A simple method for internal perfusion of mammalian central nervous system neurones in brain slices with multiple...

A simple system for internal perfusion of whole-cell patch-clamped neurones in brain slices allowing multiple, fast solution exchanges is described. With this system, perfusing solutions can be loaded immediately before use during the recording. At perfusion rates of 5-10 microliters/min, complete replacement of solutions in the patch pipette tip, as determined by changes in the pipette resistance and liquid junction potential, was achieved in less than 20 s. This occurred after a 1-min latency that was due to solution flow through the infusion tube. The effectiveness of the system was tested on rat hippocampal CA1 neurones in the slice preparation. The effects of replacement of internal 150 mM K+ by 150 mM Cs+ ions on voltage-activated K+ currents and of changing internal [Cl-] between 20 mM and 150 mM on evoked GABAA-mediated inhibitory postsynaptic currents (IPSCs) were studied. The blockade of K+ currents by Cs+ ions and the changes of IPSCs by altered internal [Cl-] ion concentration were achieved within 3.2 and 1.5 min, respectively, including the 'flow latency' of about 1 min, and recovery following solution change occurred within 5.2 and 1.5 min, respectively. More than 10 effective internal solution replacements could be performed within 1 h in a single neurone without affecting the recording stability.