大鼠中樞運(yùn)動(dòng)核團(tuán)內(nèi)P物質(zhì)受體的定位分布

1、    大鼠中樞運(yùn)動(dòng)核團(tuán)內(nèi)P物質(zhì)受體的定位分布?        【摘要】目的觀察大鼠腦干的軀體運(yùn)動(dòng)核、副交感運(yùn)動(dòng)核和脊髓前角內(nèi)P物質(zhì)(SP)受體的定位分布，闡明SP在運(yùn)動(dòng)核團(tuán)的作用部位。方法免疫組織化學(xué)染色技術(shù)。結(jié)果在動(dòng)眼神經(jīng)核、滑車神經(jīng)核、三叉神經(jīng)運(yùn)動(dòng)核、展神經(jīng)核、面神經(jīng)核、舌下神經(jīng)核和脊髓前角內(nèi)均可見SP受體樣陽性纖維和呈弱陽性的SP受體樣陽性神經(jīng)元，但疑核內(nèi)的SP受體樣陽性神經(jīng)元和纖維密集并呈強(qiáng)陽性。在動(dòng)眼神經(jīng)副交感核、上涎核和迷走神經(jīng)背核內(nèi)也可見SP受體樣陽性纖維和弱

2、陽性的SP受體樣陽性神經(jīng)元。結(jié)論SP通過SP受體的介導(dǎo)在中樞運(yùn)動(dòng)核團(tuán)內(nèi)對(duì)運(yùn)動(dòng)神經(jīng)元發(fā)揮調(diào)控作用?！娟P(guān)鍵詞】P物質(zhì)受體P物質(zhì)腦干運(yùn)動(dòng)核脊髓前角大鼠 IMMUNOCYTOCHEMICAL LOCALIZATION OF SUBSTANCE P RECEPTOR IN THE CENTRALMOTOR NUCLEI OF THE RATGao Rong, Li Yunqing, Shi Jiwu【Abstract】 Objective To observe the location of substance P receptor (SPR) in the somatic motor nuclei,

3、parasympathetic motor nuclei of the brainstem and the ventral horn of the spinal cord in the rat and to identify the functional sites of substance P (SP) in the motor nuclei of central nervous system. Method Immunocytochemical staining technique was used in the present study. Results Both SPR-like i

4、mmunoreactive (SPR-LI) fibers and weakly stained SPR-LI neurons were found in the oculomotor nucleus, trochlear nucleus, trigeminal motor nucleus, abducens nucleus, facial necleus, hypoglossal nucleus and the ventral horn of the spinal cord; but SPR-LI neurons and fibers in the ambiguous nucleus wer

5、e strongly stained with high density. SPR-Li fibers and weakly stained SPR-LI neurons were also observed in the Edinger-Westphal nucleus, superior salivary nucleus and the dorsal motor nucleus of vagus. Conclusion SP may regulate the activities of motoneurons in the central nervous system by means o

6、f SPR.【Key words】 Substance P receptor; Substance P; Immunocytochemistry; Brainstem motor nuclei; Ventral horn of spinal cord; RatP物質(zhì)(SP)廣泛分布于神經(jīng)系統(tǒng)內(nèi)，除了在神經(jīng)系統(tǒng)的感覺信息傳遞方面發(fā)揮著重要作用以外，還對(duì)運(yùn)動(dòng)神經(jīng)元具有興奮性效應(yīng)1。腦干的軀體運(yùn)動(dòng)核、副交感性內(nèi)臟運(yùn)動(dòng)核和脊髓前角的運(yùn)動(dòng)神經(jīng)元周圍，有密集的SP樣陽性終末2，SP樣陽性終末與運(yùn)動(dòng)神經(jīng)元形成突觸聯(lián)系3。近年來，隨著分子生物學(xué)技術(shù)引入神經(jīng)科學(xué)研究領(lǐng)域，已成功地克隆了SP受體并制成了SP受體的

7、特異性抗體，為研究SP受體的定位分布提供了條件4。形態(tài)學(xué)研究觀察到SP受體樣陽性結(jié)構(gòu)也廣泛地分布于中樞神經(jīng)系統(tǒng)4，但SP受體樣陽性結(jié)構(gòu)在中樞運(yùn)動(dòng)核團(tuán)內(nèi)的精確定位和分布尚未見詳細(xì)的研究報(bào)道。搞清SP受體樣陽性結(jié)構(gòu)在中樞運(yùn)動(dòng)核團(tuán)內(nèi)的定位分布對(duì)闡明SP對(duì)運(yùn)動(dòng)神經(jīng)元的影響具有重要意義，本研究用免疫細(xì)胞化學(xué)染色技術(shù)，對(duì)大鼠腦干的軀體運(yùn)動(dòng)核、副交感性內(nèi)臟運(yùn)動(dòng)核和脊髓前角內(nèi)SP受體樣陽性結(jié)構(gòu)的定位分布進(jìn)行了觀察。材料和方法雄性SD大鼠4只，體重200250g。戊巴比妥鈉經(jīng)腹腔麻醉(40 mg/kg)，開胸后插管至升主動(dòng)脈，先以0.9%鹽水100 ml沖洗血液，再用含4%多聚甲醛和0.2%苦味酸的0.1 mo

8、l/L磷酸鹽緩沖液(PB，pH7.4)灌注固定，持續(xù)45 min左右。取腦及脊髓入上述新鮮固定液中后固定24 h(4)，再移入含30%蔗糖的PB液中至沉底。恒冷箱切片機(jī)冰凍橫切腦干及脊髓，片厚30m。切片隔2張取1張，分3套收集，將第1套切片按ABC法進(jìn)行免疫組織化學(xué)染色：切片首先入兔抗SP受體血清(1mg/L9)，日本京都大學(xué)醫(yī)學(xué)部水野升先生和重本隆一先生惠贈(zèng))中孵育1824h；次入Biotin標(biāo)記的山羊抗兔IgG血清(Vector,1:200)中孵育3h；再入ABC混合液(1：50,Vector)中孵育1h；最后用二甲基聯(lián)苯胺(DAB)和H2O2呈色。孵育均在室溫下進(jìn)行，各步驟間均用PBS

9、徹底清洗切片?？贵w和復(fù)合物的稀釋均用含0.3%的Triton X-100和2%正常羊血清的PBS。將經(jīng)上述反應(yīng)的切片裱于經(jīng)明膠處理的載玻片上，脫水、透明、封片，光鏡觀察。對(duì)照實(shí)驗(yàn)用正常兔血清代替兔抗SP受體血清孵育第2套切片。將第3套切片行Nissl染色，以便觀察和定位SP受體的免疫組織化學(xué)染色結(jié)果。結(jié)果和討論SP受體樣免疫組織化學(xué)染色的陽性產(chǎn)物主要位于陽性神經(jīng)元和纖維的膜上。在動(dòng)眼神經(jīng)核內(nèi)可見少量SP受體樣陽性纖維和呈弱陽性的神經(jīng)元(1)；滑車神經(jīng)核內(nèi)可見中等密度的SP受體樣陽性纖維(2)；三叉神經(jīng)運(yùn)動(dòng)核內(nèi)可見少量散在性分布的SP受體樣陽性纖維及弱陽性神經(jīng)元，但在三叉神經(jīng)運(yùn)動(dòng)核周圍的三叉間核

10、(IntV)和三叉周區(qū)(PeV)可見較多濃染的SP受體樣陽性神經(jīng)元和纖維，部分陽性纖維伸入三叉神經(jīng)運(yùn)動(dòng)核(3)；展神經(jīng)核內(nèi)可見少量淡染的SP受體樣陽性神經(jīng)元和纖維(4)；面神經(jīng)核內(nèi)除可見到低至中等密度的SP受體樣陽性纖維外，還可見到少量弱陽性神經(jīng)元(5)；疑核內(nèi)可見密集并呈強(qiáng)陽性的SP受體樣陽性神經(jīng)元和纖維(6)；舌下神經(jīng)核的腹側(cè)部?jī)?nèi)可見密集的SP受體樣陽性終末樣結(jié)構(gòu)，而背側(cè)部?jī)?nèi)可見一些SP受體樣陽性纖維和弱陽性神經(jīng)元(7)；脊髓前角內(nèi)有中等密度的SP受體樣陽性纖維和少量弱陽性神經(jīng)元(8)。此外，在動(dòng)眼神經(jīng)副交感核(E-W核)(1)、上涎核和迷走神經(jīng)背核(7)內(nèi)也可見少量SP受體樣陽性纖維和弱

11、陽性神經(jīng)元。SP受體樣陽性神經(jīng)元多呈圓形、三角形和梭形，多極形神經(jīng)元較少，SP受體樣陽性神經(jīng)元的胞體直徑多為2030m。用正常兔血清代替兔抗SP受體血清孵育的第2套切片上，未見SP受體樣陽性結(jié)構(gòu)。最近的研究證明大鼠的大腦運(yùn)動(dòng)皮質(zhì)并不直接支配中樞的運(yùn)動(dòng)核團(tuán)，而是借助由其內(nèi)谷氨酸樣陽性的錐體細(xì)胞發(fā)出的下行投射纖維所組成的皮質(zhì)腦干束或皮質(zhì)脊髓束，分別興奮腦干的運(yùn)動(dòng)前神經(jīng)元或前角、層內(nèi)構(gòu)成局部環(huán)路的中間神經(jīng)元，再經(jīng)運(yùn)動(dòng)前神經(jīng)元或中間神經(jīng)元調(diào)控軀體運(yùn)動(dòng)核內(nèi)運(yùn)動(dòng)神經(jīng)元的活動(dòng)，實(shí)現(xiàn)不同肌群間的協(xié)同和協(xié)調(diào)，完成一系列復(fù)雜而精細(xì)的運(yùn)動(dòng)。SP不僅對(duì)腰髓前角運(yùn)動(dòng)神經(jīng)元有興奮性作用，而且可強(qiáng)化谷氨酸對(duì)腰髓前角運(yùn)動(dòng)神經(jīng)

12、元的興奮性作用，SP和谷氨酸對(duì)腰髓前角運(yùn)動(dòng)神經(jīng)元的興奮性作用基本上相似1。我們以往的研究觀察到向三叉神經(jīng)運(yùn)動(dòng)核、面神經(jīng)核和舌下神經(jīng)核等與面口部運(yùn)動(dòng)有關(guān)的核團(tuán)發(fā)出SP能投射的運(yùn)動(dòng)前神經(jīng)元主要位于臂旁外側(cè)核、延髓中縫核簇和巨細(xì)胞網(wǎng)狀核部5。我們還在電鏡下觀察到SP樣陽性終末與三叉神經(jīng)運(yùn)動(dòng)核、面神經(jīng)核和舌下神經(jīng)核內(nèi)運(yùn)動(dòng)神經(jīng)元的胞體和樹突形成以非對(duì)稱性為主的軸-體突觸和軸-樹突觸聯(lián)系3。以上結(jié)果都說明SP在腦干運(yùn)動(dòng)前神經(jīng)元和脊髓內(nèi)構(gòu)成局部環(huán)路的中間神經(jīng)元對(duì)腦干和脊髓的軀體運(yùn)動(dòng)神經(jīng)元的調(diào)控方面發(fā)揮重要作用。雖然Nakaya等4在動(dòng)眼神經(jīng)核、滑車神經(jīng)核、面神經(jīng)核、舌下神經(jīng)核、疑核及脊髓前角內(nèi)均觀察到少量S

13、P受體樣陽性纖維和胞體，但并未述及這些核團(tuán)內(nèi)SP受體樣陽性纖維和胞體的形態(tài)學(xué)特征。本研究除了在Nakaya等4所報(bào)道的運(yùn)動(dòng)核團(tuán)以外，還在三叉神經(jīng)運(yùn)動(dòng)核、展神經(jīng)核和上涎核內(nèi)觀察到SP受體樣陽性纖維和胞體，并描述了這些核團(tuán)內(nèi)SP受體樣陽性纖維和胞體的形態(tài)學(xué)特征。本研究的結(jié)果提示，從運(yùn)動(dòng)前神經(jīng)元及中間神經(jīng)元來的SP能陽性終末可能在上述運(yùn)動(dòng)核團(tuán)與SP受體樣陽性結(jié)構(gòu)形成突觸聯(lián)系，實(shí)現(xiàn)SP對(duì)軀體運(yùn)動(dòng)神經(jīng)元的調(diào)控作用，同時(shí)也說明SP能陽性終末與SP受體樣陽性結(jié)構(gòu)在中樞運(yùn)動(dòng)核團(tuán)內(nèi)互相匹配。1動(dòng)眼神經(jīng)核()和動(dòng)眼神經(jīng)副交感核(E-W核)內(nèi)的SP受體樣陽性纖維和弱陽性神經(jīng)元(箭頭)。標(biāo)尺示100m(下同)Fig.

14、1 Sparse substance P receptor-like immunoreactive (SPR-LI) fibers and weakly stained SPR-LI neurons (arrows) in the oculomotor nucleus () and Edinger-Westphal (E-W) nucleus. Bar=100 m for all Figs.18.2滑車神經(jīng)核()內(nèi)中等密度的SP受體樣陽性纖維和弱陽性神經(jīng)元(箭頭)。DR.中縫背核Fig.2 Middle density SPR-LI fibers and weakly stained SPR-

15、LI neurons (arrows) in the trochlear nucleus (). DR:dorsal raphe nucleus. 3三叉神經(jīng)運(yùn)動(dòng)核(m)內(nèi)散在性分布的SP受體樣陽性纖維及弱陽性神經(jīng)元(箭頭)。5r.三叉神經(jīng)運(yùn)動(dòng)根Int.三叉間核PeV.三叉周區(qū)Fig.3 Sparse SPR-LI fibers and weakly stained SPR-like neurons (arrows) in the trigeminal motor nucleus (Vm). 5r: motor root of trigeminal nerve; IntV:intertrige

16、minal nucleus; PeV:peritrigeminal zone.4展神經(jīng)核()內(nèi)少量淡染的SP受體樣陽性神經(jīng)元(箭頭)和纖維。g7.面神經(jīng)膝Fig.4 Sparse weakly stained SPR-LI neurons (arrows) and fibers in the abducens nucleus (). g7:genu of facial nerve.5面神經(jīng)核()內(nèi)低至中等密度的SP受體樣陽性纖維和弱陽性神經(jīng)元(箭頭)Fig.5 Low to middle density SPR-LI neurons (arrows) and fibers in the fac

17、ial nucleus ().6疑核(Amb)內(nèi)密集的強(qiáng)陽性SP受體樣陽性神經(jīng)元(箭頭)和纖維Fig.6 High density and strong stained SPR-LI neurons and fibers in the ambiguus (Amb) nucleus.7舌下神經(jīng)核()內(nèi)的SP受體樣陽性纖維、終末和弱陽性神經(jīng)元(箭頭)；迷走神經(jīng)背核()內(nèi)也有少量SP受體樣陽性纖維和弱陽性神經(jīng)元(箭頭)。4v.第四腦室NTS.孤束核Fig.7 SPR-LI neurons (arrows) and fibers in the dorsal motor nucleus of vagus

18、 () and hypoglossal nucleus (). 4v:the 4th ventricle; NTS:nucleus tractus   solitarri.8頸髓前角內(nèi)有中等密度的SP受體樣陽性纖維和少量弱陽性神經(jīng)元(箭頭)。.層.層Acs.前角副核Fig.8 Middle density SPR-LI fibers and neurons (arrows) in the ventral horn of the cervical spinal cord. :lamina ; :lamina ; Acs:accessory nucleus of the ventral horn. * 國(guó)家杰出青年科學(xué)基金資助課題(No.396250111)作者單位：高蓉李云慶施際武第四軍醫(yī)大學(xué)解剖學(xué)教研室，梁<"z1 (76 bytes)" src="/med/cano/201003/20100309160552785" 16 16>琚腦研究中心，西安710032感謝張淼麗和原悅萍同志在片制作過程中的幫助。參考文獻(xiàn)1White SR. A comparison of the effects of serotonin, s