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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

Received: 2017-02-25　　Accepted: 2017-05-05

Corresponding author: 祁金顺　　E-mail: jinshunqi2009@163.com

Citing This Article：

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).