大鼠三叉神经中脑核神经元对GABA的反应及其相关发育学研究

发布时间：2018-09-01 18:57

【摘要】： 众所周知,γ-氨基丁酸(GABA)是广泛分布于哺乳类动物中枢神经系统内的一种重要的抑制性神经递质,它可与相应的GABA受体结合从而发挥相应的生理功能。以往有诸多研究观察到GABA能神经终末围绕在三叉神经中脑核(mesencephalic trigeminal nucleus, Vme)神经元的胞体周围。本实验组以往的研究也在超微结构水平进一步证实了GABA能神经终末与Vme神经元的胞体形成突触联系;同时在Vme神经元胞体上还观察到GABAARα1和GABAARα3受体亚型的表达。虽然已有文献报道了脑片上Vme神经元对GABA的兴奋性反应,但由于方法学的局限性,GABA在Vme神经元内的反应及其可能的分子基础仍不明确,负责氯离子转运的阳离子-Cl-共转运体可能参与该反应过程,但相关的研究到目前为止尚未见报道。阳离子-Cl-共转运体在维持细胞内外Cl-浓度的平衡方面发挥主要作用。在目前报道的七种阳离子-Cl-共转运体中,神经系统内仅表达KCC1、KCC2、KCC3和NKCC1四种阳离子-Cl-共转运体。其中,KCC2负责将神经元内Cl-向外转运,而NKCC1则负责将胞外的Cl-向胞内转运,二者在神经元胞膜上构成一对平衡体,从而维持神经元内Cl-浓度的稳定和平衡。生后发育过程中大鼠Vme神经元内KCC2和NKCC1的表达及其变化,到目前为止鲜见报道,而这种表达的变化可能与氯通道开放后的作用效果有密切联系。小白蛋白(Parvalbumin, PV)是钙结合蛋白的一种。已有研究表明:发育过程中,PV特异性地表达于本体感觉初级传入神经元(如大的背根节和Vme等)胞体和突起上。目前,PV作为研究本体感觉初级传入神经元的特异性标志物已被广泛应用在神经系统的发育学研究领域。此外,有关Vme的生后发育研究亦表明,PV几乎表达于所有的Vme神经元中。但是,NKCC1、KCC2与PV在Vme的生后发育过程中是否有共存,其共存的比例是否有变化,而这种相对量上的变化与蛋白表达水平改变的实际情况是否相符也无明确的答案。基于此,本论文进行了如下研究:(1)运用机械分离带终扣的单细胞膜片钳技术对大鼠Vme神经元对GABA的反应进行观察;(2)运用Western blotting技术结合图像分析方法观察生后发育过程中Vme神经元内NKCC1和KCC2的表达变化;(3)运用免疫荧光组织化学双重标记技术对生后发育过程中Vme神经元内NKCC1和KCC2与PV之间的共存关系进行了系统观察。本论文包括以下三个部分的内容: 第一部分大鼠三叉神经中脑核神经元对GABA的反应本研究运用机械分离带终扣的单细胞膜片钳技术观察Vme神经元对GABA的反应情况,结果如下:在钳制电压为-70 mV时,给予1μM TTX、10μM AP-5和10μM CNQX情况下,在所有被检测的Vme神经元上均记录到内向的mPSCs, mPSCs的平均幅值为24±20 pA,mPSC的反转电位约为-30 mV,与运用Nernst方程计算出的Cl-平衡电位相当;同时给予GABAA受体特异性拮抗剂Bicuculline (5μM )时,已观察到的突触前GABA所引起的电流被完全阻断,洗脱后,GABA所引起的mPSCs恢复到先前水平。以上结果表明:突触前终扣内释放的GABA与分布于突触后的GABAA受体结合从而发挥对Vme神经元的调控作用,GABAA受体激活后,氯离子通道开放,Vme神经元胞体内外氯离子流动构成了GABA对Vme神经元的作用基础。第二部分大鼠生后发育过程中三叉神经中脑核神经元内NKCC1和KCC2的表达及其变化本研究分别选取生后0、4、7、10、14、21的正常雄性大鼠,应用免疫组织化学及Western blotting技术结合图像分析对Vme神经元内KCC2和NKCC1的表达变化进行了观察。结果显示:(1)NKCC1均表达于Vme神经元的胞体,突起上无表达;(2)生后0天(P0)大鼠Vme神经元内NKCC1已开始表达,但表达相对较低,随着发育时间的延长,NKCC1的表达逐渐增强,至P10时已达成年水平。运用Image-pro plus软件进行灰度分析显示,P0、P4、P7、P10、P14、P21六个不同日龄大鼠Vme内NKCC1的灰度值逐渐增大,P0、P4、P7三个日龄表达水平分别与P10、P14、P21三个日龄的表达水平相比较存在显著性差异(P 0.05);而KCC2的表达从P0至P21时均较弱,各日龄间无差异(P 0.05)。本部分研究结果表明在大鼠Vme神经元生后发育过程中,NKCC1呈逐渐高水平表达,而KCC2始终低水平表达,提示成年时Vme神经元胞体中氯离子浓度水平较高,推测胞内氯离子浓度随发育不断增高可能与成年时GABA对Vme神经元的作用密切相关。第三部分大鼠生后发育过程中三叉神经中脑核神经元内NKCC1、KCC2与PV的共存研究本研究运用免疫荧光双标技术分别观察了大鼠P4、P7、P10、P14、P21五个时间点NKCC1、KCC2与PV的共存情况,并进行了统计分析。结果显示:随着发育,NKCC1与PV的共存率逐渐升高,到成年时NKCC1几乎表达于所有的Vme神经元上。而KCC2在Vme神经元上几乎观察不到表达,在五个不同时间点,均未观察到任何双标记神经元。而在阳性对照实验中,在小脑的蒲肯野氏细胞层能观察到明显的KCC2的表达。本部分研究结果进一步提示成年后NKCC1高水平表达而KCC2的低水平表达可能是Vme神经元对GABA发生兴奋性反应的分子基础。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R338

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