Abstract
Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl−]i) and hydrogen ([H+] i) the construct, called ClopHensor, which consists of a H+- and Cl−-sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H+]i and [Cl−]i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H+]i and [Cl−]i under different experimental conditions including changing of external concentrations of ions (Ca2+, Cl−, K+, Na+) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl− and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl− and pH.
Highlights
Neuronal activity is accompanied by dynamical changes in the intra- and extracellular concentration of ions
Our preliminary observations suggest that the reason for this elimination of pH responses is a continuous strong decrease in the amplitude of evoked local field potentials upon transition to “low-Cl−” artificial cerebrospinal fluid (ACSF) conditions
We recently presented a line of transgenic mice expressing ClopHensor in neurons and obtained a detailed map of its distribution in the mouse brain [32]
Summary
Neuronal activity is accompanied by dynamical changes in the intra- and extracellular concentration of ions. Intracellular Cl− concentration ([Cl−]i) in different cell types varies from 3 mM to 60 mM, being around 5–10 mM in the majority of mammalian neurons [7] Deviations from this physiological range can alter the excitability of cells, modulate the function of a variety of proteins including ion channels [8,9,10]. Promising are genetically encoded fluorescent sensors, which allow non-invasive monitoring of intracellular ion concentrations in specific cell types. Improved variants of ClopHensor have been developed and tested in cultured brain slices using biolistical transfection [29] Another biosensor optimized for the simultaneous [Cl−]i and pHi imaging, called LSSmClopHensor, has been developed [30]. Our observations demonstrate the efficiency of transgenic mice expressing ChlopHensor for reliable non-invasive monitoring of intracellular Cl− and pH in normal and pathological conditions
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