文章基本信息

标题：Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo
本地全文：下载
作者：Sebastian Sulis Sato ; Pietro Artoni ; Silvia Landi 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：41
页码：E8770-E8779
DOI：10.1073/pnas.1702861114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Intracellular chloride ([Cl⁻]_i) and pH (pH_i) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl⁻]_i and pH_i, but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl⁻ and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl⁻]_i and pH_i at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl⁻]_i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pH_i and [Cl⁻]_i in response to hypercapnia and seizure activity.
关键词：fluorescent indicators ; neurodevelopment ; neuronal inhibition ; bumetanide ; NKCC1