大鼠孤束核微注射5羟色胺系统药物对睡眠-觉醒的影响及机制研究
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摘要
目的：睡眠-觉醒是一种生物学的昼夜节律。孤束核是内脏传入神经核团，同时作为低位脑干的睡眠诱发区，与许多内脏疾病的睡眠问题有关，但是其产生的机制尚未完全理清。5-羟色胺是作用于睡眠-觉醒的重要单胺类神经递质。故本实验采用脑立体定位法在孤束核进行微注射5-羟色胺系统的药物，研究5-羟色胺在孤束核影响睡眠-觉醒时的作用通路，同时推测孤束核在睡眠中发挥的作用，为进一步的理解睡眠机制、治疗内脏疾病引起的睡眠问题提供理论依掘，为药物研发提供靶点。
方法：1、实验动物：本实验选用体重为220～260g的健康成年雄性SD大鼠。2、动物手术：将大鼠固定于脑立体定位仪上，参照大鼠脑立体定位图谱，定位孤束核的位置，埋置导药管；同时在皮层放置2个电极记录脑电，颈下2个电极记录肌电。动物恢复5天，然后进行记录。
3、脑电记录：采用日本光电脑电机对动物进行每天4h，连续4天的脑电图记录。
4、病理切片：记录完毕，动物采用心脏灌注取脑，做石蜡切片进行HE染色完成组织定位鉴定；并对NS、5-HTP、MS三组动物做冰冻切片，进行免疫组化染色。
5、统计方法：采用SPSS10.0统计软件分析，每组动物进行自身配对t检验。α=0.05，双侧t检验
结果：1、各组自身配对检验：对照组(NS组)无统计学意义；5-HTP组、8-OH-DPAT组、H89组差别有统计学意义，均使觉醒减少，慢波睡眠增加，异相睡眠增加；MS组无显著的统计学意义，但是有明显的觉醒增加、睡眠减少的趋势；spiperone组、cAMP组差别有统计学意义，觉醒增加，慢波睡眠减少，异相睡眠减少。
2、NS组、5-HTP组、MS组进行的孤束核5-HT_(IA)R染色示：与NS组相比，5-HTP组的5-HT_(IA)R上调，MS组的5-HT_(IA)R下调。
闷成创卜花开究生学位论文
3、NS组、5一HTP组、MS组进行的孤束核5一轻色胺能神经纤维染色示:与
NS组相比，5一HTP组的5一轻色胺能神经纤维终末染色加强，MS组的5一轻色
胺神经纤维终末染色减弱。
4、NS组在中脑进行5一HTIAR的染色:5一HTIAR在中脑的分布广泛，尤其在
中缝核、网状结构LDT用PT处。
5、NS组、5一HTP组、MS组进行的中脑5一HT神经纤维染色示:与NS组相
比，5一HTP组的5一HT能阳性细胞少，MS组的5一HT能阳性细胞多。
结论:1、孤束核作为睡眠诱发区，5一轻色胺在慢波睡眠和异相睡眠时相发挥
着重要的作用。
2、5一HT在孤束核影响睡眠一觉醒时是通过作用5一HT，AR，祸联G订。蛋白，使
cAMP生成减少，PKA磷酸化减少而发挥觉醒减少、睡眠增加的生物学效应。
3、在孤束核处，5一轻色胺增多延长慢波睡眠:而在中脑处则是5一轻色胺减少
引起慢波睡眠延长:这说明了在孤束核和中脑之间存在相互抑制的网络调节
系统。
4、作为异相睡眠发生的授时因子，孤束核通过低位脑千的上行抑制系统影响
上行激动系统，产生异相睡眠。
5、5一轻色胺在睡眠一觉醒中的作用主要通过突触后靶组织发挥。从异相睡眠
看，胞外5一轻色胺含量与中缝背核的抑制是一致的，可能是通过脑桥被盖背
外侧核/脑桥被盖网状核(LDT爪PT)的胆碱能细胞功能的去抑制而引起异相
睡眠。
Objection: Sleep-wake cycle is a kind of biological rhythm. The nucleus of solitarius tractus (NST) is an important site for the intergration of visceral information and its modification by afferent neural systems. As for a sleep-inducing area of lower brain stem, it is related with sleep disturbance of visceral diseases, but these mechanisms are unclear. 5-hydroxytryptamine (serotonin, 5-HT) is an important kind of monoaminergic neurotransmitters. In the present study, a method of stereotaxic apparatus was employed to fix a guide cannula in the NST which was used as a way to microinject 5-HT 's related drugs. Then we can analyze electroencephalogram (EEG) to specify the signal transduction pathways through which 5-HT generate sleep in the NST. In addition, we furthered to understand that NST plays a role in the paradoxical sleep (PS), providing a foundation of emulating mechanism of sleep and treating some sleep disturbance involved visceral diseases and a new target point about developing a new medicine.
Methods: 1. Subjects: Male Sprague Dawley rats (220-260 mg) were purchased from military medical institution at Beijing.
2. Animal surgery: SD rats were operated under the acetylglycinamide-chloral diphosphate (AGAC) anesthesia for the implantation of chronically dwelling electrodes for recording the EEG and electromyogram (EMG) and received a fixed guide cannula in the NST, according to the atlas of Paxinos and Watson. Animals were allowed to refresh 5 days after surgery in the animal room before being placed in recording chambers for the duration of the experiment.
3. Recording EEG and EMG: The EEG and EMG signals were collected using a NIHON KOHOEN (No7213) polygragh and subsequently analyzed according to the standard regulation. Animals were recorded 4 hours every day and continually
lasted 4 days.
4.Perfusion and fixation: after recording, the animals were killed under AGAC anesthesia by intra-aortic perfusion of a fixative solution (containing 4%paraformaldehyde) and their brains were taken out using paraffin buried and HE staining; another 3 groups animals samples were used to a freezing microtome and immunohistochemical staining.
5. Statistical analysis: All the data were done by methods of pair t-test with SPSS10.0 statistical procedure. Significance level was chosen at 0.05 and all tests were two-sided.
Results: 1. Using pair t-test, normal saline (NS) group is not significantly different as a control group; 5-hydroxytryptophan (5-HTP) group, 8-OH-DPAT, H89 are significantly different on the decline of wake, increase of SWS and PS ; methysergide (MS) group is not significantly different ; spiperone group and cAMP group are significantly different on the increase of wake and the decline of SWS and PS.
2. 5-HT1A receptor (5-HT1AR) of immunohischemical data in the NST between NS group, 5-HTP group and MS group: Compared with NS group, there is uptake of 5-HT1AR in 5-HTP group and downtake of 5-HT]AR in MS group.
3. 5-HTnergic neural fibers of immunohischemical data in the NST between NS group, 5-HTP group and MS group: Compared with NS group, there is strong stain of 5-HTnergic neural terminal fibers in 5-HTP group and light stain of 5-HTnergic neural terminal fibers in MS group.
4. 5-HTiAR of immunohischemical data in the midbrain of NS group: there are widely distribution of 5-HT1AR immunoreactivity in the midbrain, especially in the raphe nuclei (RN), LDT/PPT of reticular formation.
5. 5-HTnergic neural terminal fibers of immunohischemical data in the midbrain between NS group, 5-HTP group and MS group: compared with NS group, there
are few numbers of 5-HT neuron in 5-HTP group and more in MS group. Conclusions: 1. As an inducing area, 5-HT in the NST plays an important role in the sleep-wake cycle.
2. Sleep generation is mediated by 5-HT1AR-activated signal transduction pathway involving aGi/G0-like G-protein in 5-HT microinjecting NST on sleep-wake cycle, which is caused to decline wake time and increase sleep time.
3. There is increase in SWS following the incre
方法：1、实验动物：本实验选用体重为220～260g的健康成年雄性SD大鼠。2、动物手术：将大鼠固定于脑立体定位仪上，参照大鼠脑立体定位图谱，定位孤束核的位置，埋置导药管；同时在皮层放置2个电极记录脑电，颈下2个电极记录肌电。动物恢复5天，然后进行记录。
3、脑电记录：采用日本光电脑电机对动物进行每天4h，连续4天的脑电图记录。
4、病理切片：记录完毕，动物采用心脏灌注取脑，做石蜡切片进行HE染色完成组织定位鉴定；并对NS、5-HTP、MS三组动物做冰冻切片，进行免疫组化染色。
5、统计方法：采用SPSS10.0统计软件分析，每组动物进行自身配对t检验。α=0.05，双侧t检验
结果：1、各组自身配对检验：对照组(NS组)无统计学意义；5-HTP组、8-OH-DPAT组、H89组差别有统计学意义，均使觉醒减少，慢波睡眠增加，异相睡眠增加；MS组无显著的统计学意义，但是有明显的觉醒增加、睡眠减少的趋势；spiperone组、cAMP组差别有统计学意义，觉醒增加，慢波睡眠减少，异相睡眠减少。
2、NS组、5-HTP组、MS组进行的孤束核5-HT_(IA)R染色示：与NS组相比，5-HTP组的5-HT_(IA)R上调，MS组的5-HT_(IA)R下调。
闷成创卜花开究生学位论文
3、NS组、5一HTP组、MS组进行的孤束核5一轻色胺能神经纤维染色示:与
NS组相比，5一HTP组的5一轻色胺能神经纤维终末染色加强，MS组的5一轻色
胺神经纤维终末染色减弱。
4、NS组在中脑进行5一HTIAR的染色:5一HTIAR在中脑的分布广泛，尤其在
中缝核、网状结构LDT用PT处。
5、NS组、5一HTP组、MS组进行的中脑5一HT神经纤维染色示:与NS组相
比，5一HTP组的5一HT能阳性细胞少，MS组的5一HT能阳性细胞多。
结论:1、孤束核作为睡眠诱发区，5一轻色胺在慢波睡眠和异相睡眠时相发挥
着重要的作用。
2、5一HT在孤束核影响睡眠一觉醒时是通过作用5一HT，AR，祸联G订。蛋白，使
cAMP生成减少，PKA磷酸化减少而发挥觉醒减少、睡眠增加的生物学效应。
3、在孤束核处，5一轻色胺增多延长慢波睡眠:而在中脑处则是5一轻色胺减少
引起慢波睡眠延长:这说明了在孤束核和中脑之间存在相互抑制的网络调节
系统。
4、作为异相睡眠发生的授时因子，孤束核通过低位脑千的上行抑制系统影响
上行激动系统，产生异相睡眠。
5、5一轻色胺在睡眠一觉醒中的作用主要通过突触后靶组织发挥。从异相睡眠
看，胞外5一轻色胺含量与中缝背核的抑制是一致的，可能是通过脑桥被盖背
外侧核/脑桥被盖网状核(LDT爪PT)的胆碱能细胞功能的去抑制而引起异相
睡眠。
Objection: Sleep-wake cycle is a kind of biological rhythm. The nucleus of solitarius tractus (NST) is an important site for the intergration of visceral information and its modification by afferent neural systems. As for a sleep-inducing area of lower brain stem, it is related with sleep disturbance of visceral diseases, but these mechanisms are unclear. 5-hydroxytryptamine (serotonin, 5-HT) is an important kind of monoaminergic neurotransmitters. In the present study, a method of stereotaxic apparatus was employed to fix a guide cannula in the NST which was used as a way to microinject 5-HT 's related drugs. Then we can analyze electroencephalogram (EEG) to specify the signal transduction pathways through which 5-HT generate sleep in the NST. In addition, we furthered to understand that NST plays a role in the paradoxical sleep (PS), providing a foundation of emulating mechanism of sleep and treating some sleep disturbance involved visceral diseases and a new target point about developing a new medicine.
Methods: 1. Subjects: Male Sprague Dawley rats (220-260 mg) were purchased from military medical institution at Beijing.
2. Animal surgery: SD rats were operated under the acetylglycinamide-chloral diphosphate (AGAC) anesthesia for the implantation of chronically dwelling electrodes for recording the EEG and electromyogram (EMG) and received a fixed guide cannula in the NST, according to the atlas of Paxinos and Watson. Animals were allowed to refresh 5 days after surgery in the animal room before being placed in recording chambers for the duration of the experiment.
3. Recording EEG and EMG: The EEG and EMG signals were collected using a NIHON KOHOEN (No7213) polygragh and subsequently analyzed according to the standard regulation. Animals were recorded 4 hours every day and continually
lasted 4 days.
4.Perfusion and fixation: after recording, the animals were killed under AGAC anesthesia by intra-aortic perfusion of a fixative solution (containing 4%paraformaldehyde) and their brains were taken out using paraffin buried and HE staining; another 3 groups animals samples were used to a freezing microtome and immunohistochemical staining.
5. Statistical analysis: All the data were done by methods of pair t-test with SPSS10.0 statistical procedure. Significance level was chosen at 0.05 and all tests were two-sided.
Results: 1. Using pair t-test, normal saline (NS) group is not significantly different as a control group; 5-hydroxytryptophan (5-HTP) group, 8-OH-DPAT, H89 are significantly different on the decline of wake, increase of SWS and PS ; methysergide (MS) group is not significantly different ; spiperone group and cAMP group are significantly different on the increase of wake and the decline of SWS and PS.
2. 5-HT1A receptor (5-HT1AR) of immunohischemical data in the NST between NS group, 5-HTP group and MS group: Compared with NS group, there is uptake of 5-HT1AR in 5-HTP group and downtake of 5-HT]AR in MS group.
3. 5-HTnergic neural fibers of immunohischemical data in the NST between NS group, 5-HTP group and MS group: Compared with NS group, there is strong stain of 5-HTnergic neural terminal fibers in 5-HTP group and light stain of 5-HTnergic neural terminal fibers in MS group.
4. 5-HTiAR of immunohischemical data in the midbrain of NS group: there are widely distribution of 5-HT1AR immunoreactivity in the midbrain, especially in the raphe nuclei (RN), LDT/PPT of reticular formation.
5. 5-HTnergic neural terminal fibers of immunohischemical data in the midbrain between NS group, 5-HTP group and MS group: compared with NS group, there
are few numbers of 5-HT neuron in 5-HTP group and more in MS group. Conclusions: 1. As an inducing area, 5-HT in the NST plays an important role in the sleep-wake cycle.
2. Sleep generation is mediated by 5-HT1AR-activated signal transduction pathway involving aGi/G0-like G-protein in 5-HT microinjecting NST on sleep-wake cycle, which is caused to decline wake time and increase sleep time.
3. There is increase in SWS following the incre
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62 Chen JJ, Vasko MR, WU XP, et al. Multiple subtypes of serotonin receptors are expressed
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63 Romero L, Artigas F, Preferential prtentiation of the effects of serotonin uptake inhibition by 5-HT1A receptor antagonists in the dorsal raphe pathway: role of somatodendritic autoreceptor. J Neurochem, 1997, 68:2593-2603
64 Nosjean A, Arluison M, Laguzzi RF. Increase in paradoxicla sleep after destruction of serotoninergic innervation in the nucleus tractus sotitarius of the rat. Neuroscience, 1987, 23(2):469-81
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51 Bjorvatn B, Ursin R. Changes in sleep and wakefulness following 5-HT1A ligands
given systemically and locally in different brain regions. Rev Neurosci, 1998,9(4): 265-273
52 Monti JM, Jantos H, Monti D, et al. Dorsal raphe nucleus administration of 5-HT1A receptor agonist and antaonist: Effent on rapid eye movement sleep in the rat. Sleep Res Online, 2000,3(1):29-34
53 Sorensen E, Gronli J, Bjorvatn B, et al. Sleep and waking following microdialysis perfusion of the selective 5-HT1A receptor antagonist p-MPPI into the dorsal raphe nucleus in the freely moving rat. Brain Res, 2001,897{1-2}:122-130
54 Hajos M, Hoffmann WE, Tetko IV, et al. Different tonic regulation of neuronal activity in the rat dorsal raphe and medial prefrontal cortex via 5-HT(IA) receptors. Neurosic Lett. 2001, 304(3):129-132
55 韩济生主编．神经科学原理，第2版，北京医科大学出版社，1999
56 姚泰主编．人体生理学，第3版，人民卫生出版社，2001
57 Portas CM, Bjorvatn B, Fagerland S, et al. On-line detection of extracellular levels of sreotonin in dorsal raphe nucleus and frontal cortex over the sleep/wake cycke in the freely moving rat. Neuroscience, 1998,83:807-814
58 Park SP, Lopez-Rodriguez F, Wilson CL, et al. In vivo microdialysis measures of extracellular serotonin in the rat hippocampus during sleep-wakefulness. Brain Res,1999,833:291-296
59 Gervasoni, Peyron C, Rampon C,et al. Role and origin of the GABAergic innervation of P R serotonergic neurons. J Neurosci, 2000, 20(11):4217-4225
60 Denoyer M, Sallanon M, Kitahama K,et al. Reversibility of para-chlorophenylalanine-induced insomnia by intrahypothalamic microinjection of L-5-hydroxytryptophan. Neuroscience. 1989; 28(1):83-94
61 Leonard CS, Llinas R. Serotonergic and cholinergic inhibition of mesopontine cholinergic neurons controlling REM sleep:an in-vitro electrophysiologieal study Neuroscience, 59:309-330
62 Chen JJ, Vasko MR, WU XP, et al. Multiple subtypes of serotonin receptors are expressed
in rat sensory neurons in culture. J Pharmacol Exp Ther, 1998,287:1119-1127
63 Romero L, Artigas F, Preferential prtentiation of the effects of serotonin uptake inhibition by 5-HT1A receptor antagonists in the dorsal raphe pathway: role of somatodendritic autoreceptor. J Neurochem, 1997, 68:2593-2603
64 Nosjean A, Arluison M, Laguzzi RF. Increase in paradoxicla sleep after destruction of serotoninergic innervation in the nucleus tractus sotitarius of the rat. Neuroscience, 1987, 23(2):469-81