【摘要】：目的：喉化學(xué)反射,由液體誤吸入喉誘導(dǎo),產(chǎn)生顯著的呼吸抑制。喉上神經(jīng)電刺激能誘導(dǎo)呼吸暫停反射,抑制中樞吸氣活動(dòng),激發(fā)呼氣性喉運(yùn)動(dòng)神經(jīng)元放電活動(dòng)。但是喉上神經(jīng)電刺激能誘導(dǎo)呼吸暫停反射產(chǎn)生呼吸暫停的機(jī)制并不清楚。我們目的是的是研究由喉上神經(jīng)刺激誘發(fā)呼吸暫停反射的神經(jīng)調(diào)控路徑。 方法：喉上神經(jīng)電刺激(20Hz,0.2ms)誘導(dǎo)呼吸暫停反射,繼而微量注射GABAA受體激動(dòng)劑isoguvacine (10 mM,20-40 nl)到同側(cè)包欽格復(fù)合體和對(duì)側(cè)的孤束核。經(jīng)過(guò)60mmin的注射后恢復(fù)期,僅微量注射isoguvacine到對(duì)側(cè)孤束核。喉返神經(jīng)活動(dòng)記錄用來(lái)監(jiān)測(cè)呼氣性喉運(yùn)動(dòng)神經(jīng)元的簇發(fā)放電活動(dòng)。微量注射isoguvacine兩分鐘后,記錄喉返神經(jīng)和膈神經(jīng)的電信號(hào)來(lái)評(píng)價(jià)isoguvacine的作用效果。 結(jié)果：喉上神經(jīng)刺激(20 Hz)誘發(fā)呼吸暫停反射,膈神經(jīng)放電減低到基線的12士9%。微量注射isoguvacine到同側(cè)的包欽格復(fù)合體后,呼吸暫停反射明顯減弱,然后微量注射isoguvacine到對(duì)側(cè)的孤束核,喉上神經(jīng)刺激誘發(fā)的呼吸暫停反射完全被取消。但是,喉上神經(jīng)刺激誘導(dǎo)的呼氣性喉運(yùn)動(dòng)神經(jīng)元的簇發(fā)放電活動(dòng)沒(méi)有受到影響。僅微量注射isoguvacine到對(duì)側(cè)的孤束核,呼吸暫停反射幾乎沒(méi)有影響。的包欽格復(fù)合體和對(duì)側(cè)的孤束核后,呼吸暫停反射幾乎被完全取消,但是呼氣性喉運(yùn)動(dòng)神經(jīng)元的簇發(fā)放電活動(dòng)沒(méi)有受到影響。結(jié)果提示從孤束核到雙側(cè)的疑核尾段和包欽格復(fù)合體的神經(jīng)投射可能分別介導(dǎo)呼氣性喉運(yùn)動(dòng)神經(jīng)元的簇發(fā)放電活動(dòng)和呼吸暫停反射。 目的：研究證明很多運(yùn)動(dòng)神經(jīng)元和呼吸神經(jīng)元接受酪氨酸羥化酶類免疫陽(yáng)性遞質(zhì)的傳遞,但是位于疑核尾段的喉運(yùn)動(dòng)神經(jīng)元是否接受該遞質(zhì)的投射至今并不清楚。本實(shí)驗(yàn)?zāi)康氖?)酪氨酸羥化酶免疫陽(yáng)性的神經(jīng)終端是否和呼氣性喉運(yùn)動(dòng)神經(jīng)元形成密切接觸結(jié)構(gòu)；如果結(jié)果是陽(yáng)性,2)研究大鼠喉運(yùn)動(dòng)神經(jīng)元接受的兒茶酚胺類神經(jīng)遞質(zhì)的來(lái)源。 方法：本實(shí)驗(yàn)中,通過(guò)結(jié)合細(xì)胞內(nèi)標(biāo)記和免疫組織化學(xué)的方法,我們主要研究SD大鼠的呼氣性喉運(yùn)動(dòng)神經(jīng)元是否接收酪氨酸羥化酶免疫陽(yáng)性神經(jīng)遞質(zhì)的傳遞。呼氣性喉運(yùn)動(dòng)神經(jīng)元的辨識(shí)依據(jù)它的吸氣后相放電活動(dòng)特點(diǎn)和對(duì)喉返神經(jīng)刺激產(chǎn)生的逆反射。進(jìn)一步的神經(jīng)示蹤實(shí)驗(yàn),通過(guò)注射霍亂毒素B亞單位到尾段疑核。逆向標(biāo)記的神經(jīng)元和酪氨酸羥化酶免疫陽(yáng)性結(jié)構(gòu)通過(guò)熒光雙標(biāo)記的方法來(lái)顯示。 結(jié)果：所有的呼氣性喉運(yùn)動(dòng)神經(jīng)元上均發(fā)現(xiàn)酪氨酸羥化酶免疫陽(yáng)性的神經(jīng)終端形成密切接觸結(jié)構(gòu),其平均數(shù)目是每個(gè)神經(jīng)元上約18±5(n=7,mean±SD)個(gè)密切接觸的結(jié)構(gòu)。大多數(shù)的密切接觸的結(jié)構(gòu)常在遠(yuǎn)端樹(shù)突上被發(fā)現(xiàn),近端樹(shù)突上的數(shù)目較少,但是在胞體和軸突上,沒(méi)有酪氨酸羥化酶免疫陽(yáng)性的密切接觸結(jié)構(gòu)發(fā)現(xiàn)。通過(guò)將霍亂毒素B亞單位注射到尾段疑核,進(jìn)行進(jìn)一步的示蹤方法來(lái)研究投射到喉運(yùn)動(dòng)神經(jīng)元的兒茶酚胺類神經(jīng)遞質(zhì)的來(lái)源。結(jié)果發(fā)現(xiàn),注射部位同側(cè)的孤束核和延髓最后區(qū)內(nèi)有大量的逆向標(biāo)記的兒茶酚胺類神經(jīng)元,并且密度最高的位置出現(xiàn)在閂部尾向0.2-0.4 mm。 結(jié)論：我們首先證實(shí)了呼氣性喉運(yùn)動(dòng)神經(jīng)元與酪氨酸羥化酶免疫陽(yáng)性神經(jīng)終端形成密切接觸結(jié)構(gòu),表明兒茶酚胺類神經(jīng)遞質(zhì)可能對(duì)呼氣性喉運(yùn)動(dòng)神經(jīng)元的活動(dòng)起到一定作用。逆向示蹤法研究揭示了大量的逆向標(biāo)記的兒茶酚胺類神經(jīng)元,其在孤束核的定位和喉上神經(jīng)的輸入信息投射到孤束核的水平相同,表明孤束核內(nèi)位于閂部水平的兒茶酚胺類神經(jīng)元可能對(duì)喉上神經(jīng)介導(dǎo)的氣道保護(hù)性反射起到重要作用。 目的：位于疑核尾段的喉運(yùn)動(dòng)神經(jīng)元,通過(guò)控制喉內(nèi)肌的運(yùn)動(dòng)來(lái)表達(dá)不同的喉功能,包括呼吸,發(fā)聲,和氣道的保護(hù)性反射,例如咳嗽反射,噴嚏和吞咽反射。喉運(yùn)動(dòng)神經(jīng)元接受來(lái)自不同腦部核團(tuán)的不同神經(jīng)化學(xué)物質(zhì)的傳遞,不同神經(jīng)化學(xué)物質(zhì)可能對(duì)喉運(yùn)動(dòng)神經(jīng)元有不同的作用和影響,從而引起不同的喉功能。P物質(zhì),酪氨酸羥化酶和5-羥色胺免疫陽(yáng)性神經(jīng)終端投射到到喉運(yùn)動(dòng)神經(jīng)元已經(jīng)在光鏡水平下得到研究,但是它們?cè)谝珊宋捕蔚姆植急容^還不明確。而且,這些神經(jīng)化學(xué)物質(zhì)的免疫陽(yáng)性神經(jīng)終端與喉運(yùn)動(dòng)神經(jīng)元在超微結(jié)構(gòu)水平的關(guān)系的研究,也是欠缺的。而這種超微結(jié)構(gòu)水平的研究是用來(lái)證實(shí)神經(jīng)化學(xué)突觸結(jié)構(gòu)和關(guān)系的必要方法。因此,我們研究的目的是(1)評(píng)價(jià)和比較P物質(zhì),酪氨酸羥化酶和5-羥色胺免疫陽(yáng)性神經(jīng)終端在疑核尾段的分布；(2)通過(guò)電生理細(xì)胞內(nèi)記錄,免疫組織化學(xué)和電鏡學(xué),研究P物質(zhì)免疫陽(yáng)性神經(jīng)突觸終端與喉運(yùn)動(dòng)神經(jīng)元的超微結(jié)構(gòu)關(guān)系。 方法：我們通過(guò)應(yīng)用多重免疫熒光法和共聚焦顯微鏡學(xué)方法來(lái)同時(shí)評(píng)估P物質(zhì),酪氨酸羥化酶和5-羥色胺免疫陽(yáng)性神經(jīng)終端在疑核尾段的分布。疑核尾段運(yùn)動(dòng)神經(jīng)元被膽堿乙酰轉(zhuǎn)移酶的免疫反應(yīng)陽(yáng)性標(biāo)記和辨識(shí)。突觸體素是一種突觸前結(jié)構(gòu)的蛋白。突觸體素在疑核尾段內(nèi)的表達(dá)代表了總的神經(jīng)突觸終端的數(shù)量。運(yùn)用Image J軟件共定位分析后,突觸體素陽(yáng)性和P物質(zhì),酪氨酸羥化酶或者5-羥色胺免疫陽(yáng)性共同表達(dá)的區(qū)域代表了P物質(zhì),酪氨酸羥化酶或者5-羥色胺的神經(jīng)突觸終端區(qū)域。P物質(zhì),酪氨酸羥化酶或者5-羥色胺的神經(jīng)突觸終端占總神經(jīng)突觸終端區(qū)域的比例,被用來(lái)評(píng)價(jià)和比較它們?cè)谝珊宋捕蔚耐挥|終端的分布。在比較P物質(zhì),酪氨酸羥化酶和5-羥色胺的神經(jīng)突觸在疑核尾段的分布的基礎(chǔ)上,我們進(jìn)一步研究了P物質(zhì)免疫陽(yáng)性神經(jīng)終端與喉運(yùn)動(dòng)神經(jīng)元的超微結(jié)構(gòu)關(guān)系。在一例實(shí)驗(yàn)中,一個(gè)吸氣性喉運(yùn)動(dòng)神經(jīng)元通過(guò)細(xì)胞內(nèi)記錄,頸迷走神經(jīng)刺激和它的疑核尾段的定位被辨識(shí)和確認(rèn)。然后注射神經(jīng)生物素(biotinamide,1.5%)入這個(gè)吸氣性喉運(yùn)動(dòng)神經(jīng)元。被生物胞素注射的神經(jīng)元和P物質(zhì)免疫陽(yáng)性結(jié)構(gòu),通過(guò)電鏡包埋前免疫組化染色法,同時(shí)顯示出來(lái)。包含有喉運(yùn)動(dòng)神經(jīng)元和P物質(zhì)免疫陽(yáng)性結(jié)構(gòu)的超薄切片染色并在電鏡下觀察和分析。 結(jié)果：我們研究發(fā)現(xiàn),P物質(zhì),酪氨酸羥化酶或者5-羥色胺的神經(jīng)突觸終端占疑核尾段總神經(jīng)突觸終端的強(qiáng)度比例,均不超過(guò)10%,三者合計(jì)不超過(guò)15%。和酪氨酸羥化酶或者5-羥色胺比較, P物質(zhì)神經(jīng)突觸終端在疑核尾段有相對(duì)較高的強(qiáng)度比例。在超微結(jié)構(gòu)水平,53.3%(114/206)的突觸膨大終端與該吸氣性喉運(yùn)動(dòng)神經(jīng)元的樹(shù)突形成不對(duì)稱突觸結(jié)構(gòu),22.3%(46/206)形成對(duì)稱突觸結(jié)構(gòu),其他的膨大終端與神經(jīng)元形成接觸,但是沒(méi)有清晰的特殊突觸結(jié)構(gòu)。在這206個(gè)神經(jīng)終端中,16%(33/206)是P物質(zhì)免疫陽(yáng)性神經(jīng)終端并與神經(jīng)元形成突觸結(jié)構(gòu)。29個(gè)形成不對(duì)稱突觸結(jié)構(gòu),4個(gè)形成對(duì)稱突觸結(jié)構(gòu)。少數(shù)幾個(gè)P物質(zhì)免疫陽(yáng)性神經(jīng)終端與神經(jīng)元胞體形成突觸結(jié)構(gòu),但是在神經(jīng)元軸突上沒(méi)有發(fā)現(xiàn)P物質(zhì)神經(jīng)終端形成突觸結(jié)構(gòu)。非免疫反應(yīng)性的神經(jīng)終端也在神經(jīng)元樹(shù)突棘上形成突觸結(jié)構(gòu),一些具有突觸下特殊結(jié)構(gòu)。在吸氣性喉運(yùn)動(dòng)神經(jīng)元所在的組織切片上,還可以觀察到幾個(gè)具有大胞體(直徑約為30-40μm)的,疑核非免疫陽(yáng)性神經(jīng)元。它們含有大的胞核和明顯的核仁。 結(jié)論：首先,我們的結(jié)果第一次證實(shí)了,在大鼠疑核尾段,P物質(zhì),酪氨酸羥化酶和5-羥色胺的神經(jīng)突觸終端僅占突觸體素免疫陽(yáng)性的總的神經(jīng)突觸終端的少數(shù),這間接表明了P物質(zhì),酪氨酸羥化酶和5-羥色胺在喉運(yùn)動(dòng)神經(jīng)元的功能上僅起到適中的調(diào)控作用。第二,我們證實(shí)了P物質(zhì)免疫陽(yáng)性神經(jīng)終端在喉運(yùn)動(dòng)神經(jīng)元上形成突觸結(jié)構(gòu)。在一例實(shí)驗(yàn)中,一個(gè)吸氣性神經(jīng)元,位于疑核尾段,頸迷走神經(jīng)刺激后受到激活,標(biāo)記并進(jìn)行超微結(jié)構(gòu)分析。該標(biāo)記的神經(jīng)元接受大量的對(duì)稱和不對(duì)稱的突觸傳遞。共計(jì)發(fā)現(xiàn)33個(gè)P物質(zhì)的神經(jīng)終端在吸氣性喉運(yùn)動(dòng)神經(jīng)元的樹(shù)突上形成突觸結(jié)構(gòu)。其中,大多數(shù)(29)為不對(duì)稱性突觸,少數(shù)(4)為對(duì)稱性突觸,表明表達(dá)P物質(zhì)的神經(jīng)元可能直接提高和增強(qiáng)吸氣性喉運(yùn)動(dòng)神經(jīng)元的興奮性,主要通過(guò)近端樹(shù)突上的化學(xué)突觸傳遞。第三,我們研究了包含吸氣性喉運(yùn)動(dòng)神經(jīng)元的疑核尾段內(nèi)的大胞體神經(jīng)元的總體超微結(jié)構(gòu)和突觸學(xué)。在這些神經(jīng)元上發(fā)現(xiàn)大量的對(duì)稱和不對(duì)稱的突觸結(jié)構(gòu)。 目的：在呼吸運(yùn)動(dòng)以及其他條件下和其他活動(dòng)中,例如低碳酸血癥和睡眠,喉內(nèi)肌接受不同條件的調(diào)控。以往的解剖學(xué)和藥理學(xué)研究表明,在疑核水平的乙酰膽堿起到調(diào)控喉運(yùn)動(dòng)神經(jīng)元的活動(dòng)的作用。疑核松散結(jié)構(gòu)含有吸氣性和呼氣性喉運(yùn)動(dòng)神經(jīng)元。本實(shí)驗(yàn)?zāi)康氖茄芯恳珊怂缮⒔Y(jié)構(gòu)內(nèi)的喉運(yùn)動(dòng)神經(jīng)元接受膽堿能輸入信息的解剖學(xué)特點(diǎn)。 方法：我們采用體內(nèi)細(xì)胞內(nèi)記錄,染色劑細(xì)胞內(nèi)注射,和免疫組織化學(xué)的方法來(lái)研究喉運(yùn)動(dòng)神經(jīng)元和膽堿能免疫陽(yáng)性神經(jīng)終端之間的解剖學(xué)關(guān)系。突觸體素是突觸的標(biāo)志蛋白,因此突觸體素免疫反應(yīng)陽(yáng)性的神經(jīng)終端可視為神經(jīng)突觸終端。我們采用共聚焦顯微鏡的免疫熒光學(xué)方法和共定位分析,即膽堿能免疫陽(yáng)性神經(jīng)終端與突觸體素的免疫反應(yīng)性共定位的分析,進(jìn)一步研究和評(píng)估了與喉運(yùn)動(dòng)神經(jīng)元有“密切接觸”關(guān)系的膽堿能免疫陽(yáng)性神經(jīng)終端中,同時(shí)又呈現(xiàn)突觸體素免疫陽(yáng)性的比例。 結(jié)果：結(jié)果證實(shí)了呼氣性喉運(yùn)動(dòng)神經(jīng)元與囊泡乙酰膽堿轉(zhuǎn)運(yùn)體免疫陽(yáng)性神經(jīng)終端之間存在“密切接觸”。共有12個(gè)呼氣性喉運(yùn)動(dòng)神經(jīng)元被細(xì)胞內(nèi)記錄識(shí)別和被神經(jīng)生物素注射而標(biāo)記。其中,呼氣性喉運(yùn)動(dòng)神經(jīng)元上發(fā)現(xiàn)較多的囊泡乙酰膽堿轉(zhuǎn)運(yùn)體免疫陽(yáng)性神經(jīng)終端形成“密切接觸”(mean±SD,32±9;n=8)。與遠(yuǎn)端樹(shù)突相比,喉呼氣性運(yùn)動(dòng)神經(jīng)元的細(xì)胞體和近端樹(shù)突有更多的“密切接觸”(two-way ANOVA,P0.0001).共聚焦顯微鏡熒光學(xué)方法證實(shí)了約90%的“密切接觸”結(jié)構(gòu)中囊泡乙酰膽堿轉(zhuǎn)運(yùn)體免疫陽(yáng)性神經(jīng)終端與突觸體素存在共定位結(jié)構(gòu)(突觸終端=45,呼氣性喉運(yùn)動(dòng)神經(jīng)元=4)。 結(jié)論：本實(shí)驗(yàn)的創(chuàng)新點(diǎn)在于,在體實(shí)驗(yàn)功能性識(shí)別的呼氣性喉運(yùn)動(dòng)神經(jīng)元,與囊泡乙酰膽堿轉(zhuǎn)運(yùn)體免疫陽(yáng)性神經(jīng)終端存在“密切接觸”。與遠(yuǎn)端樹(shù)突相比較,在細(xì)胞體和近段樹(shù)突發(fā)現(xiàn)較多的“密切接觸”。通過(guò)細(xì)胞內(nèi)記錄,共聚焦顯微鏡學(xué)和突觸標(biāo)志蛋白的使用,我們還評(píng)價(jià)了“密切接觸”是否代表突觸結(jié)構(gòu)。大多數(shù)囊泡乙酰膽堿轉(zhuǎn)運(yùn)體免疫陽(yáng)性神經(jīng)終端與喉運(yùn)動(dòng)神經(jīng)元之間的“密切接觸”,呈現(xiàn)突觸體素免疫陽(yáng)性�？傊�,我們的實(shí)驗(yàn)發(fā)現(xiàn)為膽堿能遞質(zhì)輸入對(duì)喉運(yùn)動(dòng)神經(jīng)元活動(dòng)存在潛在的重要作用提供了解剖學(xué)證據(jù)。但是喉運(yùn)動(dòng)神經(jīng)元上膽堿能受體的表達(dá),以及所接受的膽堿能遞質(zhì)輸入的來(lái)源,需要進(jìn)一步的研究。
[Abstract]:Objective: the laryngeal chemical reflex is induced by the larynx induced by the liquid inhalation of the larynx. The electrical stimulation of the superior laryngeal nerve can induce apnea reflex, inhibit the activity of the central nervous system and stimulate the discharge activity of the exhalation laryngeal motor neurons. However, the mechanism of the electrical stimulation of the superior laryngeal nerve can induce the respiratory suspension reflex to produce apnea. The aim is to study the neural regulation pathway of apnea reflex induced by stimulation of superior laryngeal nerve.
Methods: 20Hz (0.2ms) induced apnea reflex, and then microinjection of GABAA receptor agonist isoguvacine (10 mM, 20-40 NL) to the ipsilateral paratagal complex and contralateral nucleus of solitary tract. After 60mmin injection, only microinjection of isoguvacine to the contralateral nucleus of the solitary tract was used to monitor the recurrent laryngeal nerve activity. After two minutes of microinjection of isoguvacine, the electrical signals of the recurrent laryngeal nerve and phrenic nerve were recorded to evaluate the effect of isoguvacine.
Results: the apnea reflex was induced by the stimulation of the superior laryngeal nerve (20 Hz), and the phrenic nerve discharge decreased to the baseline of 12 isoguvacine to the same side of the pachtchin complex. The apnea reflex was markedly weakened, and then isoguvacine was injected into the contralateral nucleus of the solitary tract, and the apnea reflex induced by the upper laryngeal nerve stimulation was completely cancelled. However, the cluster discharge activity of the exhaled laryngeal motor neurons induced by the stimulation of the superior laryngeal nerve was not affected. Only microinjection of isoguvacine to the contralateral nucleus of the solitary tract had little effect on the apnea reflex. After the package Qinge complex and the contralateral nucleus of the solitary tract, the apnea was almost completely cancelled, but exhaled larynx movement The results suggest that the neural projections from the nucleus of the nucleus to the bilateral nucleus of the nucleus and the Bao Qin lattice may mediate the cluster discharge and apnea of the exhaled laryngeal motoneurons, respectively.
Objective: to demonstrate that many motor neurons and respiratory neurons accept the transfer of tyrosine hydroxylase - like immuno - positive transmitters, but it is not clear whether the projection of the laryngeal motoneurons located in the nucleus of the nucleus of the nucleus is not clear. The purpose of this experiment is 1) whether the tyrosine hydroxylase immunoreactive nerve terminal and exhalation larynx are used. If the results are positive, 2) Study the source of catecholamine neurotransmitters received by rat laryngeal motor neurons.
Methods: in this experiment, we mainly study whether the expiratory laryngeal neurons in SD rats receive the transmission of tyrosine hydroxylase immunoreactive neurotransmitters by combining the intracellular labeling and immunohistochemistry. The identification of exhaled laryngeal motoneurons is based on the characteristics of its discharge activity in the post inhalation phase of the larynx and on the recurrent laryngeal nerve spines. A further neural tracer experiment, through the injection of cholera toxin B subunit to the caudal nucleus. Reverse labeled neurons and tyrosine hydroxylase immunoreactive structures are displayed by a fluorescence double labeling method.
Results: the close contact structure of tyrosine hydroxylase immunoreactive nerve terminals was found on all exhaled laryngeal motoneurons, with the average number of close contact structures about 18 + 5 (n=7, mean + SD) on each neuron. Most of the close contact structures were often found on the distal dendrites and the number of the proximal dendrites. There is less, but there is no close contact structure of tyrosine hydroxylase immunoreactivity on the cell body and axon. The source of catecholamine neurotransmitters projecting into the laryngeal motoneurons is studied by injecting the cholera toxin B subunit into the caudal nucleus, and the results show that the isolated bundle of the injection site is on the same side. A large number of retrogradely labeled catecholamine neurons were found in the nucleus and medulla oblongata, and the highest density was found in the caudal part of the latch 0.2-0.4 mm.
Conclusion: we first confirmed the close contact structure of the exhaled laryngeal motoneurons with the tyrosine hydroxylase immunoreactive nerve terminal, indicating that the catecholamine neurotransmitters may play a role in the activity of the exhaled laryngeal motoneurons. The location of the nucleus in the nucleus of the solitary tract and the input information of the superior laryngeal nerve projected to the same level of the nucleus of the solitary tract, indicating that the catecholamines at the latch level in the nucleus of the solitary tract may play an important role in the protective reflex of the upper laryngeal nerve.
Objective: the laryngeal motor neurons, located in the nucleus of the nucleus, express different laryngeal functions by controlling the movement of the intramuscular muscles, including respiratory, vocal, and protective reflex of the airway, such as coughing reflex, sneezing and swallowing reflex. Laryngeal motor neurons accept the transfer of different neurochemicals from different brain nuclei and different neurochemistry. Substances may have different effects and effects on laryngeal motor neurons, resulting in different laryngeal function.P substances. Tyrosine hydroxylase and 5- serotonin immunoreactive nerve terminals have been projected to the laryngeal motoneurons at the level of light microscopy, but their distribution in the nucleus of the nucleus is not clear. The study of the relationship between the immunoreactive nerve terminals and the laryngeal motoneurons at the ultrastructural level is also deficient. This ultrastructural level is a necessary method to confirm the synaptic structure and relationship of the neurochemical. Therefore, the purpose of our study is to evaluate and compare P, tyrosine hydroxylase and 5- hydroxyl. The distribution of serotonin immunoreactive nerve terminals in the caudal caudal segment; (2) the ultrastructural relationship between the immunoreactive synapse terminal of substance P and the laryngeal motoneuron was studied by electrophysiological cell recording, immunohistochemistry and electron microscopy.
Methods: We used multiple immunofluorescence and confocal microscopy to evaluate the distribution of P, tyrosine hydroxylase and 5- serotonin immunoreactive nerve terminals in the nucleus caudal. The nucleus caudal motoneurons were labeled and identified by the immunoreactivity of choline acetyltransferase. Synaptophysin is a synapse. The expression of synaptophysin in the nucleus of the nucleus of the nucleus represents the number of the total synaptic terminals. After the Image J Software Co localization analysis, the regions of synaptosomal positive and substance P, tyrosine hydroxylase, or 5- HT are represented by P matter, tyrosine hydroxylase or 5- hydroxytryptamine. The synaptic terminal region.P substance, tyrosine hydroxylase or 5- serotonin synaptic terminal accounts for the proportion of the total synaptic terminal region, which is used to evaluate and compare the distribution of synaptic terminals in the nucleus caudal. Based on the comparison of the distribution of P, tyrosine hydroxylase and 5- hydroxytryptamine in the caudal segment of the nucleus of the nucleus of the nucleus, We further studied the ultrastructural relationship between the P substance immunoreactive nerve terminal and the laryngeal motoneuron. In an experiment, an inspiratory laryngeal motor neuron was identified and confirmed through intracellular recording, the cervical vagus nerve stimulation and its localization of the nucleus caudal. Then, the nerve biotin (biotinamide, 1.5%) was injected into this inhalation. Sexual laryngeal motoneurons. Immunoreactive structures of neurons and substance P injected with biotin were detected by electron microscopy before embedding immuno histochemical staining, and were displayed at the same time. Ultrathin sections of the immune positive structures of laryngeal motoneurons and substance P were stained and observed and analyzed under electron microscopy.
Results: we found that the P substance, tyrosine hydroxylase or 5- serotonin terminals accounted for no more than 10% of the total synaptic terminals in the total nucleus of the nucleus caudate, and three were not more than 15%. and tyrosine hydroxylase or 5- hydroxytryptamine. The synaptic terminal of substance P had a relatively high intensity ratio in the nucleus of the nucleus. At the ultrastructural level, the synaptic expansion terminal of 53.3% (114/206) forms an asymmetric synaptic structure with the dendrites of the inhaled laryngeal motoneuron, and 22.3% (46/206) forms a symmetric synaptic structure. The other expansion terminals are in contact with the neurons, but there is no clear special synaptic structure. In these 206 nerve terminals, 16% (33/206) is P The substance immunoreactive nerve terminal and the synapse structure.29 formed an asymmetric synaptic structure with the neurons, and 4 formed symmetric synaptic structures. A few P substance immunoreactive terminals formed synaptic structures with the neuronal cell bodies, but there was no discovery of the synaptic structure of the P matter nerve terminal on the axon of the neuron. Non immune response was not found. The sexual nerve terminal also forms a synaptic structure on the dendritic spines, some with special structures under the synapse. On the tissue section of the inspiratory laryngeal motoneurons, several large cell bodies (about 30-40 m in diameter) are also observed, and the non immunoreactive neurons of the nucleus are suspected. They contain large nuclei and obvious nucleolus.
Conclusion: first of all, our results first confirmed that in the rat nucleus of the nucleus, substance P, tyrosine hydroxylase and 5- hydroxytryptamine are only a few of the synapse terminals of synaptopsin immunoreactive, which indirectly indicates the function of P, tyrosine hydroxylase and 5- HT in the function of laryngeal motoneurons. To moderate regulation. Second, we confirmed that substance P immunoreactive nerve terminals form synaptic structures on laryngeal motor neurons. In an experiment, an inspiratory neuron, located in the nucleus of the nucleus, was activated, marked and ultrastructural after stimulation of the vagus nerve of the neck. The labeled neurons accept a large number of symmetry. And asymmetric synaptic transmission. A total of 33 P substances were found to form synaptic structures on the dendrites of the inhaled laryngeal motoneurons. Most of them (29) were asymmetric synapses, and a few (4) were symmetric synapses, indicating that neurons expressing substance P may directly increase and enhance the excitability of the inhaled laryngeal motoneurons. Third, we studied the overall ultrastructure and synapse of large cell neurons in the caudal caudal segment of the inhaled laryngeal motoneurons, and found a large number of symmetrical and asymmetric synaptic structures on these neurons.
Objective: in breathing exercises and other conditions and other activities, such as hypocapnia and sleep, and the regulation of different conditions in the laryngeal muscles. Previous anatomical and pharmacological studies have shown that acetylcholine at the nuclear level plays a role in regulating the activity of the laryngeal motoneurons. The unconsolidated structure contains inhalation and exhalation. The aim of this study is to investigate the anatomical characteristics of cholinergic input to laryngeal motor neurons in the loose structure of nucleus suspected.
Methods: We used intracellular recording, intracellular injection of dyes, and immunohistochemical methods to study the anatomical relationship between laryngeal motor neurons and cholinergic immunoreactive nerve terminals. Synaptophysin is a marker protein of synapse, so the nerve terminal of synaptophysin positive immune response can be seen as a synapse terminal. We use the immunofluorescence method and co localization analysis of confocal microscopy, that is, the analysis of the co localization of the immunoreactivity of the cholinergic positive nerve terminal and the synaptophysin, and further studies and evaluates the cholinergic immunoreactive nerve terminal which has a "close contact" with the laryngeal motoneuron. The proportion of immunopositive to the tactis.
Results: the results confirmed that there was a "close contact" between the exhaled laryngeal motoneurons and the immunoreactive terminals of the vesicular acetylcholine transporter. A total of 12 exhaled laryngeal motoneurons were identified by intracellular recording and labeled by neurobiotin injection. Among them, more vesicle acetyl was found on the exhaled laryngeal motoneurons. Choline transporter immunoreactive nerve terminals formed "close contact" (mean + SD, 32 + 9; n=8), compared with the distal dendrites.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R338

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