Neuronal hyperactivity causes Na+/H+ exchanger-induced extracellular acidification at active synapses

Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H+ concentration. The aim of this study was to describe the spatio-temporal dynamics of extracellular pH at synaptic sites during neuronal hyperexcitability. To address this issue we created ex.E2GFP, a membrane-targeted extracellular ratiometric pH indicator that is exquisitely sensitive to acidic shifts. By monitoring ex.E2GFP fluorescence in real time in primary cortical neurons, we were able to quantify pH fluctuations during network hyperexcitability induced by convulsant drugs or highfrequency electrical stimulation. Sustained hyperactivity caused a pH decrease that was reversible upon silencing of neuronal activity and located at active synapses. This acidic shift was not attributable to the outflow of synaptic vesicle H+ into the cleft nor to the activity of membrane-exposed H+ V-ATPase, but rather to the activity of the Na+/H+-exchanger. Our data demonstrate that extracellular synaptic pH shifts take place during epileptic-like activity of neural cultures, emphasizing the strict links existing between synaptic activity and synaptic pH. This evidence may contribute to the understanding of the physio-pathological mechanisms associated with hyperexcitability in the epileptic brain.