Hitherto three types of techniques have been used to changeintracellular pH (pH_i): superfusion of weak acids or bases (Jacobs, 1920), cytosolic injection of acid or alkali, or the activation of channels or transporters (e.g. the Ca²⁺:H⁺ pump, Schwiening et al. 1993) that allow transmembrane H⁺ or HCO₃^– fluxes. Here I describe a fourth technique, the regional UV-induced release ofprotons (Ciamician & Silber, 1901; George & Scaiano, 1980) that allows for extremely rapid and localized acidic pH_i shifts. This technique should provide a simple, cheap and highly controllable way of generating local pH_i signals independently of changes in other ion concentrations (e.g. Ca²⁺).

Helix aspersa neurones were isolated from the gangliafollowing enzymatic digestion and mechanical agitation (Schwiening & Willoughby, 2002). Snail Ringer solution containing the isolated neurones was placed in a superfusion chamber on a Zeiss LSM 510 confocal microscope and the cells were allowed to settle for ~1 h. A cell with an axonal projection was selected and patch clamped in the whole-cell configuration (pipettes ~1.5 µm tip filled with 110 mM CsCl, 0.5 mM of the pH-sensitive dye HPTS). pH-sensitive fluorescence was imaged (488 nm excitation, 505 nm emission) using a 63x water-immersion c-apochromat objective (1.2 NA). In some experiments ratiometric pH recordings were made using alternating 488 nmand 357 nm excitation of the dye HPTS. 2-nitrobenzaldehyde (NBA; Sigma-Aldrich) was bath applied at between 1.0 and 10 mM (made up fresh each day from a 100 mM stock solution in methanol) and uncaged using the LSM510 bleach function (AOTF-modulated illumination with 364 nm light during a whole image sweep) at maximum power.

This demonstration is, to the best of my knowledge, thefirst time acid has been both locally flash released and recorded inside a cell. Such release of intracellular acid is by far the most rapid and controllable method for altering regional pH_i. Photolysis of NBA, orits derivatives (Abbruzzetti et al. 2003), should facilitate the investigation pH_i microdomain signalling targets in normal neuronalfunction.

I thank the MRC for providing the LSM510 and their high rating of my recent grant applications. However, I deplore their failure to support any of my current work, including that presented here, which has been supported by the University.