【摘要】： 眾所周知,γ-氨基丁酸(GABA)是廣泛分布于哺乳類動(dòng)物中樞神經(jīng)系統(tǒng)內(nèi)的一種重要的抑制性神經(jīng)遞質(zhì),它可與相應(yīng)的GABA受體結(jié)合從而發(fā)揮相應(yīng)的生理功能。以往有諸多研究觀察到GABA能神經(jīng)終末圍繞在三叉神經(jīng)中腦核(mesencephalic trigeminal nucleus, Vme)神經(jīng)元的胞體周?chē)�。本�?shí)驗(yàn)組以往的研究也在超微結(jié)構(gòu)水平進(jìn)一步證實(shí)了GABA能神經(jīng)終末與Vme神經(jīng)元的胞體形成突觸聯(lián)系;同時(shí)在Vme神經(jīng)元胞體上還觀察到GABAARα1和GABAARα3受體亞型的表達(dá)。雖然已有文獻(xiàn)報(bào)道了腦片上Vme神經(jīng)元對(duì)GABA的興奮性反應(yīng),但由于方法學(xué)的局限性,GABA在Vme神經(jīng)元內(nèi)的反應(yīng)及其可能的分子基礎(chǔ)仍不明確,負(fù)責(zé)氯離子轉(zhuǎn)運(yùn)的陽(yáng)離子-Cl-共轉(zhuǎn)運(yùn)體可能參與該反應(yīng)過(guò)程,但相關(guān)的研究到目前為止尚未見(jiàn)報(bào)道。 陽(yáng)離子-Cl-共轉(zhuǎn)運(yùn)體在維持細(xì)胞內(nèi)外Cl-濃度的平衡方面發(fā)揮主要作用。在目前報(bào)道的七種陽(yáng)離子-Cl-共轉(zhuǎn)運(yùn)體中,神經(jīng)系統(tǒng)內(nèi)僅表達(dá)KCC1、KCC2、KCC3和NKCC1四種陽(yáng)離子-Cl-共轉(zhuǎn)運(yùn)體。其中,KCC2負(fù)責(zé)將神經(jīng)元內(nèi)Cl-向外轉(zhuǎn)運(yùn),而NKCC1則負(fù)責(zé)將胞外的Cl-向胞內(nèi)轉(zhuǎn)運(yùn),二者在神經(jīng)元胞膜上構(gòu)成一對(duì)平衡體,從而維持神經(jīng)元內(nèi)Cl-濃度的穩(wěn)定和平衡。生后發(fā)育過(guò)程中大鼠Vme神經(jīng)元內(nèi)KCC2和NKCC1的表達(dá)及其變化,到目前為止鮮見(jiàn)報(bào)道,而這種表達(dá)的變化可能與氯通道開(kāi)放后的作用效果有密切聯(lián)系。 小白蛋白(Parvalbumin, PV)是鈣結(jié)合蛋白的一種。已有研究表明:發(fā)育過(guò)程中,PV特異性地表達(dá)于本體感覺(jué)初級(jí)傳入神經(jīng)元(如大的背根節(jié)和Vme等)胞體和突起上。目前,PV作為研究本體感覺(jué)初級(jí)傳入神經(jīng)元的特異性標(biāo)志物已被廣泛應(yīng)用在神經(jīng)系統(tǒng)的發(fā)育學(xué)研究領(lǐng)域。此外,有關(guān)Vme的生后發(fā)育研究亦表明,PV幾乎表達(dá)于所有的Vme神經(jīng)元中。但是,NKCC1、KCC2與PV在Vme的生后發(fā)育過(guò)程中是否有共存,其共存的比例是否有變化,而這種相對(duì)量上的變化與蛋白表達(dá)水平改變的實(shí)際情況是否相符也無(wú)明確的答案。 基于此,本論文進(jìn)行了如下研究:(1)運(yùn)用機(jī)械分離帶終扣的單細(xì)胞膜片鉗技術(shù)對(duì)大鼠Vme神經(jīng)元對(duì)GABA的反應(yīng)進(jìn)行觀察;(2)運(yùn)用Western blotting技術(shù)結(jié)合圖像分析方法觀察生后發(fā)育過(guò)程中Vme神經(jīng)元內(nèi)NKCC1和KCC2的表達(dá)變化;(3)運(yùn)用免疫熒光組織化學(xué)雙重標(biāo)記技術(shù)對(duì)生后發(fā)育過(guò)程中Vme神經(jīng)元內(nèi)NKCC1和KCC2與PV之間的共存關(guān)系進(jìn)行了系統(tǒng)觀察。本論文包括以下三個(gè)部分的內(nèi)容: 第一部分大鼠三叉神經(jīng)中腦核神經(jīng)元對(duì)GABA的反應(yīng) 本研究運(yùn)用機(jī)械分離帶終扣的單細(xì)胞膜片鉗技術(shù)觀察Vme神經(jīng)元對(duì)GABA的反應(yīng)情況,結(jié)果如下:在鉗制電壓為-70 mV時(shí),給予1μM TTX、10μM AP-5和10μM CNQX情況下,在所有被檢測(cè)的Vme神經(jīng)元上均記錄到內(nèi)向的mPSCs, mPSCs的平均幅值為24±20 pA,mPSC的反轉(zhuǎn)電位約為-30 mV,與運(yùn)用Nernst方程計(jì)算出的Cl-平衡電位相當(dāng);同時(shí)給予GABAA受體特異性拮抗劑Bicuculline (5μM )時(shí),已觀察到的突觸前GABA所引起的電流被完全阻斷,洗脫后,GABA所引起的mPSCs恢復(fù)到先前水平。 以上結(jié)果表明:突觸前終扣內(nèi)釋放的GABA與分布于突觸后的GABAA受體結(jié)合從而發(fā)揮對(duì)Vme神經(jīng)元的調(diào)控作用,GABAA受體激活后,氯離子通道開(kāi)放,Vme神經(jīng)元胞體內(nèi)外氯離子流動(dòng)構(gòu)成了GABA對(duì)Vme神經(jīng)元的作用基礎(chǔ)。 第二部分大鼠生后發(fā)育過(guò)程中三叉神經(jīng)中腦核神經(jīng)元內(nèi)NKCC1和KCC2的表達(dá)及其變化 本研究分別選取生后0、4、7、10、14、21的正常雄性大鼠,應(yīng)用免疫組織化學(xué)及Western blotting技術(shù)結(jié)合圖像分析對(duì)Vme神經(jīng)元內(nèi)KCC2和NKCC1的表達(dá)變化進(jìn)行了觀察。結(jié)果顯示:(1)NKCC1均表達(dá)于Vme神經(jīng)元的胞體,突起上無(wú)表達(dá);(2)生后0天(P0)大鼠Vme神經(jīng)元內(nèi)NKCC1已開(kāi)始表達(dá),但表達(dá)相對(duì)較低,隨著發(fā)育時(shí)間的延長(zhǎng),NKCC1的表達(dá)逐漸增強(qiáng),至P10時(shí)已達(dá)成年水平。運(yùn)用Image-pro plus軟件進(jìn)行灰度分析顯示,P0、P4、P7、P10、P14、P21六個(gè)不同日齡大鼠Vme內(nèi)NKCC1的灰度值逐漸增大,P0、P4、P7三個(gè)日齡表達(dá)水平分別與P10、P14、P21三個(gè)日齡的表達(dá)水平相比較存在顯著性差異(P 0.05);而KCC2的表達(dá)從P0至P21時(shí)均較弱,各日齡間無(wú)差異(P 0.05)。 本部分研究結(jié)果表明在大鼠Vme神經(jīng)元生后發(fā)育過(guò)程中,NKCC1呈逐漸高水平表達(dá),而KCC2始終低水平表達(dá),提示成年時(shí)Vme神經(jīng)元胞體中氯離子濃度水平較高,推測(cè)胞內(nèi)氯離子濃度隨發(fā)育不斷增高可能與成年時(shí)GABA對(duì)Vme神經(jīng)元的作用密切相關(guān)。 第三部分大鼠生后發(fā)育過(guò)程中三叉神經(jīng)中腦核神經(jīng)元內(nèi)NKCC1、KCC2與PV的共存研究 本研究運(yùn)用免疫熒光雙標(biāo)技術(shù)分別觀察了大鼠P4、P7、P10、P14、P21五個(gè)時(shí)間點(diǎn)NKCC1、KCC2與PV的共存情況,并進(jìn)行了統(tǒng)計(jì)分析。結(jié)果顯示:隨著發(fā)育,NKCC1與PV的共存率逐漸升高,到成年時(shí)NKCC1幾乎表達(dá)于所有的Vme神經(jīng)元上。而KCC2在Vme神經(jīng)元上幾乎觀察不到表達(dá),在五個(gè)不同時(shí)間點(diǎn),均未觀察到任何雙標(biāo)記神經(jīng)元。而在陽(yáng)性對(duì)照實(shí)驗(yàn)中,在小腦的蒲肯野氏細(xì)胞層能觀察到明顯的KCC2的表達(dá)。本部分研究結(jié)果進(jìn)一步提示成年后NKCC1高水平表達(dá)而KCC2的低水平表達(dá)可能是Vme神經(jīng)元對(duì)GABA發(fā)生興奮性反應(yīng)的分子基礎(chǔ)。
[Abstract]:It is well known that gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter widely distributed in the mammalian central nervous system. It can bind to the corresponding GABA receptors to perform the corresponding physiological functions. In addition, the expression of GABAAR alpha 1 and GABAAR alpha 3 receptor subtypes was also observed in Vme neurons. Although Vme God was reported on brain slices, the expression of GABAAR alpha 1 and GABAAR alpha 3 receptor subtypes was also observed in Vme neurons. The excitatory response of meridians to GABA is still unclear due to methodological limitations. Cationic-Cl-cotransporters responsible for chloride transport may be involved in the excitatory response of GABA in Vme neurons. However, the related studies have not been reported so far.
Cationic-Cl-cotransporters play a major role in maintaining the balance of intracellular and extracellular Cl-concentrations. Of the seven reported cationic-Cl-cotransporters, only four cationic-Cl-cotransporters KCC1, KCC2, KCC3 and NKCC1 are expressed in the nervous system. Among them, KCC2 transports Cl-outward in neurons, while NKCC1 transports Cl-outward in neurons. The expression and change of KCC2 and NKCC1 in Vme neurons during postnatal development are rarely reported, and this change may be closely related to the effect of chloride channel opening. Contact.
Parvalbumin (PV) is a kind of calcium-binding protein. Previous studies have shown that PV is specifically expressed in the somas and processes of primary afferent neurons (such as large dorsal root ganglia and Vme) during development. At present, PV has been widely used as a specific marker of primary afferent neurons of proprioception. In addition, studies on postnatal development of Vme have also shown that PV is expressed in almost all Vme neurons. However, whether NKCC1, KCC2 and PV coexist in the postnatal development of Vme, and whether the coexistence ratio has changed, are these changes in relative quantity related to the changes in protein expression If there is no agreement, there is no definite answer.
Based on this, the following studies were carried out in this paper: (1) Single-cell patch clamp technique with end-buckle of mechanical separation band was used to observe the response of Vme neurons to GABA; (2) Western blotting and image analysis were used to observe the expression of NKCC 1 and KCC 2 in Vme neurons during postnatal development; (3) Immunofluorescence was used to observe the expression of NKCC 1 and KCC 2 in Vme neurons; (3) Immunofluores The coexistence of NKCC1 and KCC2 with PV in Vme neurons during postnatal development was observed by histochemical double labeling technique.
The first part is the response of neurons in the trigeminal mesencephalic nucleus to GABA in rats.
In this study, we observed the response of Vme neurons to GABA by single-cell patch clamp technique with end-buckle of mechanical separation. The results were as follows: when the clamping voltage was - 70 mV, the inward mPSCs were recorded on all Vme neurons, the average amplitude of mPSCs was 24 6550 The inversion potential was about - 30 mV, which was equivalent to the Cl-equilibrium potential calculated by Nernst equation, and the current induced by presynaptic GABA was completely blocked when the GABAA receptor-specific antagonist Bicuculline (5 mu M) was administered. After elution, the mPSCs induced by GABA returned to the previous level.
These results indicate that GABA released from the presynaptic terminal cingulum binds to GABAA receptors distributed in the postsynaptic region and plays a regulatory role in Vme neurons. After GABAA receptors are activated, chloride channels are opened and chloride flow in Vme neurons in vivo and in vitro forms the basis of GABA's effect on Vme neurons.
Part 2 Expression and changes of NKCC1 and KCC2 in trigeminal mesencephalic nucleus neurons during postnatal development in rats
The expression of KCC 2 and NKCC1 in Vme neurons was observed by immunohistochemistry and Western blotting combined with image analysis in 0,4,7,10,14,21 postnatal male rats. The expression of NKCC1 began to express in the meta-cells, but the expression was relatively low. The expression of NKCC1 increased gradually with the development time, and reached the adult level at P10. Image-pro plus software was used to analyze the gray level of NKCC1 in Vme of six rats of different ages: P 0, P 4, P 7, P 10, P 14, P 21. Compared with P10, P14 and P21, the expression of KCC2 was weaker from P0 to P21, and there was no significant difference among different ages (P 0.05).
These results suggest that NKCC1 is gradually expressed at a high level during the postnatal development of Vme neurons, while KCC2 is always expressed at a low level, suggesting a high level of chloride ion in Vme neurons during adulthood. It is speculated that the increase of intracellular chloride concentration with the development of Vme neurons may be closely related to the effect of GABA on Vme neurons in adulthood.
The coexistence of NKCC1, KCC2 and PV in trigeminal mesencephalic nucleus neurons during postnatal development in rats
In this study, the coexistence of NKCC1, KCC2 and PV in P4, P7, P10, P14 and P21 was observed by immunofluorescence double labeling technique. The results showed that the coexistence rate of NKCC1 and PV increased gradually with development, and NKCC1 was almost expressed in all Vme neurons in adulthood. No double-labeled neurons were observed at any of the five different time points. In the positive control experiment, the expression of KCC2 was observed in the Purkinje cell layer of the cerebellum. The molecular basis of excitatory response.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R338

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