本文關(guān)鍵詞： 孤束核 腦啡肽 γ-氨基丁酸 阿片μ受體 味覺 免疫熒光雙重標(biāo)記 免疫電鏡雙標(biāo) 大鼠　出處：《河北醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

【摘要】： 孤束核吻側(cè)段(rostral nucleus of the solitary tract,rNST)與味覺信息的傳遞及整合有密切關(guān)系。rNST不僅接受來自于面、舌咽及迷走神經(jīng)的味覺傳入,而且與味覺調(diào)控關(guān)系密切的核團(臂旁核、下丘腦、丘腦、中央杏仁核、味覺皮質(zhì)等)之間具有往返的纖維聯(lián)系。rNST內(nèi)含有多種神經(jīng)遞質(zhì)和調(diào)質(zhì)。其中,谷氨酸和P物質(zhì)主要發(fā)揮興奮性調(diào)節(jié)作用,而腦啡肽(enkephalin,ENK)和γ-氨基丁酸(γ-aminobutyric acid,GABA)主要對味覺感受神經(jīng)元產(chǎn)生抑制作用。rNST內(nèi)分布有密集的ENK陽性(ENK-ir)纖維和終末及大量的μ型和δ型阿片受體(μopioid receptor,MOR和δopioid receptor, DOR)。GABA陽性(GABA-ir)神經(jīng)元也大量存在于rNST內(nèi)。Malanga等發(fā)現(xiàn)GABA的活性受到阿片類物質(zhì)的抑制,而Echo等則證實阿片與GABA存在功能上的協(xié)同作用,但其作用機制的形態(tài)學(xué)基礎(chǔ)目前仍未見報道。且孤束核內(nèi)的GABA能神經(jīng)元是否呈MOR陽性,這些尚都缺乏直接的形態(tài)學(xué)證據(jù)。為此,本研究利用腦立體定位儀并應(yīng)用光、電鏡免疫組織化學(xué)方法,對大鼠攝食變化及rNST內(nèi)ENK-ir與GABA-ir結(jié)構(gòu)之間的相互聯(lián)系以及MOR-ir神經(jīng)元與ENK-ir終末的聯(lián)系進行了觀察。 第一部分孤束核內(nèi)微量注射腦啡肽和γ-氨基丁酸引起大鼠攝食改變的行為學(xué)研究 目的 利用腦立體定位儀向孤束核內(nèi)微量注射腦啡肽和γ-氨基丁酸,觀察大鼠攝食變化。 方法 1.測量基礎(chǔ)攝食量SD大鼠22只。分為三組,GABA組10只、ENK組10只和對照組2只。均單籠飼養(yǎng)在消毒后的籠子中,自由進水、飲食,室溫22±2℃,12 h光照/黑暗轉(zhuǎn)換。連續(xù)喂養(yǎng)5日,以適應(yīng)環(huán)境。并測量累積攝食量。 2.外科手術(shù) 2.1用2%戊巴比妥鈉(45 mg/kg)腹腔麻醉后,將動物頭部固定在立體定位儀上,根據(jù)Paxinos和Watson[Paxinos and Watson, 1999]圖譜經(jīng)顱骨在rNST將一帶芯不銹鋼導(dǎo)管(外徑0.9mm)插入rNTS上方2.0 mm處(Bregma向尾段12.80mm,中線旁開1.2mm,Bregma點水平向下6mm。Bregma點是大鼠的前囟門位置)手術(shù)后,每天給予肌肉注射青霉素20萬單位,連續(xù)四天,以防傷口和顱內(nèi)感染。2.2腦內(nèi)注射:動物輕微麻醉后,將一微量注射器針頭(外徑0.4 mm)經(jīng)導(dǎo)管插入腦內(nèi),使其尖端在導(dǎo)管尖端下方2.0 mm處,以到達rNTS(Bregma向尾段12.80mm,中線旁開1.2mm,Bregma點水平向下8mm)。將0.2(每側(cè))μl的藥物或生理鹽水緩慢地注入rNTS。 3.術(shù)后測量基礎(chǔ)攝食量 在注藥后4小時、8小時、12小時按同一方法記錄大鼠累積進食量。 4.注射區(qū)的組織學(xué)定位 實驗結(jié)束時,在深度麻醉下,將動物用生理鹽水和4%多聚甲醛液中相繼經(jīng)升主動脈進行灌注。取出腦并將其在4%多聚甲醛溶液中后固定1-2 d。用冰凍切片機切成大約60μm厚的切片,HE染色。然后將注射位點按組織學(xué)圖譜進行組織學(xué)定位。 結(jié)果 1.注藥部位檢查:切片檢查顯示,rNST微量注射的部位(即針道的頂端)處于孤束核吻端內(nèi),雙側(cè)對稱。 2.GABA組10只,其中有1只出現(xiàn)了術(shù)后感染,棄去不用。從分別在注藥后4小時、8小時、12小時測量攝食量,剩余9只與對照組相比,均出現(xiàn)攝食量降低。 3.ENK組10只分別在注藥后4小時、8小時、12小時測量攝食量,與對照組相比,均出現(xiàn)攝食量降低。 結(jié)論 分別向孤束核內(nèi)微量注射GABA和ENK,均可引起大鼠基礎(chǔ)攝食量降低,這主要是由于二者均可抑制孤束核內(nèi)的味覺神經(jīng)元活性,從而影響大鼠的基礎(chǔ)攝食量。 第二部分大鼠延髓孤束核吻側(cè)段內(nèi)腦啡肽陽性終末與γ-氨基丁酸陽性神經(jīng)元聯(lián)系的形態(tài)學(xué)研究 目的 觀察大鼠孤束核吻側(cè)段(rNTS)內(nèi)腦啡肽陽性(ENK-ir)終末與γ-氨基丁酸陽性(GABA-ir)神經(jīng)元之間的聯(lián)系。 方法 1.組織材料的處理 SD大鼠10只。將大鼠在腹腔內(nèi)注射過量戊巴比妥鈉(100 mg/kg)的深麻醉狀態(tài)下開胸,經(jīng)升主動脈插管,先用80 ml生理鹽水沖凈血液,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磷酸緩沖液(PB,pH 7.4)。灌注完畢立即取腦并置于上述新鮮固定液中后固定4h,再移入含30%蔗糖的0.1 mol/L PB(4℃)內(nèi)至沉底。將材料切塊,分離出低位腦干并切片。 2.免疫熒光染色法 將光鏡組切片置于含小鼠抗ENK(1:500)及兔抗GABA(1:5000)的反應(yīng)液內(nèi)(含5%正常山羊血清及0.5% Triton X-100)室溫孵育24小時。之后浸入含Biotin標(biāo)記的綿羊抗小鼠IgG(1:200)的反應(yīng)液內(nèi)于室溫下孵育8小時。最后入含Texas Red標(biāo)記的Avidin(1:200)及Fluorescein標(biāo)記的驢抗兔IgG(1:200)反應(yīng)液,室溫下避光孵育6小時。將上述切片裱于載玻片上,使用SlowFade抗熒光衰減劑封片后于激光掃描共聚焦顯微鏡(Leica TCS-SP2)下觀察。 3.包埋前染色免疫電鏡法 電鏡組切片在進行免疫組化染色之前,置于液氮中速凍數(shù)秒,放置前、后將切片浸于冰凍保護液中。將切片置于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)的反應(yīng)液內(nèi)(含5%正常山羊血清),室溫孵育24 h。隨后浸入含Biotin標(biāo)記的綿羊抗小鼠IgG(1:200)及納米金標(biāo)記的山羊抗兔IgG的反應(yīng)液內(nèi),室溫下過夜。在1%戊二醛內(nèi)固定10 min后將切片用銀加強試劑盒進行銀加強染色,染色前后使用雙蒸水清洗。之后使用ABC復(fù)合物孵育2 h并進行常規(guī)二氨基聯(lián)苯胺(DAB)反應(yīng)。上述各步驟之間均用PBS徹底清洗。隨后將經(jīng)上述免疫組化雙重染色的切片置入1%鋨酸溶液固定1 h,在70%酒鈾中浸泡4 h,梯度酒精及環(huán)氧丙烷脫水,Epon-812平板包埋。取rNTS位置的組織片做超薄切片,枸櫞酸鉛染色后于電鏡(H-7500,Hitachi)下觀察。 結(jié)果 1.在激光共聚焦掃描顯微鏡下 1.1 rNTS內(nèi)分布著紅色標(biāo)示的ENK-ir結(jié)構(gòu),以密集分布的終末為主,陽性纖維呈中等密度分布。 1.2 rNTS內(nèi)分布著綠色標(biāo)示的GABA-ir結(jié)構(gòu),以散在分布且直徑為10~15μm的小神經(jīng)元為主,纖維和終末樣結(jié)構(gòu)則稀疏地分布于陽性胞體之間。 1.3部分ENK-ir終末與GABA-ir胞體以及陰性的胞體(直徑10~35μm)之間形成密切接觸。 2.在電鏡下 2.1 rNTS內(nèi)存在許多ENK-ir軸突和終末。在ENK-ir終末內(nèi),可見DAB反應(yīng)產(chǎn)物主要沉積于圓形清亮囊泡表面及線粒體等細(xì)胞器表面,在部分終末內(nèi)還可見到陽性的大顆粒囊泡。 2.2 rNTS內(nèi)存在GABA-ir結(jié)構(gòu),可見數(shù)目及大小不等的黑色金顆粒散在分布于胞體、樹突及少量軸突內(nèi)。GABA-ir產(chǎn)物主要分布于粗面內(nèi)質(zhì)網(wǎng)及核糖體表面等結(jié)構(gòu)。 2.3電鏡下可見到ENK-ir軸突終末形成以下突觸關(guān)系: 2.3.1與GABA-ir陽性胞體、樹突及樹突棘形成對稱(62%)及非對稱性(38%)軸-體(26%)及軸-樹(74%)突觸。 2.3.2與GABA-ir陰性的胞體、樹突及樹突棘形成對稱(73%)及非對稱性(27%)軸-體(18%)及軸-樹(82%)突觸; 2.3.3與GABA-ir陰性的軸突之間還可見到少量對稱性軸-軸突觸,ENK-ir軸突終末為突觸前或后成分。 結(jié)論 rNTS內(nèi)的ENK-ir終末可能通過抑制或增強GABA能神經(jīng)元活性或者直接抑制味覺感受神經(jīng)元活性的方式參與NTS內(nèi)味覺信息的感受和調(diào)節(jié)。 第三部分大鼠延髓孤束核吻側(cè)段內(nèi)GABA和MOR共存神經(jīng)元及ENK陽性終末與MOR陽性神經(jīng)元聯(lián)系的實驗研究 目的 觀察延髓孤束核吻側(cè)段(rNST)內(nèi)是否存在γ-氨基丁酸(GABA)與阿片μ受體(MOR)共存的神經(jīng)元,以及MOR陽性(MOR-ir)神經(jīng)元與腦啡肽陽性(ENK-ir)終末的突觸聯(lián)系。 方法 1.組織材料的處理 SD大鼠22只。將大鼠在腹腔內(nèi)注射過量戊巴比妥鈉(100 mg/kg)的深麻醉狀態(tài)下灌注,取材。方法同上。 2免疫熒光染色法 方法同上。光鏡組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標(biāo)記的驢抗豚鼠IgG(1:500)和Cy3標(biāo)記的綿羊抗兔IgG(1:500)代替原二抗。光鏡組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標(biāo)記的綿羊抗小鼠IgG(1:200)及Texas Red標(biāo)記的Avidin(1:200)及Fluorescein標(biāo)記的驢抗豚鼠IgG(1:500)代替原二抗。余反應(yīng)步驟同。 3.包埋前染色免疫電鏡法 將電鏡組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標(biāo)記的綿羊抗小鼠IgG(1:200)及納米金標(biāo)記的山羊抗豚鼠IgG代替原二抗。將電鏡組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標(biāo)記的綿羊抗兔IgG(1:200)及納米金標(biāo)記的山羊抗豚鼠IgG代替原二抗。余反應(yīng)步驟同。 結(jié)果 1.在激光共聚焦掃描顯微鏡下 1.1 GABA/MOR雙標(biāo)反應(yīng)可見:可見rNTS內(nèi)分布著GABA-ir、MOR-ir結(jié)構(gòu)。GABA-ir(紅色)和MOR-ir(綠色)結(jié)構(gòu)均以散在分布且直徑為10-15μm的小型神經(jīng)元為主�？梢娡瑫r呈GABA和MOR免疫反應(yīng)陽性的神經(jīng)元。 1.2 ENK/MOR雙標(biāo)反應(yīng)可見:可見rNTS內(nèi)分布著ENK-ir(紅色)終末與MOR-ir神經(jīng)元(綠色)。ENK-ir終末與MOR-ir神經(jīng)元的胞體及突起形成密切接觸。 2.在電鏡下 2.1 GABA/MOR雙標(biāo)反應(yīng)可見:GABA與MOR共存于胞體及樹突內(nèi),GABA陽性(GABA-ir)產(chǎn)物主要分布于粗面內(nèi)質(zhì)網(wǎng)及核糖體表面等結(jié)構(gòu),MOR陽性(MOR-ir)產(chǎn)物位于樹突膜、線粒體膜、內(nèi)質(zhì)網(wǎng)膜等結(jié)構(gòu)表面。GABA-ir與MOR-ir共存神經(jīng)元與免疫反應(yīng)陰性的終末形成對稱性及非對稱性突觸,以對稱性突觸為主。 2.2 ENK/MOR雙標(biāo)反應(yīng)可見:可見到大量的ENK-ir軸突和終末和MOR-ir胞體和樹突。在ENK-ir終末內(nèi),可見DAB反應(yīng)產(chǎn)物主要沉積于圓形清亮囊泡表面及線粒體等細(xì)胞器表面,在部分終末內(nèi)還可見到陽性的大顆粒囊泡。ENK-ir終末與MOR-ir神經(jīng)元的胞體及樹突形成以對稱性為主的突觸聯(lián)系。部分MOR-ir產(chǎn)物存在于軸突末端,且與ENK-ir產(chǎn)物共存,并且與免疫反應(yīng)陰性樹突形成對稱性突觸。 結(jié)論 rNST內(nèi)的GABA能神經(jīng)元表達MOR,而ENK-ir終末可能通過與MOR結(jié)合調(diào)節(jié)GABA能神經(jīng)元的活性,從而參與味覺信息的感受和調(diào)節(jié)。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R33

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 李鵬飛;阿片肽在束縛—浸水應(yīng)激致大鼠胃黏膜損傷中的作用的研究[D];山東師范大學(xué);2011年

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本文編號：1521819