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非损伤微测技术实时检测海马脑片跨膜钙离子流

李甜, 原丽, 张军, 焦娟娟, 祁金顺

山西医科大学生理学系，细胞生理学省部共建教育部重点实验室，太原 030001；山西长治医学院生理教研室，长治 046000

摘要

脑内β-淀粉样蛋白(amyloid-β protein, Aβ)的聚集是阿尔茨海默病(Alzheimer’s disease, AD)的重要病理特征。Aβ的神经毒性作用机制与其扰乱神经元Ca2+稳态有密切关系。非损伤微测技术(non-invasive micro-test technique, NMT)是近年发展起来的一种利用Fick第一扩散定律和Nernst方程，通过非接触方式检测膜外扩散电位获取离子跨膜流速的最新技术手段。本研究在C57BL/6小鼠海马脑片，利用NMT首次检测了Aβ对谷氨酸(Glu)诱发的Ca2+内流以及细胞外低钙引起的Ca2+外排的影响，并初步探讨了Aβ扰乱神经元Ca2+稳态的相关机制。结果显示：(1)急性给予Glu可诱发海马脑片CA1区神经元产生起始快、继而缓慢衰减的持续性内向Ca2+流；(2) Aβ预处理浓度依赖性地增强海马神经元对Glu的反应性，显著提高给药后5 min内Ca2+内流的平均流速，而NMDA受体拮抗剂D-APV可有效阻断Aβ对神经元Glu反应的这种易化作用；(3)用低钙人工脑脊液急性灌流脑片可引起海马CA1区神经元产生持续的外向跨膜Ca2+流，其大部分可被特异性Na+/Ca2+交换体抑制剂KB-R7943所阻断；(4) Aβ预处理可部分抑制低钙人工脑脊液引起的Ca2+外排。这些结果表明：Aβ引起的细胞内Ca2+超载不仅涉及到Ca2+内流增加，也与其对Ca2+外排的抑制有关；Aβ易化Glu的兴奋毒作用主要是通过NMDA受体介导的，其抑制Ca2+外排的靶点主要是Na+/Ca2+交换体。NMT具有操作相对简单、实时获取结果、非损伤的优点，适用于脑片Ca2+内流和Ca2+外排的长时间测定。因此，本研究不仅为解释Aβ所致Ca2+超载的神经毒性机制提供了新的实验证据，也为开展跨膜Ca2+信号转导机制的脑研究提供了新的技术方法。

关键词： 非损伤微测技术; 海马脑片 ; Ca2+流 ; 淀粉样β蛋白 ; 谷氨酸

分类号：Q4-33

Real-time measurement of Ca2+ flux in hippocampal slice with non-invasive micro- test technique

LI Tian, YUAN Li, ZHANG Jun, JIAO Juan-Juan, QI Jin-Shun

Department of Physiology, Key Laboratory for Cellular Physiology of Ministry of Education, Shanxi Medical University, Taiyuan 030001, China； Department of Physiology, Changzhi Medical College, Changzhi 046000, China

Abstract

The deposition of amyloid-β protein (Aβ) in the brain is the most important pathological feature of Alzheimer’s disease (AD). The mechanism of Aβ neurotoxicity may be closely related to the disturbance of intracellular Ca2+ homeostasis. Non-invasive micro-test technique (NMT) is a novel technique developed in recent years, which can be used to directly record transmembrane ion influx and efflux in a non-contact way by detecting the diffusion potentials outside of the membrane. The present study examined the effects of Aβ31-35 pretreatment on glutamate (Glu)-induced Ca2+ influx and low [Ca2+] solution-induced Ca2+ efflux in the hippocampal slices of C57BL/6 mice using NMT. The results showed that: (1) acute administration of Glu (2.5, 5, 10 mmol/L) evoked a persistent transmembrane Ca2+ influx in hippocampal CA1 neurons, with a rapid onset and subsequent decay; (2) pretreatment with Aβ dose- dependently increased the average rate of Ca2+ influx induced by Glu during the initial 5 min, which was blocked by NMDA receptor antagonist D-APV; (3) perfusion with low [Ca2+] artificial cerebrospinal fluid (aCSF) induced a continuous Ca2+ efflux, which was mostly blocked by KB-R7943, a specific antagonist of Na+/Ca2+ exchanger; (4) Aβ31-35 pretreatment partially inhibited the low [Ca2+] aCSF- induced Ca2+ efflux. These results indicate that Aβ not only facilitates Ca2+ influx but also inhibits Ca2+ efflux, which jointly contribute to the Aβ-induced intracellular Ca2+ overload; the potentiation of Aβ on Glu excitotoxicity is mainly mediated by NMDA receptors, while the target for Aβ to affect Ca2+ efflux was mainly Na+/Ca2+ exchanger. NMT showed multiple advantages in detecting transmembrane Ca2+ flux in brain slices, such as non-invasiveness to target cells, fast, convenient and real-time acquisition of Ca2+ flux. Therefore, this study provided new experimental evidence for Aβ-induced Ca2+ overload, as well as a novel application for NMT in measuring transmembrane Ca2+ flux of neurons in the brain.

Key words: non-invasive micro-test technique; hippocampal slice ; Ca2+ flux ; amyloid-β protein ; glutamate

收稿日期：2017-02-25　　录用日期：2017-05-05

通讯作者：祁金顺　　E-mail: jinshunqi2009@163.com

引用本文：

李甜, 原丽, 张军, 焦娟娟, 祁金顺. 非损伤微测技术实时检测海马脑片跨膜钙离子流[J]. 生理学报 2017; 69 (4): 467-476.

LI Tian, YUAN Li, ZHANG Jun, JIAO Juan-Juan, QI Jin-Shun. Real-time measurement of Ca2+ flux in hippocampal slice with non-invasive micro- test technique. Acta Physiol Sin 2017; 69 (4): 467-476 (in Chinese with English abstract).