大鼠下丘神经元上NMDA和GABAA受体之间的功能交互作用

发布时间：2018-04-01 07:05

  本文选题：下丘　切入点：NMDA受体　出处：《中国科学技术大学》2011年硕士论文

【摘要】：背景:当细胞膜上两个不同的离子受体同时被激活时,他们之间往往会存在一种功能性的相互调节,即一种受体的激活会抑制或增强另一种受体的反应,这种受体间的相互调节作用被叫做受体间的相互对话。这种受体间的相互对话对神经元的兴奋性和神经信号的加工整合等具有重要的作用和生理意义。N-甲基-D-门冬氨酸(NMDA)型谷氨酸受体和γ-氨基丁酸A(GABA_A)型受体分别是中枢神经系统中主要的兴奋性和抑制性受体。至今为止,关于这两种受体之间的功能相互作用还缺乏系统和详尽的研究,特别在中枢听觉系统中,关于这两种受体之间的功能交互作用至今还未见相关的报道。下丘是中枢听觉通路中重要的信息整合中心,NMDA受体和GABA_A受体在该神经核团上均有丰富的表达。本论文旨在探讨大鼠下丘神经元上NMDA受体和GABA_A受体之间的功能交互作用及可能的机制。 方法:本研究在原代培养的新生大鼠下丘神经元上采用传统的全细胞膜片钳的方法研究NMDA受体和GABA_A受体之间的交互作用。在对膜电流的记录过程中采用的是电压钳模式,并且保持被记录细胞的膜电压始终被钳制在-60 mV。在本实验中药物的施加是通过一种被称为“Y管给药”的快速给药系统。 结果:(1)100μM的GABA可抑制100μM Asp在下丘神经元激活的全细胞电流(IAsp),表明GABA_A受体的激活能够抑制NMDA受体的功能。GABA_A受体的竞争性拮抗剂bicuculline(10μM)可完全阻断GABA激活的电流,并可消除GABA对IAsp的抑制作用;(2)100μM的Asp对高浓度(100μM )GABA在下丘神经元激活的全细胞电流(IGABA)没有影响,但对低浓度(3μM)GABA激活的IGABA有抑制作用,表明NMDA受体的激活对GABA_A受体的抑制作用是浓度依赖性的。NMDA受体的竞争性拮抗剂APV(100μM)可完全阻断Asp激活的电流,并可消除Asp对IGABA的抑制作用;(3)细胞外液中加入电压依赖的钙通道(VDCC_s)的阻断剂CdCl_2不影响Asp对IGABA的抑制作用,但是当外液中无钙或在电极内液中加入钙的螯合剂BAPTA时,Asp对IGABA的抑制作用消失,说明这种抑制作用是由钙离子介导的,并且是钙离子通过NMDA受体通道内流而非经由VDCCs内流而发挥作用的;(4)Asp对IGABA的抑制作用可被钙-钙调素依赖性的蛋白激酶Ⅱ(CaMKⅡ)的抑制剂KN-62所阻断,说明在NMDA受体和GABA_A受体的功能交互作用中,CaMKⅡ起到了重要的作用。 结论:本论文的研究结果表明,在培养的下丘神经元上,NMDA受体和GABA_A受体之间存在相互抑制的作用。这两种受体间的交互作用是一个钙依赖性的过程,但并不是通过激活电压依赖的钙通道来起作用的,其下游机制与细胞内的CaMKⅡ有关。NMDA和GABA_A受体在下丘的交互作用提示了中枢听觉系统中信息加工方式的复杂性。
[Abstract]:Background: when two different ion receptors on the cell membrane are activated at the same time, there is often a functional interaction between them, that is, the activation of one receptor inhibits or enhances the response of the other. The interaction between the receptors is called the interaction between the receptors. The interaction between the receptors plays an important role in neuronal excitability and neural signal processing and integration. Aspartate NMDA-type glutamate receptors and 纬 -aminobutyric acid (GABA) type receptors are the main excitatory and inhibitory receptors in the central nervous system. There is a lack of systematic and detailed research on the functional interactions between these two receptors, especially in the central auditory system. The functional interaction between these two receptors has not been reported yet. The inferior colliculus is an important information integration center in the central auditory pathway. Both NMDA receptor and GABA_A receptor are expressed in the neuronal nuclei. The purpose of this study was to investigate the functional interaction between NMDA receptor and GABA_A receptor in rat inferior colliculus neurons and its possible mechanism. Methods: in this study, the interaction between NMDA receptor and GABA_A receptor was studied by traditional whole-cell patch clamp method on primary cultured rat inferior colliculus neurons. The membrane voltage of the recorded cells was maintained at -60 MV. In this experiment, the drug was applied through a rapid delivery system called "Y tube delivery". Results 100 渭 M GABA could inhibit the whole cell current activated by 100 渭 M Asp in the inferior colliculus neurons, suggesting that the activation of GABA_A receptor could inhibit the function of NMDA receptor. Bicuculline(10 渭 M, a competitive antagonist of GABA A receptor, could completely block the GABA activated current. The inhibitory effect of GABA on IAsp was also eliminated. The inhibitory effect of 100 渭 M Asp on the whole cell current (IGABA) activated by 100 渭 M GABA in the inferior colliculus neurons was not affected, but it inhibited the IGABA activated by 3 渭 M)GABA at low concentration. It was suggested that the inhibitory effect of NMDA receptor activation on GABA_A receptor was that APV(100 渭 M, a competitive antagonist of. NMDA receptor in a concentration dependent manner, could completely block the current activated by Asp. The inhibitory effect of Asp on IGABA was also eliminated. The addition of voltage-dependent calcium channel (VDCCs) blocker CdCl_2 into extracellular fluid did not affect the inhibitory effect of Asp on IGABA. However, the inhibitory effect of Asp on IGABA disappeared when there was no calcium in the external solution or when the calcium chelator BAPTA was added to the electrode solution, indicating that the inhibition was mediated by calcium ion. Moreover, the inhibition of IGABA by calcium acting through the influx of NMDA receptor channel rather than through the influx of VDCCs can be blocked by KN-62, an inhibitor of calcium calmodulin dependent protein kinase 鈪，

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