孤束核味觉区脑啡肽和γ-氨基丁酸对味觉信息的调制作用

发布时间：2018-02-21 11:10

本文关键词： 孤束核脑啡肽 γ-氨基丁酸阿片μ受体味觉免疫荧光双重标记免疫电镜双标大鼠　出处：《河北医科大学》2008年博士论文　论文类型：学位论文

【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。
[Abstract]:The nucleus of the solitary tract (rostral nucleus of in the rostral portion of the the solitary tract, rNST) and taste information transfer and integration of.RNST is closely related not only to accept the facial, glossopharyngeal and vagus nerves gustatory afferents, and close relationship with taste regulation nuclei (parabrachial nucleus, thalamus, hypothalamus, central amygdaloid nucleus, gustatory cortex etc.) reciprocal connections between the.RNST contains many neurotransmitters and neuromodulators. Among them, glutamate and substance P mainly play a role in regulating the excitability, and enkephalin (enkephalin, ENK) and GABA (gamma -aminobutyric acid, GABA) of gustatory neurons produce inhibitory effects of.RNST are distributed in the intensive ENK (ENK-ir) positive fibers and terminals and a large number of Mu and delta opioid receptor (opioid receptor, MOR opioid and delta receptor, DOR).GABA (GABA-ir) positive neurons also exist in the rNST.Malanga found GABA inhibited the activity of opioids, and Echo confirmed the synergistic effect of opioid and GABA function, but the morphological basis of the mechanisms are still not reported. And in NTS GABA neurons are positive for MOR, which is the lack of direct evidence form this. In this study, using stereotaxic apparatus and application of light microscopy, immunohistochemical method, the relationship between rats feeding and rNST change of ENK-ir and GABA-ir structure and MOR-ir neurons and ENK-ir terminal connections were observed.
The behavioral study of feeding changes in rats induced by microinjection of enkephalin and gamma aminobutyric acid in the nucleus of solitary tract in the first part of the nucleus of solitary tract
objective
Brain stereotaxis was used to microinjection of enkephalin and gamma aminobutyric acid into the nucleus of the solitary tract to observe the change of feeding in rats.
Method
1., we measured the basal intake of SD in 22 rats. They were divided into three groups, 10 in group GABA, 10 in group ENK and 2 in control group. They were kept in a cage after being sterilized in a cage, and fed freely, at room temperature 22 + 2 degrees, 12 h light / dark conversion.
2. surgery
2.1 with 2% pentobarbital sodium (45 mg/kg) after intraperitoneal anesthesia, the animal's head was fixed on stereotaxic apparatus, according to Paxinos Watson[Paxinos and and Watson in rNST 1999] maps by skull along the core of stainless steel pipe (diameter 0.9mm) into the rNTS at 2 mm above the tail section (Bregma to 12.80mm, adjacent to the center line of open 1.2mm Bregma 6mm.Bregma is the level down the anterior fontanelle rats) position after surgery, given daily intramuscular injection of penicillin 200 thousand units, for four consecutive days, the wound to prevent intracranial infection and.2.2 intracerebral injection: mild animal anesthesia, a micro syringe needle (diameter 0.4 mm) after catheter insertion in the brain, making it in the tip of the catheter tip below 2 mm, to reach rNTS (Bregma to 12.80mm tail section, adjacent to the center line of open 1.2mm, Bregma 8mm. The 0.2 level down) (per side) rNTS. slowly injected l drug or saline
Measurement of basic food intake after 3.
The cumulative intake of the rats was recorded in the same way at 4 hours, 8 hours, and 12 hours after the injection.
Histological orientation of 4. injection area
At the end of the experiment, in the depth of anesthesia, the animal with saline and 4% paraformaldehyde solution successively through ascending aorta perfusion. Remove the brain and the 4% poly Formaldehyde Solution fixed in 1-2 D. with frozen section machine cut into about 60 m thick slices and HE staining. Then according to the injection site the histological Atlas of histological localization.
Result
1. the site examination of drug injection: the section examination showed that the site of rNST microinjection (the top of the needle canal) was in the nucleus of the nucleus of the solitary tract, and the bilateral symmetry was bilateral.
In group 2.GABA, there were 10 cases, 1 of which had postoperative infection and no need to discard. From 4 hours, 8 hours, 12 hours after injection, the amount of food intake was measured. The remaining 9 showed a decrease in food intake compared with the control group.
In group 3.ENK, food intake was measured at 4 hours, 8 hours and 12 hours after injection, and the food intake decreased compared with the control group, compared with the control group.
conclusion
Microinjection of GABA and ENK into the nucleus of solitary tract can reduce the basal food intake in rats. This is mainly due to the two factors that can inhibit the activity of taste neurons in the nucleus of solitary tract, thereby affecting the basal food intake in rats.
The morphological study of the relationship between enkephalin positive terminal and gamma aminobutyric acid positive neurons in the second part of the rat medulla nucleus of the nucleus tractus soliton
objective
The relationship between enkephalin positive (ENK-ir) end and gamma aminobutyric acid positive (GABA-ir) neurons in the rat nucleus of the nucleus of the nucleus tractus soliton (rNTS) was observed.
Method
The treatment of 1. tissue materials
10 SD rats. The rats with intraperitoneal injection of an overdose of sodium pentobarbital (100 mg/kg) deep anesthesia, thoracotomy, by ascending aorta cannulation, first with 80 ml saline rinse blood reperfusion at 500 ml with 4% paraformaldehyde, 0.1 mol/L phosphate buffered 0.05% glutaraldehyde and 2% picric acid (PB, pH 7.4). Immediately after the cerebral perfusion and arranged in the fresh fixative fixed after 4h, and then into the containing 30% sucrose 0.1 mol/L PB (4 DEG C) to sink to the bottom. The material cut, isolated and lower brainstem slices.
2. immunofluorescence staining
The light was placed in groups containing mouse anti ENK (1:500) and Rabbit anti GABA (1:5000) in the reaction solution (containing 5% normal goat serum and 0.5% Triton X-100) and incubated at room temperature for 24 hours. After immersed with Biotin labeled Sheep anti mouse IgG (1:200) in the reaction solution at room temperature were incubated for 8 the last hour. In Texas containing Red labeled Avidin (1:200) and Fluorescein labeled donkey anti rabbit IgG (1:200) reaction liquid at room temperature, lucifugal incubation for 6 hours. The sections were mounted on slides using SlowFade, fluorescence decay agent after mounting anti laser scanning confocal microscope (Leica TCS-SP2) under observation.
3. packets of pre embedding immunoelectron microscopy
Electron microscope before group immunohistochemical staining, frozen in liquid nitrogen medium a few seconds, placed before, after the slices immersed in freezing protection liquid. The slice was placed in 30% normal goat serum in 30 min after immersion with mouse anti ENK (1:500) and Rabbit anti GABA (1:5000) in the reaction solution (containing 5% normal goat serum), incubated at room temperature for 24 h. and then immersed with Biotin labeled Sheep anti mouse IgG (1:200) reaction liquid and nano gold labeled Goat anti rabbit IgG, at room temperature overnight. In 1% glutaraldehyde fixation after 10 min of the sections with silver silver staining and strengthening kit. Staining before and after using double distilled water cleaning. After using ABC complexes were incubated for 2 h and two conventional diaminobenzidine (DAB) reaction. Among the above steps by PBS will be followed by thorough washing. The immunohistochemical double staining slice into 1% osmic acid solution fixed 1 h, in 70% uranium soaked in wine 4 H, gradient alcohol and epoxy propane dehydration, embedding rNTS Epon-812 tablet. The position of the tissue slices and ultrathin sections, stained in lead citrate (H-7500, Hitachi) under electron microscope observation.
Result
1. under laser confocal scanning microscope
The ENK-ir structure marked in red was distributed in 1.1 rNTS, which was dominated by dense distribution of the end, and the positive fiber was in a medium density distribution.
1.2, the green labeled GABA-ir structure was distributed in rNTS, mainly distributed in small distributed 10~15 and small m neurons. Fibrous and terminal like structures were distributed between positive cells.
The 1.3 part of ENK-ir ends closely with the GABA-ir cell body and the negative cell body (diameter 10~35 m).
2. under electron microscope
2.1 rNTS is stored in many ENK-ir axons and terminals. At the end of ENK-ir, DAB reaction products are mainly deposited on the surface of the circular vesicles, mitochondria and other organelles.
2.2 rNTS was stored in GABA-ir structure. The number and size of black gold particles distributed in the cell body, and the.GABA-ir products in dendrites and small axons were mainly distributed in rough endoplasmic reticulum and ribosome surface.
2.3 the synaptic relationship between the end of the axon of ENK-ir can be seen under electron microscope.
2.3.1 and GABA-ir positive cells, dendrites and dendritic spines formed symmetrical (62%) and asymmetric (38%) axisymmetric (26%) and axis - tree (74%) synapses.
2.3.2 and GABA-ir negative cells, dendrites and dendritic spines formed symmetrical (73%) and asymmetric (27%) axono - body (18%) and axis - tree (82%) synapses.
A small amount of symmetrical axonaxic synapses can be seen between 2.3.3 and GABA-ir negative axons, and the end of ENK-ir axon is a presynaptic or postsynaptic component.
conclusion
The ENK-ir terminal in rNTS may participate in the sense and regulation of taste information in NTS by inhibiting or enhancing the activity of GABA neurons or directly inhibiting the activity of gustatory sensory neurons.
Experimental study on the relationship between GABA and MOR coexisting neurons and ENK positive terminals and MOR positive neurons in the third part of the rat medullary nucleus of the nucleus tractus soliton
objective
The presence of GABA (GABA) coexisted with opioid receptor (MOR) in the rostral part of the nucleus tractus soliton (rNST) and the synaptic connections between MOR positive (MOR-ir) neurons and enkephalin positive (ENK-ir) terminals were observed.
Method
The treatment of 1. tissue materials
SD rats were 22. The rats were injected into the abdominal cavity with excess pentobarbital sodium (100 mg/kg) in the deep anesthetic state. The methods were the same as in the same way.
2 immunofluorescence staining
The same method. Light microscope sections were used for guinea pig anti MOR (1:500) and Rabbit anti GABA (1:5000) to replace the original anti Fluorescein labeled donkey anti guinea pig IgG (1:500) and Cy3 labeled Sheep anti rabbit IgG (1:500) instead of the original two. Second sets of anti light group with anti ENK slice mice (1:500) and guinea pig anti MOR (1:500) to replace the original anti Biotin labeled Sheep anti mouse IgG (1:200) and Texas Red (1:200) labeled Avidin and Fluorescein labeled donkey anti guinea pig IgG (1:500) instead of the original two. More than anti reaction steps.
3. packets of pre embedding immunoelectron microscopy
The electron microscope sections were using mouse anti ENK (1:500) and guinea pig anti MOR (1:500) to replace the original anti Biotin labeled Sheep anti mouse IgG (1:200) and nano gold labeled Goat anti guinea pig IgG instead of the original two. The anti electron microscope group second sets of sections with guinea pig anti MOR (1:500) and the Rabbit anti GABA (1:5000) to replace the original anti Biotin labeled Sheep anti rabbit IgG (1:200) and nano gold labeled Goat anti guinea pig IgG. More than two instead of the original anti reaction steps.
Result
1. under laser confocal scanning microscope
1.1 GABA/MOR double labeling reaction showed that GABA-ir was distributed in rNTS, and MOR-ir structure.GABA-ir (red) and MOR-ir (green) structure were mainly distributed in small size neurons with a diameter of 10-15 m, showing GABA and MOR immunoreactive neurons at the same time.
1.2 ENK/MOR double labeling reaction is visible: visible in rNTS, ENK-ir (red) terminals and MOR-ir neurons (green).ENK-ir terminals are closely related to MOR-ir cell body and protuberance.
2. under electron microscope
2.1 GABA/MOR double reaction visible: GABA and MOR coexist in the soma and dendrites, GABA positive (GABA-ir) products are mainly distributed in the rough endoplasmic reticulum and ribosome surface structure, MOR positive (MOR-ir) product is located in the mitochondrial membrane surface, dendritic membrane, endoplasmic reticulum.GABA-ir structure and MOR-ir neurons and immune response to negative end at the end of the formation of symmetric and asymmetric synapses, with symmetric synapses.
2.2 ENK/MOR double reaction: visible visible to a large number of ENK-ir axons and terminals and MOR-ir cell bodies and dendrites. At ENK-ir end, visible DAB reaction product was mainly deposited on the surface of spherical vesicles and mitochondria in the inner part of the end surface, can also see the positive large granular vesicles.ENK-ir at the end of MOR-ir and neuronal cell bodies and dendrites form synapses with symmetry based. Part of the MOR-ir product is in the axons end and coexists with ENK-ir products, and formed symmetric synapses and dendrites of negative immune response.
conclusion
The GABA neurons in rNST express MOR, and the end of ENK-ir may regulate the activity of GABA energy neurons by binding to MOR.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R33

【引证文献】