Calibration of agonist concentrations applied by pressure pulses or via rapid solution exchanger

Desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) can develop so rapidly to preclude functional studies when agonists are applied under steady-state conditions. To minimize receptor desensitization, we recorded from rat cultured chromaffin cells currents evoked by nicotine applied by pressure (via a fine tipped pipette) or through a multibarreled rapid solution exchanger. By comparing equi-amplitude, non-fading responses on the same cells, we constructed log 'dose'-response curves with the amount of nicotine expressed as either pulse duration (ms) or initial concentration (microM) inside the perfusion tubes. The linear part of these plots (10-50 ms or 20-100 microM) allowed extrapolating the initial nicotine concentration for a certain pulse duration within these limits. Potential dilution of nicotine in the bath was calculated with diffusion equations for a continuous point source: the maximal concentration of nicotine attainable by pressure application from a 100 microM containing pipette was 92 microM. We also calculated how diluted the same nicotine solution became following rapid superfusion and found it to be 46%. These results indicate that the amount of agonist applied by pressure to a relatively close cell could be calibrated in terms of effective concentration at membrane level and, with the present experimental arrangement, underwent limited dilution in the extracellular microenvironment.