The control of intracellular Cl − and pH plays a crucial role in several neuronal functions, and the study of these processes would be helped by tools for their noninvasive optical measurement in vivo. In this study, we have performed combined measurements of Cl − and pH of individual pyramidal neurons by means of in vivo two-photon imaging, and we provide direct experimental demonstration for the presence of the postnatal developmental shift to lower intraneuronal Cl −. Moreover, we introduce an approach for dynamic and simultaneous monitoring of intraneuronal Cl − and pH in vivo. These methods will open a window for the study of the roles of intraneuronal pH and Cl − in neuronal signaling, plasticity, and disease.
Intracellular chloride ([Cl −] i) and pH (pH i) 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 pH i, 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 pH i 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 pH i and [Cl −] i in response to hypercapnia and seizure activity.