A sustained elevation of calcium accompanies the slow current. (A) Membrane current was measured concomitantly with Ca fluorescence of the rhabdomeric lobe in a cell loaded with the indicator Fluo 4 (83 µM). Recordings were made every 3 s; after four baseline measurements, excitation flashes (90 ms in duration) were delivered repetitively. The top graph shows the development of the slow current. The bottom graph plots the fluorescence signal, which decayed partially after the initial large increase, but remained significantly elevated with respect to the basal level. The open square and dotted line mark the basal fluorescence level during the first flash, and the highest point represents the peak fluorescence level attained during the same flash (in subsequent flashes the trace was flat during the light, as there was no further release; therefore, the “foot” vs. “peak” distinction is not made). (B) To estimate whether dye bleaching might contribute significantly to the decay of the fluorescence signal, owing to the extended light exposure, photoreceptor cells were loaded with the Ca indicator for ≈4 min and the pipette was withdrawn to prevent any further dye exchange. (C) The epi-fluorescence beam was activated for 5 s, evoking a large optical signal that decayed to a plateau (basal fluorescence is indicated by the dotted line). (D) After 10 min, the excitation light was turned on again, and the basal fluorescence was found not to be noticeably depressed with respect to the initial level (i.e., immediately after the first opening of the shutter). The initial portion of both fluorescence traces is displayed (on the left they were displaced vertically for clarity; the arrowheads mark the basal fluorescence levels). The slower rise of the light-induced Ca2+ increase on the second trial indicates physiological rundown. On the right, the same traces are superimposed and shown on an expanded time scale. Because no replenishment of dye was possible under these conditions, the results indicate that a 5-s exposure to the excitation light did not result in significant bleaching of the calcium probe.
