Whole-cell recording of intracellular pH with silanized and oiled patch-type single or double-barreled microelectrodes.
Conventional ion-sensitive microelectrodes cannot be used in small cells, since they create too large an electrical leak at the site of penetration. Membrane potentials can be measured in such cells with the whole-cell configuration of the patch-clamp technique, after obtaining a high-resistance seal (giga-seal) to the cell membrane. Achieving such seals with patch-type microelectrodes silanized and filled with ion-sensitive cocktails has proved very difficult. Since ion-sensitive microelectrodes offer advantages over fluorescent techniques, we have developed a method which enables whole-cell recordings of membrane potential and intracellular pH to be achieved with silanized microelectrodes. We have been able to obtain high-resistance seals with silanized tips by dipping them in mineral oil. We describe the method for both single-barrel and theta-glass double-barreled microelectrodes. Double-barreled microelectrodes can be used to measure and control membrane potential in the whole-cell patch-clamp configuration while also measuring ionic activities with the adjacent barrel. We present illustrative experiments showing intracellular pH recordings in snail neurones and rat dorsal root ganglion cells, and we suggest the method can also be applied to other liquid-sensor ion-sensitive microelectrodes.
Thomas
Roger C
RC
Department of Physiology, University of Cambridge, Downing Site, Cambridge CB2 3EG, UK. rct26@cam.ac.uk
Pagnotta
Sara E
SE
Nistri
Andrea
A
D000818:Animals; D002021:Buffers; D002245:Carbon Dioxide / Q000494:pharmacology; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D004947:Esophagus / Q000294:innervation; D005724:Ganglia / Q000166:cytology / Q000378:metabolism; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism; D006372:Helix, Snails; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008839:Microelectrodes; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D009821:Oils; D011522:Protons; D051381:Rats; D012821:Silanes
D002021:Buffers; D009821:Oils; D011522:Protons; D012821:Silanes; D002245:Carbon Dioxide