剂引起的睡眠抑制
效应有无影响，同时用免疫组化的方法，观察应用NOS抑制剂后大鼠额叶皮质2
区(FrZ区)GABA免疫反应(GABA一IR)阳性细胞表达的变化，从而探讨NO
和GABA在睡一醒周期调控中的作用。
本实验采用健康成年雄性SD大鼠，体重2509士209。36只大鼠随机分为6
组，每组6只，分别为:1.对照组;2.L一NAME组;3.muscimol组;4.baclofen
组;5.muc加of+L一NAME组;6.bacfofen+L一NAME组。动物常规安装记录电极，
术后恢复5天，于第6天行睡眠描记。实验日和对照日分别经皮下注射药物和同
体积生理盐水，注射完毕即行睡眠描记，连续记录6小时(11:00一17:oo)睡眠
多导图，进行睡眠分析。并在睡眠描记结束后，取1和2组动物，灌注取脑，石
蜡包埋，冠状切片，片厚4协m，做免疫组化染色，观察大鼠FrZ区GABA一IR
阳性细胞数，并进行统计学处理。
结果显示:1.L~NAME组大鼠的慢波睡眠(S WS)和快眼动睡眠(REMS)
明显减少(尸<0.01，尸<0 .05)，SWS潜伏期明显延长(尸<0.01)，REMS出现次数
无显著变化(尸，0.05)。2.musclinol组和baclofen组大鼠的SWS和REMS出现
次数无显著变化(P>0.05)，而REMS减少伊<0 .05)，Sws潜伏期则明显延长
(P<0.01)。3.预先应用museimol或baclofen，再用L一NAME后，与L一NAME单
郑州大学2003届硕士毕业论文
GABA受体激动剂消除NO合成酶抑制剂对大鼠喻醒周期的影响
独作用时相比，大鼠的SWS(P<0.01)增加，SWS潜伏期也较L一NAME单独作
用时缩短(尸<0 .01)，而对L.NAME引起的REMS及其出现次数的减少并无明显
影响(P>0.05)。4.免疫组化染色显示，L.NAME组大鼠FrZ区GABA-IR阳性细
胞表达较对照组明显减少(P<0 .01)。
结论:1.应用NOS抑制剂，可明显增加大鼠的觉醒(W)，抑制SWS和REMS。
2.应用GABA受体激动剂能够抑制大鼠REMS，而SWS不受影响。3.预先应用
GABA受体激动剂后，能够消除NOS抑制剂单独作用时对大鼠SWS的抑制效应。
4.应用NOS抑制剂后，大鼠FrZ区GABA-IR阳性细胞表达明显减少。
Since the first suggestion by Garthwait et al. in 1980s that the free radical nitric oxide (NO) might play a role in the brain of mammals, more and more people have paid close attention to the biological functions of NO. NO is an intercellular messenger synthesized from L-arginine by NO synthase (NOS). As a liposoluble gas, NO can affect other tissues by diffusion after produced in local tissue in the brain. Neuronal cells containing NOS have been found in basal forebrain, pedunculopontine tegmental nuclei, dorsal raphe nuclei, olfactory bulb and hippocampus which have been proved to participate in the regulation of sleep/waking cycle. Kapds and Krueger found that NOS inhibitors reducing the production of NO in the brain suppressed both slow wave sleep (SWS) and rapid eye movement sleep (REMS) in rats and rabbits, but NO donors promoted their SWS. These results suggest NO can promote sleep. Administration of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) to rabbits or rats by s.c., i.v. or Lc.v. suppressed both SWS and REMS but increased W, and the effects showed dose dependence. Moreover, the relatively smaller dose of icv injected L-NAME suppressed sleep and a higher systemically admistered dose was effective (20 times more than the former), which suggested that the site of the sleep-suppressing action of L-NAME is central. In the same time, Dzoljic studied the NOS inhibitor 7-nitro indazole (7-NI) and found that 7-NI decreased the duration of SWS and REMS. All of these support that NO can promote sleep.
Sleep/waking cycle is a complex network modulation and many factors such as
interleukin-1 (IL-1), tumor necrosis factor (TNF), growth hormone releasing hormone (GHRH), vasoactive intestina polypeptide (VIP) and many conventional neurotransmitters such as serotonin (5-HT), acetylcholine (ACh), norepinephrine (NE), dopamine (DA) and gamma-aminobutyric acid (GABA) were involved in it. Recent evidence has shown that NO synthesized in neurons in several areas of the brain can induce the release of neurotransmitters. In the rat central nervous system, the anatomical distribution of NOS -containing neurons is now well established, and it was reported that NOS is co-localized with neurotransmitters well known for their involvement in sleep mechanisms, i.e. 5-HT, ACh, DA and GABA. In vivo or in vitro, NO donor facilitated the release of GABA, while NOS inhibitor inhibited it, which indicated that the release of GABA changed with NO in the brain. So we can suppose that the sleep suppression resulting from administration of the NOS inhibitor by s.c. or i.c.v. be probably related with the change of GABA releasing in the brain. In order to testify the effect of NOS inhibitor and GABA on the regulation of the sleep/waking cycle in adult rats implanted for chronic sleep recordings, the polysomnogram was recorded. We observed whether the sleep-suppressing action of NOS inhibitor was affected after the administration of the agonists of GABA receptors by subcutaneous route.
Experiments were performed on adult male Sprague-Dawley rats weighing 230-270g. The rats were divided into six groups by random and each had 6 ones, they are: l.The control group, 2.The L-NAME group, S.The muscimol group, 4.The baclofen group, S.The muscimol + L-NAME group, 6.The baclofen + L-NAME group. Animals were implanted for chronic sleep recordings. After five-day recovery postoperatively the animals were given either drugs or control solution subcutaneously on the sixth day, then the six-hour electroencephalogram (fome 11:00 to 17:00) was obtained. At the end of recording, the animals of group 1 and group 2 were perfused with paraformaldehyde, then removed the brain and fixed for 24 hours with paraformaldehyde, paraffin sections were prepared at 4 u m, histochemical staining for counting the GABA positive neurons in the frontal cortex (Fr2).
Results: 1. In the group 2, following the administration of L-NAME SWS and
REMS were significantly reduced (P<0.01, P<0.05), while the SWS latency showed a significant increment
郑州大学2003届硕士毕业论文
GABA受体激动剂消除NO合成酶抑制剂对大鼠睡/醒周期的影响
睡/醒周期的形成是一个复杂的网络调控的结果，体内许多因子都参与了这一
调控网络，这些因子如白介素一1(IL一1)、肿瘤坏死因子(TNF)、生长激素释放激
素(GHRH)、血管活性肠肤(VIP)以及经典的神经递质如5一轻色氨(5一HT)、
乙酞胆碱(ACh)、去甲肾上腺素(NE)、多巴胺(DA)和卜氨基丁酸(GABA)
等，它们在睡眠的发生和调节中也发挥着重要作用。近年来的研究表明，NO在
中枢发挥信使作用，可通过调节神经递质的释放而实现。组织学研究也发现，大
鼠部分脑区内NOS与经典神经递质如5~HT、ACh、GABA等共存，提示NO与
这些递质之间具有广泛的相互调节作用。在体或离体的一些研究表明，应用NO
供体后，大鼠脑内GABA的基础释放量增加，而应用NOS抑制剂后则发生相反
的变化。这提示改变脑内NO的含量可引起脑内GABA释放量的改变。由此推测
系统应用或侧脑室局部应用NOS抑制剂引起的睡眠抑制效应可能与脑内GABA
的释放量的减少有关。这方面的研究少有报道。本研究采用多导睡眠描记技术和
皮下给药方式，观察应用GABA受体激动剂后，对NOS抑制剂引起的睡眠抑制
效应有无影响，同时用免疫组化的方法，观察应用NOS抑制剂后大鼠额叶皮质2
区(FrZ区)GABA免疫反应(GABA一IR)阳性细胞表达的变化，从而探讨NO
和GABA在睡一醒周期调控中的作用。
本实验采用健康成年雄性SD大鼠，体重2509士209。36只大鼠随机分为6
组，每组6只，分别为:1.对照组;2.L一NAME组;3.muscimol组;4.baclofen
组;5.muc加of+L一NAME组;6.bacfofen+L一NAME组。动物常规安装记录电极，
术后恢复5天，于第6天行睡眠描记。实验日和对照日分别经皮下注射药物和同
体积生理盐水，注射完毕即行睡眠描记，连续记录6小时(11:00一17:oo)睡眠
多导图，进行睡眠分析。并在睡眠描记结束后，取1和2组动物，灌注取脑，石
蜡包埋，冠状切片，片厚4协m，做免疫组化染色，观察大鼠FrZ区GABA一IR
阳性细胞数，并进行统计学处理。
结果显示:1.L~NAME组大鼠的慢波睡眠(S WS)和快眼动睡眠(REMS)
明显减少(尸<0.01，尸<0 .05)，SWS潜伏期明显延长(尸<0.01)，REMS出现次数
无显著变化(尸，0.05)。2.musclinol组和baclofen组大鼠的SWS和REMS出现
次数无显著变化(P>0.05)，而REMS减少伊<0 .05)，Sws潜伏期则明显延长
(P<0.01)。3.预先应用museimol或baclofen，再用L一NAME后，与L一NAME单
郑州大学2003届硕士毕业论文
GABA受体激动剂消除NO合成酶抑制剂对大鼠喻醒周期的影响
独作用时相比，大鼠的SWS(P<0.01)增加，SWS潜伏期也较L一NAME单独作
用时缩短(尸<0 .01)，而对L.NAME引起的REMS及其出现次数的减少并无明显
影响(P>0.05)。4.免疫组化染色显示，L.NAME组大鼠FrZ区GABA-IR阳性细
胞表达较对照组明显减少(P<0 .01)。
结论:1.应用NOS抑制剂，可明显增加大鼠的觉醒(W)，抑制SWS和REMS。
2.应用GABA受体激动剂能够抑制大鼠REMS，而SWS不受影响。3.预先应用
GABA受体激动剂后，能够消除NOS抑制剂单独作用时对大鼠SWS的抑制效应。
4.应用NOS抑制剂后，大鼠FrZ区GABA-IR阳性细胞表达明显减少。
Since the first suggestion by Garthwait et al. in 1980s that the free radical nitric oxide (NO) might play a role in the brain of mammals, more and more people have paid close attention to the biological functions of NO. NO is an intercellular messenger synthesized from L-arginine by NO synthase (NOS). As a liposoluble gas, NO can affect other tissues by diffusion after produced in local tissue in the brain. Neuronal cells containing NOS have been found in basal forebrain, pedunculopontine tegmental nuclei, dorsal raphe nuclei, olfactory bulb and hippocampus which have been proved to participate in the regulation of sleep/waking cycle. Kapds and Krueger found that NOS inhibitors reducing the production of NO in the brain suppressed both slow wave sleep (SWS) and rapid eye movement sleep (REMS) in rats and rabbits, but NO donors promoted their SWS. These results suggest NO can promote sleep. Administration of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) to rabbits or rats by s.c., i.v. or Lc.v. suppressed both SWS and REMS but increased W, and the effects showed dose dependence. Moreover, the relatively smaller dose of icv injected L-NAME suppressed sleep and a higher systemically admistered dose was effective (20 times more than the former), which suggested that the site of the sleep-suppressing action of L-NAME is central. In the same time, Dzoljic studied the NOS inhibitor 7-nitro indazole (7-NI) and found that 7-NI decreased the duration of SWS and REMS. All of these support that NO can promote sleep.
Sleep/waking cycle is a complex network modulation and many factors such as
interleukin-1 (IL-1), tumor necrosis factor (TNF), growth hormone releasing hormone (GHRH), vasoactive intestina polypeptide (VIP) and many conventional neurotransmitters such as serotonin (5-HT), acetylcholine (ACh), norepinephrine (NE), dopamine (DA) and gamma-aminobutyric acid (GABA) were involved in it. Recent evidence has shown that NO synthesized in neurons in several areas of the brain can induce the release of neurotransmitters. In the rat central nervous system, the anatomical distribution of NOS -containing neurons is now well established, and it was reported that NOS is co-localized with neurotransmitters well known for their involvement in sleep mechanisms, i.e. 5-HT, ACh, DA and GABA. In vivo or in vitro, NO donor facilitated the release of GABA, while NOS inhibitor inhibited it, which indicated that the release of GABA changed with NO in the brain. So we can suppose that the sleep suppression resulting from administration of the NOS inhibitor by s.c. or i.c.v. be probably related with the change of GABA releasing in the brain. In order to testify the effect of NOS inhibitor and GABA on the regulation of the sleep/waking cycle in adult rats implanted for chronic sleep recordings, the polysomnogram was recorded. We observed whether the sleep-suppressing action of NOS inhibitor was affected after the administration of the agonists of GABA receptors by subcutaneous route.
Experiments were performed on adult male Sprague-Dawley rats weighing 230-270g. The rats were divided into six groups by random and each had 6 ones, they are: l.The control group, 2.The L-NAME group, S.The muscimol group, 4.The baclofen group, S.The muscimol + L-NAME group, 6.The baclofen + L-NAME group. Animals were implanted for chronic sleep recordings. After five-day recovery postoperatively the animals were given either drugs or control solution subcutaneously on the sixth day, then the six-hour electroencephalogram (fome 11:00 to 17:00) was obtained. At the end of recording, the animals of group 1 and group 2 were perfused with paraformaldehyde, then removed the brain and fixed for 24 hours with paraformaldehyde, paraffin sections were prepared at 4 u m, histochemical staining for counting the GABA positive neurons in the frontal cortex (Fr2).
Results: 1. In the group 2, following the administration of L-NAME SWS and
REMS were significantly reduced (P<0.01, P<0.05), while the SWS latency showed a significant increment
引文
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36 Par D, Smith Y. GABAergic projection from the intercalated cell masses of the dala to the basal forebrain in cats. J Comp Neurol, 1994; 344:33-49
37 Nitz D, Siegel J M. GABA release in posterior hypothalamus across sleep-wake cycle. Am J Physiol, 1996; 271:R1707-R1712
38 Lancel M, Cronlein TAM, Faulhaber J. Role of GABAA receptors in sleep
regulation differential effects of muscimol and midazolam on sleep in rats. Neuropsychopharmacology, 1996; 15: 63-64
39 Mendelson WB, Martin JV. Effects of muscimol and flurazepam on the sleep EEG in rat. Life Sci, 1990; 47: PL81-87
40 Lancel M, Faulhaber J, Schiffelholz T, Mathias S. Muscimol and midazolam do not potentiate each other's effects on sleep EEG in the rat. J Neurophysiol, 1997; 77: 1624-29
41 Monti JM, Jantos H, Monti D. Increase of waking and regulation of NREM and REM sleep after nitric oxide synthase inhibition: prevention with GABAA or adenosine A1 receptor agonists. Behav Brain Res, 2001; 123; 23-35
42 Xi MC, Morales FR, Chase MH. Induction of wakefulness and inhibition of active (REM) sleep by GABAergic processes in the nucleus pontis oralis. Arch Ital Biol, 2001; 139 (1-2) : 125-145
43 Camacho-Arroyo I, Alvarado R, Manjarrez J, Tapia R. Microinjection of muscimol and bicuculline into the pontine reticular formation modify the sleep-waking cycle in the rat. Neurosci Lett, 1991; 129 (1) :95-97
44 Torterolo P, Morales FR, Chase MH. GABAergic mechanisms in the pedunculopontine tegmental nucleus of the cat promote active (REM) sleep. Brain Res, 2002; 944 (1-2) : 1-9
45 Lin JS, Sakai K. A critical role of the posterior hypothalamus in the mechanism of wakefulness determined by microinjection of muscimol in freely moving cats. Brain Res, 1989; 479: 225-240
46 关新民，医学神经生物学(第一版)，北京，人民卫生出版社，2002，128
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48 Kaehler S T, Singewald N, Sinner C, Philippu A. Nitric oxide modulates the release of serotonin in the rat hypothalamus. Brain Res, 1999; 835: 346-349
49 Segieth J, Fowler L, Whitton P, Pearce B. Nitric oxide-mediated regulation of dopamine release in the hippocampus in vivo. Neuropharmacology, 2000; 39:
571-577
50 Wegener G, Volke V, Rosenberg R. Endogenous nitric oxide decrease hippocampal levels of serotonin and dopamine in vivo. Br J Pharmacol, 2000, 130: 575-580
51 Fischer H, Prast H, Philippu A. Adenosine release in the ventral striatum of the rat is modulated by endogenous nitric oxide. Eur J Pharmacol, 1995; 275: R5-6
52 Leonard T O, Lydic R. Nitric oxide synthase inhibition decreases pontine acetylcholine release. NeuroReport, 1995; 6: 1525-1529
53 Prast H, Philippu A. Nitric oxide releases acetylcholine in the basal forebrain. Eur J Phamacol, 1992; 216: 139-140
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34 Manfridi A, Brambilla D, Mancia M. Sleep is differently modulated by basal forebrain GABA_A and GABA_B receptors. Am J Physiol Rrgul Intrgr Comp Physiol, 2001; 281:R170-R175
35 Zaborszky C, Heimer L, Eckenstein F, Leranth C. GABAergic input to cholinergic forebrain neurons: an ultrastructural study using retrograde tracing of HRP and double immunolabelling. J Comp Neurol, 1996; 250:282-295
36 Par D, Smith Y. GABAergic projection from the intercalated cell masses of the dala to the basal forebrain in cats. J Comp Neurol, 1994; 344:33-49
37 Nitz D, Siegel J M. GABA release in posterior hypothalamus across sleep-wake cycle. Am J Physiol, 1996; 271:R1707-R1712
38 Lancel M, Cronlein TAM, Faulhaber J. Role of GABAA receptors in sleep
regulation differential effects of muscimol and midazolam on sleep in rats. Neuropsychopharmacology, 1996; 15: 63-64
39 Mendelson WB, Martin JV. Effects of muscimol and flurazepam on the sleep EEG in rat. Life Sci, 1990; 47: PL81-87
40 Lancel M, Faulhaber J, Schiffelholz T, Mathias S. Muscimol and midazolam do not potentiate each other's effects on sleep EEG in the rat. J Neurophysiol, 1997; 77: 1624-29
41 Monti JM, Jantos H, Monti D. Increase of waking and regulation of NREM and REM sleep after nitric oxide synthase inhibition: prevention with GABAA or adenosine A1 receptor agonists. Behav Brain Res, 2001; 123; 23-35
42 Xi MC, Morales FR, Chase MH. Induction of wakefulness and inhibition of active (REM) sleep by GABAergic processes in the nucleus pontis oralis. Arch Ital Biol, 2001; 139 (1-2) : 125-145
43 Camacho-Arroyo I, Alvarado R, Manjarrez J, Tapia R. Microinjection of muscimol and bicuculline into the pontine reticular formation modify the sleep-waking cycle in the rat. Neurosci Lett, 1991; 129 (1) :95-97
44 Torterolo P, Morales FR, Chase MH. GABAergic mechanisms in the pedunculopontine tegmental nucleus of the cat promote active (REM) sleep. Brain Res, 2002; 944 (1-2) : 1-9
45 Lin JS, Sakai K. A critical role of the posterior hypothalamus in the mechanism of wakefulness determined by microinjection of muscimol in freely moving cats. Brain Res, 1989; 479: 225-240
46 关新民，医学神经生物学(第一版)，北京，人民卫生出版社，2002，128
47 Lonart G, Wang J, Johnson K M. Nitric oxide induces neurotransmitter release from hippocampal slices. Eur J Pharmacol, 1992; 220: 271-272
48 Kaehler S T, Singewald N, Sinner C, Philippu A. Nitric oxide modulates the release of serotonin in the rat hypothalamus. Brain Res, 1999; 835: 346-349
49 Segieth J, Fowler L, Whitton P, Pearce B. Nitric oxide-mediated regulation of dopamine release in the hippocampus in vivo. Neuropharmacology, 2000; 39:
571-577
50 Wegener G, Volke V, Rosenberg R. Endogenous nitric oxide decrease hippocampal levels of serotonin and dopamine in vivo. Br J Pharmacol, 2000, 130: 575-580
51 Fischer H, Prast H, Philippu A. Adenosine release in the ventral striatum of the rat is modulated by endogenous nitric oxide. Eur J Pharmacol, 1995; 275: R5-6
52 Leonard T O, Lydic R. Nitric oxide synthase inhibition decreases pontine acetylcholine release. NeuroReport, 1995; 6: 1525-1529
53 Prast H, Philippu A. Nitric oxide releases acetylcholine in the basal forebrain. Eur J Phamacol, 1992; 216: 139-140
54 Scilicovich A, Lasaga M, Befumo M. Nitric oxide inhibits the release of norepinephrine and dopamine from the medial basal hypothalamus of the rat. Proc Natl Acad Sci USA, 1995, 92: 11299-302