Rapid regionally restricted pHi shifts inneurones induced by the UV photolysis of 2-nitrobenzaldehyde

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 mm 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; SigmaAldrich) 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.