Abstract
Within the nervous system, intracellular Cl− and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl− and pH are often co-regulated, and network activity results in the movement of both Cl− and H+. Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl− and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN—a new genetically-encoded ratiometric Cl− and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl− and H+ concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons.
Highlights
Chloride (Cl−) and hydrogen (H+) ions are fundamental to a wide range of processes within the nervous system including cell division, volume regulation, migration, metabolism, synaptic vesicle loading, network excitability, and fast synaptic inhibition (Tabb et al, 1992; Denker and Barber, 2002; Putney and Barber, 2003; Farrant and Kaila, 2007)
Expression of ClopHensor or PalmPalm-ClopHensor in hippocampal pyramidal neurons often resulted in uniform E2GFP expression, but highly heterogeneous DsRed expression that occurred as dense intracellular aggregations (Figures 1B,E,F)
Here we present ClopHensorN—a genetically-encoded ratiometric Cl− and pH sensor that is optimized for use in the nervous system
Summary
Chloride (Cl−) and hydrogen (H+) ions are fundamental to a wide range of processes within the nervous system including cell division, volume regulation, migration, metabolism, synaptic vesicle loading, network excitability, and fast synaptic inhibition (Tabb et al, 1992; Denker and Barber, 2002; Putney and Barber, 2003; Farrant and Kaila, 2007). Whilst intracellular Cl− concentration ([Cl−]i) and the negative logarithm of intracellular H+ ion concentration (pHi) are known to affect network excitability, network activity itself can generate shifts in the intracellular concentrations of these two ions (Isomura et al, 2003; Raimondo et al, 2012b). This reciprocal relationship means that tools to accurately and independently measure [Cl−]i and pHi are important for understanding the separate and combined roles that these ions play during physiological and pathological network states.
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