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  • 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
  • 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 (pHi) 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 pHi, 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 pHi 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 pHi and [Cl]i in response to hypercapnia and seizure activity.
  • 关键词:fluorescent indicators ; neurodevelopment ; neuronal inhibition ; bumetanide ; NKCC1
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