Bleached pigment produces a maintained decrease in outer segment Ca2+ in salamander rods

Abstract

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 +/- 250 nM in a dark-adapted rod and 30 +/- 10 nM during response saturation after exposure to bright light (mean +/- SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Figure 1

Measurement of Ca²⁺ _i from an isolated salamander rod using the spot confocal technique. The beam from an argon ion laser (514 nm line) illuminates the pinhole of the spatial filter, is recollimated, and enters the inverted microscope through the epi-fluorescence port. A dichroic mirror (525 nm) redirects the beam through the oil-immersion objective lens (40×, 1.3 NA) to illuminate a spot (diameter 7.7 μm) on the outer segment of a rod loaded with fluo-3 AM whose inner segment is held in a suction pipette. The fluo-3 fluorescence evoked by the laser spot passes through the emission filter (530 nm long-pass) and is collected confocally by a PIN photodiode. (inset) Calibration curve measured in situ using fluo-3-free acid solutions of known Ca²⁺ concentration in a haemocytometer. Solid curve is the Michaelis-Menten relation (Eq. 1) fitted using a Marquardt-Levenberg least-squares algorithm (K _d = 400 nM).

Figure 2

Exposure of a dark-adapted rod to the laser spot. (A) Circulating current measured by the suction pipette in response to the first laser exposure in a dark-adapted rod. (B) Normalized fluo-3 fluorescence signal recorded by the photodiode. (trace 1) First laser exposure in the dark-adapted rod; (trace 2) second laser exposure presented 60 s later while the circulating current remained completely suppressed. Fluorescence has been normalized by dividing the signal by the initial photodiode current in trace 1. The declining fluorescence signal in trace 1 was fitted with two exponentials with time constants of 380 and 2,000 ms. (top) Laser light monitor.

Figure 3

Histogram plotting the short (A) and long (B) time constants of the two exponentials fitted to the decline in fluo-3 fluorescence in response to laser illumination of a dark-adapted rod. Filled bars, rods loaded with fluo-3 AM (86 cells); open bars, rods loaded with fluo-3-free acid from a patch pipette (eight cells). Solid curves are Gaussian distributions fitted to the filled bars, yielding mean time constants of 260 and 2,200 ms and standard deviations of 80 and 500 ms, respectively.

Figure 4

In situ calibration of Ca²⁺-dependent fluorescence from a rod loaded with fluo-3 AM. (top) Laser monitor; laser illumination delivered as a sequence of four 20-ms pulses. Left, fluo-3 fluorescence; right, suction pipette current. (A) First laser exposure of a dark-adapted rod. (B) Second laser exposure presented shortly thereafter while the circulating current remained completely suppressed. (C) Outer segment exposed to 0 Ca²⁺ solution containing 25 μM ionomycin to determine minimum fluo-3 fluorescence (F _min). The arrow indicates the clipping level of the suction pipette amplifier. (D) Outer segment exposed to isotonic Ca²⁺ solution containing 25 μM ionomycin to determine maximum fluo-3 fluorescence (F _max). These parameters allow the initial and final fluorescence levels from this experiment to be translated into values for Ca²⁺ _i of 474 nM in darkness and 35 nM when the circulating current was completely suppressed.

Figure 5

Comparison of normalized fluorescence responses from dark-adapted rods loaded with fluo-3 AM and fluo-3-free acid. (A) Rod loaded by incubation for 30 min with 10 μM fluo-3 AM. (B) Rod loaded from a patch pipette containing a pseudo-intracellular solution that included 100 μM fluo-3-free acid; 4 min of whole-cell recording before first laser pulse. Traces 1, first laser exposure in the dark-adapted rod; traces 2, second laser exposure presented shortly thereafter while the circulating current remained completely suppressed. Fluorescence has been normalized in each case by dividing the signal by the initial photodiode current in trace 1. (top) Laser light monitor.

Figure 6

Changes in fluo-3 fluorescence during the photopigment cycle. Rod loaded by incubation with 10 μM fluo-3 AM. (left) Fluo-3 fluorescence; (right) suction pipette current. (top) Laser light monitor; laser illumination delivered as a sequence of four 20-ms pulses. (A) First laser exposure of the dark-adapted rod; this and a subsequent train of laser pulses bleached the rod by >99%. (B) Laser exposure 53 min after bleaching once the circulating current and sensitivity had stabilized (see Fig. 7). (C) Laser exposure after regeneration of the photopigment by superfusion with phospholipid vesicles containing 11-cis-retinal.

Figure 7

Changes in circulating current, sensitivity, and fluo-3 fluorescence during the photopigment cycle. (A) Circulating current recorded by the suction pipette measured as the current suppressed by the laser exposure or by a saturating flash of light. (B) Response sensitivity measured using trains of dim flashes delivering 0.74 (dark-adapted, before the first laser exposure), 11 (bleached), and 2.6 (regenerated) photon μm⁻² at 570 nm. (C) Fluo-3 fluorescence in response to trains of laser pulses presented to the rod when dark adapted, after bleaching, and after regeneration. Under each condition, two successive trains of laser pulses were presented in rapid succession; the values plotted represent the maximum and minimum fluorescence levels in darkness and after complete suppression of the circulating current. In each case, the column representing the second fluorescence measurement has been displaced slightly to the right for clarity. Top traces denote the delivery of bright flashes (Light), trains of laser pulses (Laser), and superfusion with phospholipid vesicles containing 11-cis- retinal (11-cis).