Comparing melatonin and 5-HT permeation using quenching of an intracellular fluorescent dye. (A) Schematic of a single cell on a coverslip locally perfused by a pipette containing control Ringer or Ringer supplemented by 10 mM melatonin (Mel) or 5-HT. The epi-illumination and fluorescence recordings use a 40× objective below the coverslip with a rectangular field of view stopped down to include just the one cell. (B) Mean fluorescence of fura-2-AM–loaded single cells as indoleamine solutions are perfused rapidly. The trace in B is derived from original records as follows. Separate single-cell fluorescence traces for 14 perfused coverslips were averaged and corrected for photobleaching. Traces for 12 perfused cell-free areas were treated similarly and subtracted from the with cell traces to correct for a small additional fluorescence contributed by the indoleamine compounds applied in the bath. This correction subtracted a step of fluorescence of 0.5 × 10−3 from the melatonin period and of 4.6 × 10−3 from the 5-HT period. Excitation wavelength was 370 nm.
