Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Guillaume Luxardi; Brian Reid; Fernando Ferreira; Pauline Maillard; Min Zhao

doi:10.3791/52782

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Method Article

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

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DOI:

10.3791/52782

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May 3rd, 2015

Guillaume Luxardi¹ , Brian Reid¹ , Fernando Ferreira¹^,² , Pauline Maillard³ , Min Zhao¹^,⁴

¹Department of Dermatology, Institute for Regenerative Cures, University of California, Davis, ²Departamento de Biologia, Centro de Biologia Molecular e Ambiental, Universidade do Minho, ³Department of Neurology and Center for Neuroscience, University of California, Davis Imaging of Dementia and Aging Laboratory, ⁴Department of Ophthalmology, Institute for Regenerative Cures, University of California, Davis

Summary

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Abstract

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick’s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Introduction

All animal cells are surrounded by a lipid bilayer membrane which separates the cytoplasm from the outside environment. The cell maintains an electrical membrane potential, negative inside, by active transport of ions¹. The membrane potential is a stored energy source which the cell can utilize to operate various molecular devices in the membrane². Neurons and other excitable cells have large membrane potentials. Rapid opening of sodium channels collapses the membrane potential (depolarization) and produces the action potential which is transported along the length of the neuron². Aside from these rapid electrical changes, many tissues....