Quantitative Analysis of Presynaptic Vesicle Luminal pH in Cultured Neurons

Newly generated synaptic vesicles (SVs) are re-acidified by the activity of the vacuolar-type H⁺-ATPases. Since H⁺ gradient across SV membrane drives neurotransmitter uptake into SVs, precise measurements of steady-state vesicular pH and dynamics of re-acidification process will provide important information concerning the H⁺-driven neurotransmitter uptake. Indeed, we recently demonstrated distinct features of steady state and dynamics of vesicular pH between glutamatergic vesicles and GABAergic vesicles in cultured hippocampal neurons. In this article, we focus on an experimental protocol and setup required to determine steady-state luminal pH of SVs in living neurons. This protocol is composed of efficient expression of a pH-sensitive fluorescent protein in the lumen of SVs in cultured neurons, and recordings of its fluorescence changes under a conventional fluorescent microscope during local applications of acidic buffer and ionophores-containing solution at a given pH. The method described here can be easily applied for measuring luminal pH of different types of secretory organelles and other acidic organelles such as lysosomes and endosomes in cultured cell preparations.