【作者】 刘玲；

【导师】 闫蕴力；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2008， 博士

【摘要】 孤束核吻侧段(rostral nucleus of the solitary tract,rNST)与味觉信息的传递及整合有密切关系。rNST不仅接受来自于面、舌咽及迷走神经的味觉传入,而且与味觉调控关系密切的核团(臂旁核、下丘脑、丘脑、中央杏仁核、味觉皮质等)之间具有往返的纤维联系。rNST内含有多种神经递质和调质。其中,谷氨酸和P物质主要发挥兴奋性调节作用,而脑啡肽(enkephalin,ENK)和γ-氨基丁酸(γ-aminobutyric acid,GABA)主要对味觉感受神经元产生抑制作用。rNST内分布有密集的ENK阳性(ENK-ir)纤维和终末及大量的μ型和δ型阿片受体(μopioid receptor,MOR和δopioid receptor, DOR)。GABA阳性(GABA-ir)神经元也大量存在于rNST内。Malanga等发现GABA的活性受到阿片类物质的抑制,而Echo等则证实阿片与GABA存在功能上的协同作用,但其作用机制的形态学基础目前仍未见报道。且孤束核内的GABA能神经元是否呈MOR阳性,这些尚都缺乏直接的形态学证据。为此,本研究利用脑立体定位仪并应用光、电镜免疫组织化学方法,对大鼠摄食变化及rNST内ENK-ir与GABA-ir结构之间的相互联系以及MOR-ir神经元与ENK-ir终末的联系进行了观察。第一部分孤束核内微量注射脑啡肽和γ-氨基丁酸引起大鼠摄食改变的行为学研究目的利用脑立体定位仪向孤束核内微量注射脑啡肽和γ-氨基丁酸,观察大鼠摄食变化。方法1.测量基础摄食量SD大鼠22只。分为三组,GABA组10只、ENK组10只和对照组2只。均单笼饲养在消毒后的笼子中,自由进水、饮食,室温22±2℃,12 h光照/黑暗转换。连续喂养5日,以适应环境。并测量累积摄食量。2.外科手术2.1用2%戊巴比妥钠(45 mg/kg)腹腔麻醉后,将动物头部固定在立体定位仪上,根据Paxinos和Watson[Paxinos and Watson, 1999]图谱经颅骨在rNST将一带芯不锈钢导管(外径0.9mm)插入rNTS上方2.0 mm处(Bregma向尾段12.80mm,中线旁开1.2mm,Bregma点水平向下6mm。Bregma点是大鼠的前囟门位置)手术后,每天给予肌肉注射青霉素20万单位,连续四天,以防伤口和颅内感染。2.2脑内注射:动物轻微麻醉后,将一微量注射器针头(外径0.4 mm)经导管插入脑内,使其尖端在导管尖端下方2.0 mm处,以到达rNTS(Bregma向尾段12.80mm,中线旁开1.2mm,Bregma点水平向下8mm)。将0.2(每侧)μl的药物或生理盐水缓慢地注入rNTS。3.术后测量基础摄食量在注药后4小时、8小时、12小时按同一方法记录大鼠累积进食量。4.注射区的组织学定位实验结束时,在深度麻醉下,将动物用生理盐水和4%多聚甲醛液中相继经升主动脉进行灌注。取出脑并将其在4%多聚甲醛溶液中后固定1-2 d。用冰冻切片机切成大约60μm厚的切片,HE染色。然后将注射位点按组织学图谱进行组织学定位。结果1.注药部位检查:切片检查显示,rNST微量注射的部位(即针道的顶端)处于孤束核吻端内,双侧对称。2.GABA组10只,其中有1只出现了术后感染,弃去不用。从分别在注药后4小时、8小时、12小时测量摄食量,剩余9只与对照组相比,均出现摄食量降低。3.ENK组10只分别在注药后4小时、8小时、12小时测量摄食量,与对照组相比,均出现摄食量降低。结论分别向孤束核内微量注射GABA和ENK,均可引起大鼠基础摄食量降低,这主要是由于二者均可抑制孤束核内的味觉神经元活性,从而影响大鼠的基础摄食量。第二部分大鼠延髓孤束核吻侧段内脑啡肽阳性终末与γ-氨基丁酸阳性神经元联系的形态学研究目的观察大鼠孤束核吻侧段(rNTS)内脑啡肽阳性(ENK-ir)终末与γ-氨基丁酸阳性(GABA-ir)神经元之间的联系。方法1.组织材料的处理SD大鼠10只。将大鼠在腹腔内注射过量戊巴比妥钠(100 mg/kg)的深麻醉状态下开胸,经升主动脉插管,先用80 ml生理盐水冲净血液,再灌注以500 ml含4%多聚甲醛、0.05%戊二醛和2%苦味酸的0.1 mol/L磷酸缓冲液(PB,pH 7.4)。灌注完毕立即取脑并置于上述新鲜固定液中后固定4h,再移入含30%蔗糖的0.1 mol/L PB(4℃)内至沉底。将材料切块,分离出低位脑干并切片。2.免疫荧光染色法将光镜组切片置于含小鼠抗ENK(1:500)及兔抗GABA(1:5000)的反应液内(含5%正常山羊血清及0.5% Triton X-100)室温孵育24小时。之后浸入含Biotin标记的绵羊抗小鼠IgG(1:200)的反应液内于室温下孵育8小时。最后入含Texas Red标记的Avidin(1:200)及Fluorescein标记的驴抗兔IgG(1:200)反应液,室温下避光孵育6小时。将上述切片裱于载玻片上,使用SlowFade抗荧光衰减剂封片后于激光扫描共聚焦显微镜(Leica TCS-SP2)下观察。3.包埋前染色免疫电镜法电镜组切片在进行免疫组化染色之前,置于液氮中速冻数秒,放置前、后将切片浸于冰冻保护液中。将切片置于30%正常山羊血清中30 min,之后浸入含小鼠抗ENK(1:500)及兔抗GABA(1:5000)的反应液内(含5%正常山羊血清),室温孵育24 h。随后浸入含Biotin标记的绵羊抗小鼠IgG(1:200)及纳米金标记的山羊抗兔IgG的反应液内,室温下过夜。在1%戊二醛内固定10 min后将切片用银加强试剂盒进行银加强染色,染色前后使用双蒸水清洗。之后使用ABC复合物孵育2 h并进行常规二氨基联苯胺(DAB)反应。上述各步骤之间均用PBS彻底清洗。随后将经上述免疫组化双重染色的切片置入1%锇酸溶液固定1 h,在70%酒铀中浸泡4 h,梯度酒精及环氧丙烷脱水,Epon-812平板包埋。取rNTS位置的组织片做超薄切片,枸橼酸铅染色后于电镜(H-7500,Hitachi)下观察。结果1.在激光共聚焦扫描显微镜下1.1 rNTS内分布着红色标示的ENK-ir结构,以密集分布的终末为主,阳性纤维呈中等密度分布。1.2 rNTS内分布着绿色标示的GABA-ir结构,以散在分布且直径为10~15μm的小神经元为主,纤维和终末样结构则稀疏地分布于阳性胞体之间。1.3部分ENK-ir终末与GABA-ir胞体以及阴性的胞体(直径10~35μm)之间形成密切接触。2.在电镜下2.1 rNTS内存在许多ENK-ir轴突和终末。在ENK-ir终末内,可见DAB反应产物主要沉积于圆形清亮囊泡表面及线粒体等细胞器表面,在部分终末内还可见到阳性的大颗粒囊泡。2.2 rNTS内存在GABA-ir结构,可见数目及大小不等的黑色金颗粒散在分布于胞体、树突及少量轴突内。GABA-ir产物主要分布于粗面内质网及核糖体表面等结构。2.3电镜下可见到ENK-ir轴突终末形成以下突触关系:2.3.1与GABA-ir阳性胞体、树突及树突棘形成对称(62%)及非对称性(38%)轴-体(26%)及轴-树(74%)突触。2.3.2与GABA-ir阴性的胞体、树突及树突棘形成对称(73%)及非对称性(27%)轴-体(18%)及轴-树(82%)突触;2.3.3与GABA-ir阴性的轴突之间还可见到少量对称性轴-轴突触,ENK-ir轴突终末为突触前或后成分。结论rNTS内的ENK-ir终末可能通过抑制或增强GABA能神经元活性或者直接抑制味觉感受神经元活性的方式参与NTS内味觉信息的感受和调节。第三部分大鼠延髓孤束核吻侧段内GABA和MOR共存神经元及ENK阳性终末与MOR阳性神经元联系的实验研究目的观察延髓孤束核吻侧段(rNST)内是否存在γ-氨基丁酸(GABA)与阿片μ受体(MOR)共存的神经元,以及MOR阳性(MOR-ir)神经元与脑啡肽阳性(ENK-ir)终末的突触联系。方法1.组织材料的处理SD大鼠22只。将大鼠在腹腔内注射过量戊巴比妥钠(100 mg/kg)的深麻醉状态下灌注,取材。方法同上。2免疫荧光染色法方法同上。光镜组第一套切片用豚鼠抗MOR(1:500)及兔抗GABA(1:5000)代替原一抗,用Fluorescein标记的驴抗豚鼠IgG(1:500)和Cy3标记的绵羊抗兔IgG(1:500)代替原二抗。光镜组第二套切片用小鼠抗ENK(1:500)及豚鼠抗MOR(1:500)代替原一抗,用Biotin标记的绵羊抗小鼠IgG(1:200)及Texas Red标记的Avidin(1:200)及Fluorescein标记的驴抗豚鼠IgG(1:500)代替原二抗。余反应步骤同。3.包埋前染色免疫电镜法将电镜组第一套切片用小鼠抗ENK(1:500)及豚鼠抗MOR(1:500)代替原一抗,用Biotin标记的绵羊抗小鼠IgG(1:200)及纳米金标记的山羊抗豚鼠IgG代替原二抗。将电镜组第二套切片用豚鼠抗MOR(1:500)及兔抗GABA(1:5000)代替原一抗,用Biotin标记的绵羊抗兔IgG(1:200)及纳米金标记的山羊抗豚鼠IgG代替原二抗。余反应步骤同。结果1.在激光共聚焦扫描显微镜下1.1 GABA/MOR双标反应可见:可见rNTS内分布着GABA-ir、MOR-ir结构。GABA-ir(红色)和MOR-ir(绿色)结构均以散在分布且直径为10-15μm的小型神经元为主。可见同时呈GABA和MOR免疫反应阳性的神经元。1.2 ENK/MOR双标反应可见:可见rNTS内分布着ENK-ir(红色)终末与MOR-ir神经元(绿色)。ENK-ir终末与MOR-ir神经元的胞体及突起形成密切接触。2.在电镜下2.1 GABA/MOR双标反应可见:GABA与MOR共存于胞体及树突内,GABA阳性(GABA-ir)产物主要分布于粗面内质网及核糖体表面等结构,MOR阳性(MOR-ir)产物位于树突膜、线粒体膜、内质网膜等结构表面。GABA-ir与MOR-ir共存神经元与免疫反应阴性的终末形成对称性及非对称性突触,以对称性突触为主。2.2 ENK/MOR双标反应可见:可见到大量的ENK-ir轴突和终末和MOR-ir胞体和树突。在ENK-ir终末内,可见DAB反应产物主要沉积于圆形清亮囊泡表面及线粒体等细胞器表面,在部分终末内还可见到阳性的大颗粒囊泡。ENK-ir终末与MOR-ir神经元的胞体及树突形成以对称性为主的突触联系。部分MOR-ir产物存在于轴突末端,且与ENK-ir产物共存,并且与免疫反应阴性树突形成对称性突触。结论rNST内的GABA能神经元表达MOR,而ENK-ir终末可能通过与MOR结合调节GABA能神经元的活性,从而参与味觉信息的感受和调节。更多还原

【Abstract】 The rostral portion of the nucleus of the solitary tract (rNST) is closely related to process and regulate effects on gustatory input. rNST receives visceral afferent fibers consisting of the primary afferent fibers from the facial, glossopharyngeal and vagus nerve, and gustatory information from NST is further converyed to the parabrachial nuclei,the hypothalamus, the thalamus, central nucleus of the amygdale and insular cortex.The NST is rich in neuroaction substance. Some experiments have demonstrated the excitatory influence of glutamate, SP and the inhibitory effect of GABA, ENK on cells in gustatory region of the NST.The NST is a region known to express GABA. Previous studies have shown an extensive network of ENK-immunoreactive fibers and terminal within the gustatory zone of NST,μ-opoid receptors andδ- opoid receptors have also shown intense expression in NST.Malanga proposed opoids inhibit GABA-mediated synaptic transmission .But Echo confirmed there is synergism between opoids and GABA. Both the modulatory effects of opioid peptides on GABA-induced inhibition and the studies that GABA-immunopositive nearons also exhibited MOR in NST are still deficient of morphological eviedence.According to these hints, the present study was principally designed to observe the change of food intake following administration of GABA and ENK into rNST rigion by stereotaxic apparatus and investigate the connection among ENK-ir terminals, GABA-ir neurons and MOR-ir nearons in NST by using immunoflouorescence histochemical double-staining and immuno-electronmicroscrope double- staining technique. Part 1 Alterations in food intake elicited by GABA and ENK administered into the rostral portion of the nucleus tractus solitarious of ratsObjectiveTo observed the change of food intake following administration of GABA and ENK into rNST rigion by stereotaxic apparatus.Methods1. Food intake measurements: 22 adults Sprague-Dawley rats weighing 200-300g were divided into 3 groups: There were 10 in GABA group, 10 in ENK group and 2 in control group. The rats were individually housed in wire-mesh cages and maintained on a 12-hour light/ 12- hour dark cycle with Durine rat chow and water available ad libitum.All animals were allowed at least 5 days to accommodate the environment, and calculate food intake.2. SurgeryEach rat was pretreated with 2% pentobarbital sodium (45mg/kg.ip). The animal was mounted in a stereotaxic instrument with the use of a non-traumatic head holder. Bilateral stainless steel guide was aimed stereotaxically at the rNST region using the following coordinates: 12.80mm posteriorto the bregma suture, 1.2 mm lateral to either side of the sagittal suture and 6.0 mm from the top of the skull. Each rat was treated with Penicillin (20u/kg.i.m.) for 4 days. All animals were allowed at least 1 week to recover from stereotaxic surgery before behavioral testing began.GABA and ENK were dissolved in buffered physiological saline microinjections were administered bilaterally into the NST in 0.2 ul volumes over 30s. Physiological saline was initially used as a control injection for these experiments.3. Food intake measurements after surgeryCumulative intakes were assessed 4, 8 and 12h following the microinjection.4. HistologyAt the end of each experiment,males were deeply anesthetized with sodium pentobarbital (80mg/kg.i.p.) and perfused transcardially with 4% paraformadehyde in 0.1 M sodium phosphate-buffered saline (PBS,PH7.4).the brainstem were separated from the whole brain, post-fixed in the same fresh fixative for 1-2 days. The lower brainstem was cut into 20um thick frontal sections with a freezing microtome, and the sections were used in HE staining.Result1. Histological examination showed that the cannulae placements of all animals participating in the protocols were found to be within the region of the rNST.2. One rat of group GABA appeared postoperative infection, and was disused. GABA significantly decreased food intake.3. Rats of group ENK significantly decreased food intake.ConclusionFood intake was significantly decreased by local microinjection of GABA and ENK, because taste-responsive neurons of the NST were inhibited by GABA and ENK.Part 2 A morphological studies of conection between ENK immunoreactive terminals and GABA immunoreactive neurons in the rostral portion of the nucleus tractus solitarious of the ratObjectiveTo observe the connections between enkephalin immunoreactive (ENK-ir) terminals andγ-aminobutyric acid immunoreactive (GABA-ir) neurons in the rostral portion of the nucleus tractus solitarious (rNTS) of the rat.Methods1. Materials and methods:10 adult Sprague-Dawley rats weighing 200-300g were used. All rats were performed under anesthesia by intraperitoned injection of sodium pentobarbital (80mg/kg,i.p.), and perfused transcardially with 500ml of 0.1 M phosphate butter (PB, PH 7.4) containing 4% (W/V) paraformaldehyde and 75%(V/V)–saturated picric acid. After the perfueion, the brains were removed, post-fixed in the same fresh fixative and placed in 0.1 M PB containing 30%(W/V) sucrose overnight at 4℃. The lower brainstems were cut into 20um thick frontal sections with a freezing microtome and cut into 50um thick frontal sections with a microslicer.2. Double-immunofluorescent labelingThe sections were used for immunofluorescence histochemical double-staining for ENK/GABA: Mouse anti ENK (1:500) and rabbit anti GABA (1:5000) in PBS over night; biotinylated sheep anti-moouse IgG (1:200) in PBS for 8 hours; a mixture of Texas Red-labled avidin and Fluorescein- labled donkey anti-ribbit IgG in PBS for 6 hours; the sections were mounted onto clean glass slides, air-dried and cover slipped with PBS containing anti-fading reagent. All sections were observed with a confocal laser-scaning microscope.3. Electron microscopic immunohistochemical double-stainingSections were placed in 0.05 M PB containing 25% sucrose and 10% glycerol for 30 mintues for cryoprotection,followed by freeze-thawing with liquid nitrogen.Subsequently, the sections were incubated with 0.05M Tris-Hcl buffered-saline containing 30% normal goat serum for 30 mintues, and then processed for immunohistochemical double-staining, the immunogold-silver method for GABA was combined with the ABC method for ENK. The sections were incubation with first anti bodies for 24 hours.The sections incubated over night at room temperature with a mixture of coat anti-rabbit IgG conjugated to gold particle and biotinylated sheep anti-mouse IgG.The sections were postfixation with glutaraldehyde, silver enhancement with Silver kit, incubation with ABC Elite Kit, visualization of GABA and ENK-immunoreactivity with DAB, osmification ,counterstaining with uranyl acetate, flat-embeddingin Epon812 after dehydration and mounting onto silicon-coated glass slides.Then The sections were prepared for electron microscopy.Results1. Under the laser scanning confocal microscope1.1 There were dense ENK-ir fibers and terminals in the rNTS, they presented red.1.2 Some GABA-ir neurons were observed in the rNTS, they presented green.1.3 Close connections between ENK-ir terminals and GABA-ir cell bodies were also detected.2. By using the electron microscopic technique2.1 ENK-ir reaction products were mainly localized on the surface of round clear vesicles and dense-cored vesicles in the axon terminals.2.2 GABA-ir reaction products were mainly localized on the surface of rough endoplasmic reticulum and ribosome in dendrites.2.3 Synaptic connections between DAB staining ENK- immunoposi- tive terminals and nanogold labeling GABA- immunopositive or immunonegative dendrites or terminal in the rNTS:2.3.1 Symmetric (62%) and asymmetric synaptic (38%) connections were observed between ENK-immunopositive terminals and GABA-immunopositive cell bodies (26%) and dendrites (74%).2.3.2 Symmetric (73%) and asymmetric synaptic (27%) connections were observed between ENK-immunopositive terminals and GABA -imunonegative cell bodies (18%) and dendrites (82%).2.3.3 Slight symmetric synaptic connections were observed between ENK-ir terminals and GABA-imunonegative terminals, ENK-immunopositive terminals were presynaptic or postsynaptic components.ConclusionThe ENK-ir terminals might take part in the transmission and regulation of the taste information in the rNTS through inhibiting, exciting the GABAergic neurons or inhibit directly the activity of neurons within the rNTS.Part 3 An experimental study on GABA / MOR co-existed neurons and the connections between ENK immunoreactive terminals and MOR immunoreactive neurons in the rostral portion of the nucleus tractus solitarious of the ratObjectiveTo observe whetherγ-aminobutyric aicd (GABA) andμopioid receptor (MOR) co-exist in neurons of the rostral segment of the nucleus tractus solitarious (rNTS) in the rat, and the connections between Enkephalin immunoreactive (ENK-ir) terminals and MOR immunoreactive (MOR-ir) neurons.Methods1. Materials and methodsAll rats were performed under anesthesia by intraperitoned injection of sodium pentobarbital (80mg/kg, i.p.), and transcardially perfused.It was same with the above-mentioned method.2. Double-immunofluorescent labelingThe sections were used for immunofluorescence histochemical double-staining for MOR/GABA: It was same with the above-mentioned method. Guinea pig anti MOR (1:500) and rabbit anti GABA (1:5000) replace virgin first anti bodies. Cy3-labled donkey anti- guinea pig IgG(1:500) And Fluorescein- labled sheep anti-ribbit IgG(1:500) replace virgin second anti bodies.The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: It was same with the above-mentioned method. Mouse anti ENK (1:500) and Guinea pig anti MOR (1:500) replaced virgin first anti bodies. Biotinylated sheep anti-mouse IgG (1:200), Texas Red-labled avidin (1:200), Fluorescein-labled donkey anti-guinea pig IgG (1:500) replaced virgin second anti bodies.3. Electron microscopic immunohistochemical double-stainingThe sections were used for Electron microscopic immunohisto-chemical double-stainingfor MOR/GABA: It was same with the above-mentioned method. Guinea pig anti MOR (1:500) and rabbit anti GABA (1:5000) replace virgin first anti bodies. Biotinylated sheep anti-rabbit IgG and gold particle-labled coat anti-guinea pig IgG replaced virgin second anti bodies.The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: It was same with the above-mentioned method. Mouse anti ENK (1:500) and Guinea pig anti MOR (1:500) replaced virgin first anti bodies. Biotinylated sheep anti-mouse IgG and gold particle-labled coat anti-guinea pig IgG eplaced virgin second anti bodies.Results1. Under laser scanning confocal microscope1.1 The sections were used for immunofluorescence histochemical double-staining for MOR/GABA: Some GABA-ir neurons (red) and MOR-ir neurons (green) were observed in the rNTS; GABA/MOR co-existed neurons were also observed, and they were small-sized neurons.1.2 The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: There were dense ENK-ir fibers and terminals in the rNTS, they presented red; Close connections between ENK-ir terminals and MOR-ir cell bodies or dendrites were also detected.2. By using the electron microscopic technique2.1 The sections were used for Electron microscopic immunohisto- chemical double-stainingfor MOR/GABA : GABA and MOR co-exist in cell body and dendrites, GABA-ir reaction products were mainly localized on the surface of rough endoplasmic reticulum and ribosome in dendrites, MOR-ir reaction products were mainly localized on the membrane of dendrites and mitochondria, and GABA and MOR co-existed neurons formed symmetric synaptic(mainly) and asymmetric synaptic connections with the imunonegative terminals. 2.2 The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: ENK-ir reaction products were mainly localized on the surface of round clear vesicles and dense-cored vesicles in the axon terminals; ENK-ir terminals formed symmetric synaptic (mainly) and asymmetric synaptic connections with MOR-ir cell bodies and dendrites; Slight MOR-ir reaction products were localized in the axon terminal, ENK-ir and MOR-ir reaction products co-existed,and formed symmetric synaptic connections with the imunonegative dendrite.ConclusionThese results indicate that ENK might have regulatory effects on GABAergic neurons in rNTS by bonding with MOR.更多还原