【作者】 姜岩岩；

【导师】 徐淑梅；

【作者基本信息】 天津医科大学 ， 神经生理学， 2004， 硕士

【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_IAR染色示：与NS组相比，5-HTP组的5-HT_IAR上调，MS组的5-HT_IAR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。更多还原

【Abstract】 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 continuallylasted 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, thereare 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更多还原