Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo.

Intracellular chloride ([Cl]) and pH (pH) 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] and pH, 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] and pH at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl] 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 and [Cl] in response to hypercapnia and seizure activity.