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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

A 10 μm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo-3, fluo-4 or fluo-5F, to estimate dark, resting free Ca2+ concentration ([Ca2+]i) and changes in [Ca2+]i upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild-type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin α-subunit knock-out (Trα–/–) animals showed no light-dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (τ∼1–4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre-exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca2+]i. A similar rapid increase in fluorescence was also seen in the rods of wild-type mice just preceding the fall in fluorescence produced by the light-dependent decrease in [Ca2+]i. Dissociation constants were measured in vitro for fluo-3, fluo-4 and fluo-5F with and without 1 mm Mg2+ from 20 to 37 °C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3–4 over this range. Values at 37 °C were used to estimate absolute levels of rod [Ca2+]i. All three dyes gave similar values for [Ca2+]i in wild-type rods of 250 ± 20 nm in darkness and 23 ± 2 nm after exposure to saturating light. There was no significant difference in dark [Ca2+]i between wild-type and Trα–/– animals.

The increasing availability of mice whose proteins have been eliminated or altered by genetic manipulation has provided an opportunity to understand the physiology of vertebrate photoreceptors in considerable detail. Animals are now available lacking rhodopsin (Humphries et al. 1997; Lem et al. 1999), the α subunit of transducin (Calvert et al. 2000), rhodopsin kinase (Chen et al. 1999), arrestin (Xu et al. 1997), the γ-subunit of phosphodiesterase (Tsang et al. 1996), recoverin (Dodd, 1998), GCAP (guanylyl cyclase activating protein; Hurley & Chen, 2001), and RGS-9 (regulator of G-protein signalling; Chen et al. 2000). Increasing effort is being made to use these knock-out animals as a background for substituting proteins of somewhat altered properties (see for example Tsang et al. 2001) to understand the role of these molecules in photoreceptor excitation and adaptation.

Since Ca2+ plays an important role in sensitivity regulation in rods and cones (recently reviewed by Pugh et al. 1999; Fain et al. 2001), and perhaps also in photoreceptor pathogenesis (Fain & Lisman, 1999), it would be useful to be able to measure the free Ca2+ concentration ([Ca2+]i) in normal and genetically altered mouse outer segments. Measurements of [Ca2+]i have previously been made from single rod photoreceptors of gecko (Gray-Keller & Detwiler, 1994) and salamander (Sampath et al. 1998a; Matthews & Fain, 2001), but rods in these species have outer segments nearly 100 times larger in volume than those of mouse. It was therefore initially unclear whether sufficient dye could be loaded into mouse outer segments to permit measurement of [Ca2+]i without altering the physiological responses of the receptor, at an exciting intensity for the calcium indicator that produces minimal bleaching of the dye fluorophore.

We now show that by appropriately adjusting dye concentration and laser illumination, it is possible to modify for mouse rods a laser spot method previously used with success for salamander (Sampath et al. 1998a; Matthews & Fain, 2000). We find that light produces a decrease in [Ca2+]i in rods of wild-type mice but not in those lacking the α subunit of transducin. We also show that the dark resting [Ca2+]i is the same in normal and transducin knock-out animals but is lower in mammalian rods than in the rods of salamander or gecko. This may have the important consequence that [Ca2+]i is modulated over a smaller range in mammalian rods than in lower vertebrates. Preliminary results of this study have been reported to the Association for Research in Vision and Ophthalmology (Sampath et al. 1998b; Woodruff et al. 2001).

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

Chemicals and solutions

The fluorescent Ca2+ dyes fluo-3, fluo-4 and fluo-5F as pentapotassium salts or acetoxymethyl (AM) esters, as well as the calcium standards used to measure the dissociation constants of these dyes were purchased from Molecular Probes (Eugene, OR, USA). All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA). The dissection and incubation with AM ester dye was carried out in Locke solution (including mm): 140 NaCl, 3.6 KCl, 2.4 MgCl2, 1.2 CaCl2, 3 Hepes, 10 glucose, 5 Na ascorbate and 20 μm EDTA, pH 7.4). During fluorescence and suction pipette measurements, photoreceptors were continuously superfused with oxygenated Dulbecco's modified Eagle's medium (DMEM) without bicarbonate or phenol red (Sigma Chemical Co., D-2902), supplemented with (mm): 20 Hepes as well as 5 succinic acid, 0.5 glutamic acid and 5 gluconic acid (as Na+ salts). The pH was adjusted to pH 7.4, and temperature was maintained near 37 °C as described previously (Matthews, 1991). The suction pipette and bath ground electrode were filled with Locke solution without glucose or ascorbate.

Dissection and dye loading

All procedures were approved by the Chancellor's Animal Research Committee (ARC no. 93-230).

Mice previously maintained in total darkness for 3 h were killed by cervical dislocation. The eyes were removed from the head and trimmed of fat and extra-ocular muscle, and they were then washed with 1–2 ml of cold Locke solution. Killing, eye removal, trimming and washing were done in dim red light. The rest of the dissection was done under infrared illumination with an IR-sensitive TV camera (CCD camera model OS-40D, Mintron USA, Fremont, CA, USA) or an IR-sensitive viewer (FJW Optical Systems, Inc., Palatine, IL, USA).

Each eye was hemisected with a razor blade. The front of the eye (with lens) was discarded, and the eyecup (with retina) was cut in two by slicing through or close to the optic nerve head. Three pieces of eye were reserved on ice in total darkness, and one was placed in Locke solution in a 35 mm petri dish whose bottom had been coated with Sylgard (Dow Corning, Midland, MI, USA). From this piece the retina was isolated and finely chopped with a piece of razor blade. The resulting suspension was incubated for 30 min at room temperature in Locke solution containing fluo-3 AM, fluo-4 AM or fluo-5F AM (see Results). After completion of the incubation period, the cells were superfused at 37 °C for 20 min with DMEM, supplemented as described above, to wash out excess dye and to allow completion of the intracellular hydrolysis of the AM ester.

Fluorescence measurement

Methods for measuring calcium have been described previously (Sampath et al. 1998a, 1999; Matthews & Fain, 2000, 2001). Calcium dye fluorescence was excited by an intense 10 μm spot of light imaged on the rod outer segment through a 40 × oil-immersion fluorescence objective (1.3 NA). Excitation light was provided by an argon laser (American Laser Corporation, Salt Lake City, UT, USA), which for fluo-3 was tuned to 514 nm, and for fluo-4 and fluo-5F, to 488 nm. For fluo-3 we used a 525 nm dichroic filter and a 530 nm long pass filter, but for fluo-4 and fluo-5F, a 505 nm dichroic and a 510 nm long pass filter (Omega Optical, Brattleboro, VT, USA). Ca2+ dye fluorescence was recorded from the outer segments of single mouse rods either fully isolated or protruding from small chunks of retina; the saturated electrical response to the intense light of the laser was normally not recorded.

In control experiments, fluorescence was measured for the pentapotassium salt of each of the dyes immobilised in agarose gel (see Fig. 3A). To prevent dye-bleaching, the unattenuated intensity of the laser was normally reduced to around 5–6 × 1010 photons μm−2 s−1 by reflective neutral density filters (Newport Corporation, Irvine, CA, USA). Fluorescence was measured with a low-dark-count photomultiplier tube with a restricted photocathode (Model 9130/100A, Electron Tubes Ltd, Ruislip, UK), and the current was amplified by a low-noise current-to-voltage converter (PDA-700, Terahertz Technology, Oriskany, NY, USA), low-pass filtered with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA, USA) and digitised (PCLAMP, Axon Instruments, Foster City, CA, USA) with a PC-compatible computer over the bandwidths and at the sampling rates specified in individual figure legends.

image

Figure 3. Bright light evokes a rapid decline in fluo-4 fluorescence in wild-type mouse rods without dye bleaching

A, photomultiplier current elicited by fluorescence from a film of fluo-4 pentapotasssium salt immobilised in a high-Ca2+ agarose gel, to a 30 s exposure at a laser intensity comparable to that used for measurements from mouse rods. Trace is the average of four records obtained from different areas of the dye film. B, fluorescence response from a dark-adapted mouse rod loaded with fluo-4 evoked by the first exposure to laser light. C, the same data on an expanded time base illustrating the initial decline in fluorescence, together with a superimposed trace evoked by a second laser exposure 60 s later. The decay in fluorescence was fitted with a double exponential decay (eqn 2) with time constants for this cell of τ1/ 159 ms and τ2= 508 ms. Inset, mean change in circulating current as a function of time for 12 rods recorded with a suction pipette and exposed to the same laser intensity used for the calcium measurements. For clarity, only the response to the first laser exposure is shown; there was no response to the second, indicating that the circulating current remains suppressed between the two laser presentations. The arrow indicates Na+/Ca2+-K+ exchange current, which could be fitted with two constants of about 40 and 690 ms (continuous curve).

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Measurement of dye dissociation constant

Dissociation constants for the pentapotassium salts of fluo-3, fluo-4 and fluo-5F were measured as described by Tsien & Pozzan (1989). Two sets of standards were used, one without Mg2+ and one containing 1 mm Mg2+. The standards also both contained 100 mm KCl and 10 mm Mops buffer (pH 7.2). The free Ca2+ concentration in these solutions was estimated by the Max Chelator program (version 4.62, Chris Patton, Hopkins Marine Station, Stanford University, CA, USA) following well-established procedures (Martell & Smith, 1974; Fabiato & Fabiato, 1979; Harrison & Bers, 1989), to correct estimates of free Ca2+ for temperature, Mg2+ concentration and ionic strength. The concentration of Ca2+ in the standards was confirmed by Ca2+ electrode measurements. The final fluorescent dye concentration in all calibration standards was 10 μm. All dye calibration measurements were carried out with the same microscope, laser wavelength, filters and spot diameter as for measurements from photoreceptors.

To determine the temperature dependence of the dissociation constant, a special chamber was constructed. A triple layer of Parafilm (American National Can, Neenah, WI, USA) was pressed onto both sides of a rectangular glass coverslip (45 mm × 50 mm × 150 μm thick). A 10 mm hole was cut in the parafilm on the underside of the coverslip, and a second, smaller coverslip was placed over this hole, forming a chamber that contained the dye solution, held in place by surface tension. Slits were cut in the parafilm to either side to permit the passage of two small-diameter (0.051 mm) thermocouple wires (T-type, Cu-CuNi; Omega Engineering, Stamford, CT, USA). The thermocouple junction was positioned in the middle of the chamber, and melted paraffin wax held the wires in place and prevented leakage of solution. The temperature of the dye solution was monitored with a digital thermometer at 0.1 °C resolution (Model HH-25TC, Omega Engineering, Stamford, CT, USA).

The other side of the coverslip facing upward on the microscope stage contained a larger, 14 mm diameter well for the perfusion of distilled water to control the temperature of the dye chamber. Two streams of water fed into the upper well, one from an ice bath and the other from a heated water bath also passing through a heated aluminium block. Solution inflow from these two inlets was controlled by solenoid values (Lee Company, Westbrook, NJ, USA). The path length of the dye-containing solution in the lower well was approximately 0.4 mm, much larger than the vertical dimension of the laser spot focused at the image plane of the microscope.

Fluorescence was recorded via a 40 × dry objective (0.65 NA) while moving the plane of focus smoothly up and down through the sample by rotating the fine focusing control of the microscope with a small DC motor (Motor Mike, Oriel, Stratford, CT, USA). The fluorescence was initially low but increased to a plateau as the focal plane of the laser entered the dye solution (Fig. 1A, inset). The motor was then reversed and the spot moved back through the solution. This procedure was repeated to measure fluorescence from the same sample at four temperatures: 20, 22, 30 and 37 °C. All measurements were taken within 1 mm of the thermocouple junction to ensure that the temperature at the laser spot would be close to the measured temperature. At the higher free Ca2+ concentrations (from 500 nm to 40 μm), when the fluorescence signal was relatively high, a measurable change in fluorescence could be detected for a change of temperature of only 0.1 °C. For each dye at each temperature, three independent determinations were made, each with freshly made standard solutions.

image

Figure 1. Temperature changes within the physiological range alter the Ca2+ affinity of the fluorescent indicator dyes fluo-3, fluo-4 and fluo-5F

A, effect of temperature on the titration curve for Ca2+ binding to fluo-4 measured at 20 (×), 22 (□), 30 (▵) and 37 °C (□). Ordinate plots photomultiplier current evoked by dye fluorescence as a function of Ca2+ concentration (see Methods). Ca2+ concentrations have been corrected for effects of temperature on Ca2+-EGTA binding, leading to a slight displacement of the values at each temperature for individual solutions. Continuous curves determined according to a sigmoidal logistic model (eqn 1) fitted to the data at each temperature by a least-squares algorithm. Inset, superimposed photomultiplier current traces during fluorescence measurements with fluo-5F and nominally 60 nm Ca2+ at 20, 30 and 37 °C as the plane of focus was moved upward (first maximum) and then downward (second maximum) through the liquid dye film (see Methods). B, plot of dissociation constant against temperature determined for each of the three dyes as in A in the presence and absence of 1 mm Mg2+. Each point represents the mean of three independent determinations; error bars are s.e.m. Thermodynamic parameters calculated from the regression lines fitted to these data are collected in Table 1.

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Increasing temperature shifted the dissociation constant (Kd) of the dye to a lower Ca2+ concentration and decreased the maximum fluorescence (Fmax see Fig. 1A). Little effect of temperature was observed on minimum fluorescence (Fmin). Data at each temperature were fitted with a sigmoidal logistic model (eqn 1):

  • image(1)

The apparent cooperativity (n) of Ca2+ binding was not significantly different from unity for any of the three dyes used. Values for both Kd and Fmax are summarised in Table 1. The addition of Mg2+ caused an increase in the Kd of about 100–200 nm for all of the dyes at all temperatures.

Table 1.  In vitro dissociation constants, fluorescence ratios and thermodynamic constants for fluo indicator dyes
DyeMg2+TemperatureKds.e.m.FminFmaxFmax/FminΔHΔGΔS
  1. Dye concentration was 10 μm; Mg2+ when present was at 1 mM. Kd given in nM was determined in 100 mM KCl, 10 mM Mops (pH 7.2). Means of triplicate samples (n / 3) are given with standard error of the mean (S.E.M.). Fluorescence minimum (Fmin) and fluorescence maximum (Fmax) are in units of photomultiplier current (nA). ΔH and ΔG are in calories per mole, and ΔS is in calories per mole per degree kelvin.

Fluo-5F201130187.6236032013700–798074
 221000197.62260310 –810074
 3054067.21860260 –869074
 37310116.91570230 –923074
Fluo-4206201014.7156011012450–833071
 225201414.81530100 –848071
 30310913.81430100 –903071
 37190913.11300100 –954071
Fluo-320690411.842026012940–826072
 22590231.9400260 –841072
 30330241.8350230 –899072
 37200121.8320200 –950072
Fluo-5F+2014701159.3233025013810–782074
 +2212801179.32230240 –796074
 +30700559.61850190 –854074
 +374002510.41560150 908074
Fluo-4+207702114.2149010012760820072
 +226701814.31510110 –834072
 +303701014.31460100 –892072
 +372301214.41350100 –941072
Fluo-3+20842921.945728412170–815069
 +22723571.9437272 –829069
 +30420502.1387225 –884069
 +37266302.2344176 –933069

In Fig. 1B, the logarithm of the Kd is plotted against temperature for each dye, with and without Mg2+. The slopes of the regression lines through the data are virtually the same for all three dyes and were unaffected by Mg2+. The average enthalpy (ΔH) of Ca2+ binding was determined from a van't Hoff analysis (log Kavs. 1/T) and is given in Table 1. The enthalpy was about +12 to +13 kcal mol−1 (50 to 54 kJ mol−1) for all of the three fluo dyes tested. Table 1 also gives calculated values for the changes in free energy (ΔG) and entropy (ΔS).

Determination of free Ca2+ concentration

We estimated the absolute value of [Ca2+]i in darkness and after exposure to bright light in the same way as previously for salamander rods (Sampath et al. 1998a). The minimum fluorescence Fmin was determined by exposing the rod to a zero Ca2+/ionomycin solution (mm): 140 NaCl, 3.6 KCl, 3.08 MgCl2, 2.0 EGTA, 3.0 Hepes, with 10 μm ionomycin, pH 7.4). When the fluorescence reached a steady minimum value (Fmin), the rod was exposed to a high Ca2+/ionomycin solution (mm): 96.9 CaCl2, 3.6 KCl, 3.0 Hepes with 10 μm ionomycin, pH 7.4) to estimate Fmax. The calcium concentration was then calculated from the previously determined value of dye Kd and the Michaelis-Menten equation (Grynkiewicz et al. 1985; Sampath et al. 1998a).

Light stimulation and electrical recording

In experiments to determine the effects of dye loading on the electrical responses of wild-type rods or to investigate the degree of stimulation by the laser, the photocurrent was recorded using the suction pipette technique. Suction pipette currents were recorded with a patch-clamp amplifier (Model PC501-A, Warner Instruments Co, Hamden, CT, USA), low-pass filtered at 35 Hz and sampled at 100 Hz with the same apparatus used for recording the dye signals. In these experiments dye fluorescence was typically not recorded, since the outer segment was normally drawn into the suction pipette precluding spatially precise fluorescence measurements.

Light stimuli were delivered from a dual-beam optical bench via a 500 nm interference filter; stimulus intensity was controlled with absorptive neutral density filters and calibrated with a calibrated silicon photodiode (Graseby Optronics, Orlando, FL, USA). In some experiments, instead of 500 nm light rods were stimulated with white light, whose effective intensity was estimated by comparing the relative sensitivity of the electrical response to dim flashes of white and 500 nm light. Rhodopsin bleaching was estimated from the photosensitivity for a vitamin-A2-based pigment in solution (Dartnall, 1972), corrected for the difference in dichroism in free solution and in disk membranes (Jones et al. 1993).

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

Our initial attempts to measure [Ca2+]i in mouse rods were made in much the same way as previously for salamander (Sampath et al. 1998a, 1999). The isolated retina was incubated in 10 μm fluo-3 AM for 30 min, and fluorescence measurements were made from the outer segments either of isolated rods or of cells in small pieces of intact retina. As a control, suction pipette recordings were made from rods loaded with dye according to this protocol, either separately or simultaneously with measurements of dye fluorescence. We found, however, that both the sensitivity and the wave form of the light response were affected by the presence of the dye (Sampath, 1999), and concluded that, for this dye concentration and incubation period, sufficient fluo-3 had entered the rod to perturb the Ca2+ buffering of the outer segment, much as when rods are filled with the Ca2+ chelator BAPTA (Torre et al. 1986; Matthews, 1991).

The concentration of dye in the incubation solution was therefore varied in a series of experiments to determine the maximum concentration for each of these fluo dyes that left the photoreceptor wave form and sensitivity unaltered. The results of these experiments are shown in Fig. 2. Effects of the dye on response sensitivity were investigated by comparing the averaged response-intensity relationship after incubation with dye (open symbols) with that measured from controls in the absence of the dye (filled symbols). No systematic displacement of the response- intensity relation was observed when the retina was incubated for 30 min with fluo-3 AM at a concentration of 2 μm, fluo-4 AM at 2.5 μm or fluo-5F AM at the greater concentration of 10 μm, reflecting its higher Kd (see Table 1). The inset illustrates that 10 μm fluo-5F also had no detectable effect on the kinetics of the averaged dim flash response, and similar results were obtained for the other two dyes at a concentration of 2 μm. In the following measurements of [Ca2+]i, we accordingly used AM ester concentrations of 2 μm for fluo-3 and fluo-4, and 10 μm for fluo-5F.

image

Figure 2. Effect of dye loading on electrical responses of wild-type mouse rods

Mean response-intensity relations measured at the peak of responses to 20 ms light flashes of wavelength 500 nm under control conditions (•, 20 cells) and after incubation for 30 min with 2 μm fluo-3 AM (○, 9 cells), 2.5 μm fluo-4 AM (⋄, 11 cells) and 10 μm fluo-5F AM (□, 20 cells). Error bars denote s.e.m. Inset, superimposed, normalised dim flash responses for control rods (thin trace, 20 cells) and rods pre-incubated with 10 μm fluo-5F AM (thick trace, 25 cells). Mean responses were normalised for each cell before averaging between cells; flash intensity ∼0.75 photons μm−2.

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Light-induced [Ca2+]i decrease in wild-type mouse rods

When a dark-adapted mouse rod outer segment was first exposed to the laser, there was a rapid and complete suppression of the circulating current entering the rod outer segment (Fig. 3B, inset), induced by excitation of rhodopsin by the visible light of the argon ion laser leading to the closure of the cyclic-nucleotide-gated channels. This was then accompanied by a rapid decrease in dye fluorescence (Fig. 3B). The time course of the decline in fluorescence was not influenced by bleaching of the dye by laser light, since the laser intensity used in these experiments (around 5–6 × 1010 photons μm−2 s−1, see Methods) did not induce detectable bleaching for any of the three dyes in a gel film over a 30 s period, the longest employed in our measurements (Fig. 3A). The decrease in fluorescence in Fig. 3B was comparable in relative magnitude to that seen in single gecko (Gray-Keller & Detwiler, 1994) and salamander rods (Sampath et al. 1998a) but was considerably faster, being virtually complete within 2 s.

The upper trace in Fig. 3C illustrates the light-dependent decrease in fluorescence upon laser illumination from the same rod on a faster time scale. The time course of this decline could be well fitted over the first 5 s with the sum of two exponentials:

  • image(2)

with time constants τ1/ 159 ms and τ2= 508 ms. Fitting was not extended over a longer time window to avoid contamination by any slow movements of the outer segment. In similar experiments on a total of 10 rods these individually fitted time constants had mean values of τ1= 154 ± 31 ms and τ2= 540 ± 80 ms (mean ±s.e.m.). The ratio of their amplitudes, A1/A2, was approximately 0.6. The lower trace shows the fluorescence signal evoked from the same rod by a second laser exposure delivered 60 s after the first. This comparison illustrates that once a rod had reached steady state after exposure to the bright light of the laser, no further change in fluorescence occurred during at least this 60 s period (see also Fig. 6A). These results are consistent with those obtained previously from salamander rods (Sampath et al. 1998a; Matthews & Fain, 2001) and with suction pipette recordings (Fig. 3B, inset), that show that the laser was sufficiently intense to close all of the cyclic nucleotide-gated channels in the outer segment and to allow [Ca2+]i to fall to a minimum value.

image

Figure 6. Calibration of the dye fluorescence signals in a wild-type rod pre-incubated with 2 μm fluo-4 AM

A, dark-adapted rod exposed to five 30 ms laser flashes at 1 s intervals; sequence repeated four times at 30 s intervals. Fluorescence intensity evoked by the first laser flash in the first sequence was used to estimate dark-adapted [Ca2+]i, while the fluorescence from subsequent laser flashes was used to estimate [Ca2+]i after saturating light (see text and eqn 3). B, bath perfused with an EGTA-buffered zero Ca2+/ionomycin solution. Single laser flashes were delivered at 5 s intervals until a stable low level of fluorescence (Fmin) was attained (∼10 min in this rod). C, bath perfused with an isotonic Ca2+/ionomycin solution until a stable high level of fluorescence (Fmax) was attained (∼5 min in this rod). In some experiments, ionomycin was omitted from the high Ca2+ solution, since sufficient ionophore seemed to remain in the membrane from the preceding low Ca2+/ionomycin exposure. Estimates of dark-adapted and light-adapted [Ca2+]i derived from such measurements with all three dyes are collected in Table 2.

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Fluorescence recordings from transducin knock-out animals

Transduction of an electrical signal in rod photoreceptors requires the activation of the heterotrimeric G protein transducin (Fung et al. 1981), and in mice genetically altered so that the gene for the α subunit of transducin is knocked out, the electrical response of the rods over the normal physiological range is abolished (Calvert et al. 2000). Dark-adapted rods from homozygous transducin knock-out mice (Trα–/–) showed no decrease in dye fluorescence when exposed to laser light (Fig. 4A), indicating that the normal light-induced decline in [Ca2+]i was also abolished. However, when the fluorescence signal was examined on a more rapid time base (Fig. 4B), an initial increase in fluorescence could be detected in transducin knock-out mice, which was also seen at the very beginning of the fluorescence response in rods from wild-type animals.

image

Figure 4. The light-induced decline in dye fluorescence is not present in Trα–/– rods

A, comparison of the time course of the fluorescence signal evoked by laser illumination from dark-adapted Trα–/– and wild-type rods. Lower trace is the average of the responses of eight dark-adapted wild-type rods, each normalised to its initial value; upper trace is the average of the responses of 20 rods from Trα–/– animals individually normalised to the maximum value of the photocurrent. Rods were pre-incubated with 10 μm fluo-5F/AM. Control responses were low-pass filtered at 2 or 50 Hz and acquired at 5 or 1 kHz respectively; Trα–/– responses were filtered at 1 kHz and acquired at 2 kHz. B, time course of fluorescence signal soon after the onset of laser illumination. Note rapid initial fluorescence increase in both wild-type and Trα–/– rods. Data were all filtered over the wider bandwidth of 2-kHz and sampled at 5-kHz; traces are the averages of 46 (transducin knock-out) and 19 (wild-type) normalised responses.

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A similar rapid increase in the dye fluorescence signal from normal salamander rods has been described in some detail (Matthews & Fain, 2002). In salamander, exposure of a fluo-5F-loaded rod to bright laser light produces an exponential rise in fluorescence with a time constant of 1–2 ms, which is triggered by rhodopsin bleaching but which appears not to reflect an actual change in [Ca2+]i. Our initial observations suggest that this is also true for mouse rods. In Trα–/– animals, a rapid rise in fluorescence could only be evoked by the first of a series of bright laser exposures (Fig. 5A). Since the first laser exposure was sufficiently bright to have nominally bleached in excess of 99 % of the rhodopsin in the area illuminated by the laser, comprising most of the outer segment of the rod, the failure of the second exposure to evoke a rapid increase in fluorescence may have resulted from the considerable reduction in the concentration of rhodopsin remaining to be bleached. This conclusion is reinforced by the result of Fig. 5B, for which rods from a Trα–/– animal were pre-exposed to diffuse white light of an intensity and duration sufficient to bleach approximately 40 % of the rhodopsin in the outer segment. Subsequent exposure to the laser evoked a greatly reduced rapid component of fluorescence increase. Furthermore, the kinetics of the rapid fluorescence increase were found to depend upon the rate of rhodopsin bleaching (Fig. 5C). At a laser intensity of 4.6 × 1010 photons μm−2 s−1, the value of the best-fitting time constant for the averaged exponential rise of fluorescence was 4.0 ms, whereas for a laser intensity of 4.6 × 1011 photons μm−2 s−1, the time constant decreased to 1.5 ms.

image

Figure 5. The rapid initial increase in fluorescence of Trα–/– rods is associated with rhodopsin bleaching, but not with an increase in [Ca2+]i

All measurements were from Trα–/– rods pre-incubated with 10 μm fluo-5F/AM. A, comparison of the early fluorescence signal evoked by the first (thin trace) and second (thick trace) exposures to the laser in dark-adapted Trα–/– rods. The first laser exposure was of 4.5 s duration; the second exposure took place 90 s after the first. Traces are the means of the responses of 18 cells, each individually normalised to the initial fluorescence signal immediately after the opening of the laser shutter before the onset of the subsequent rapid increase in fluorescence. Note the presence of the rapid fluorescence increase in response to the first but not subsequent laser exposures in the same rod. B, comparison of the early fluorescence signal in dark-adapted Trα–/– rods (thin trace) and Trα–/– rods pre-exposed to bright white light calculated to bleach ∼40 % of the photopigment (thick trace). Data are the average of 14 (dark-adapted) and 46 (prebleached) normalised responses from rods in the same retinal preparation; the prebleached trace is from the same data as the Trα–/– trace of Fig. 4B on a faster time base. Data low-pass filtered at 2 kHz and sampled at 5 kHz. C, comparison of the rapid initial increase in fluorescence evoked from Trα–/– rods by the standard laser intensity (thin trace, 4.6 × 1010 photons μm−2 s−1; same data as dark-adapted trace in B) and a 10 times greater laser intensity (thick trace, 4.6 × 1011 photons μm−2 s−1). The trace obtained at the higher laser intensity represents averaged normalised data from 14 Trα–/–rods, and has been constrained to unity near time zero and magnified by a factor of 1.08 to aid comparison with the rapid fluorescence increase at the lower laser intensity. Note the more rapid rise in fluorescence at the higher laser intensity. Data low-pass filtered at 2 kHz and sampled at 5 kHz. D, comparison of the rapid initial increase in fluorescence from dark-adapted Trα–/– rods after BAPTA loading (thick trace, BAPTA) and under control conditions (thin trace, Control). For BAPTA loading the dye incubation solution contained either 50 or 100 μm BAPTA-AM; incubation duration, 30 min. Data are the average of 19 (BAPTA) and 18 (control, same data as first pulse trace in A) normalised responses. No difference was seen between the low and high BAPTA concentrations, so these data have been pooled. Note the persistence of the early fluorescence increase after BAPTA loading. Data low-pass filtered at 1 kHz and sampled at 2 kHz.

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In mouse as in salamander (Matthews & Fain, 2002) the rapid rise appears not to reflect an actual rise in [Ca2+]i. In salamander, the incorporation of the calcium chelator BAPTA into the outer segment had no effect on the rapid component of fluorescence increase (Matthews & Fain, 2002) but abolished a subsequent slow increase in dye fluorescence (with two time constants of ∼200 ms and ∼6 s), observed in rods superfused with 0Ca2+/0Na+ solution to minimise plasma membrane Ca2+ fluxes and representing a release of Ca2+ within the outer segment (Matthews & Fain, 2001). Similarly, when Trα–/– mouse rods were incubated for 30 min in either 50 or 100 μm BAPTA-AM, a concentration sufficient to effect a pronounced alteration in the waveform of mammalian rod photoresponses (Matthews, 1991) and to greatly slow light-dependent changes in [Ca2+]i in salamander rods (Matthews & Fain, 2001), the rapid component of fluorescence increase was not reduced from that in Trα–/– mouse rods without BAPTA incorporation (Fig. 5D). Furthermore, when rods were exposed in darkness to high Ca2+/ionomycin solution (see Methods), which would have produced a sizeable increase in [Ca2+]i within the outer segment, there was no observable change in the proportion of the amplitude of the response due to the fast component or in the normalised time course of the rise of the response during the first few milliseconds (data not shown). This result would not be expected if the rapid component were the result of a change in [Ca2+]i.

In situ calibration of [Ca2+]i

To estimate the absolute level of [Ca2+]i in mouse rods, the fluorescence signals were calibrated by artificially manipulating [Ca2+]i to minimise or maximise dye fluorescence. First, the outer segment was exposed to a 30 ms laser flash to estimate the initial fluorescence in darkness (Fig. 6A). This was then followed by a series of 30 ms flashes to determine the steady-state fluorescence following exposure to light and the complete suppression of the circulating current. Brief laser flashes were used in these determinations to minimise dye bleaching during the long procedure required to perform the calibration.

The same rod was then superfused with zero Ca2+/ ionomycin solution (see Methods) to artificially reduce [Ca2+]i (Fig. 6B). Dye fluorescence was measured repeatedly at 5 s intervals with 30 ms laser flashes, and outer segment position and laser spot focus were checked regularly and adjusted as required. Once the value of the fluorescence had reached a steady state, 10–20 successive measurements were averaged to determine Fmin. The outer segment was then exposed to high Ca2+/ionomycin solution to determine Fmax (Fig. 6C). Fluorescence intensities were translated into values for [Ca2+]i according to the equation:

  • image(3)

where F is the fluorescence from the rods either in darkness or at steady-state after exposure to bright light, and Kd is the dissociation constant of the dye in the presence of 1 mm Mg2+ and at 37 °C, taken from Table 1. The resulting estimates of [Ca2+]i are given in Table 2. There was no significant difference in the estimate of dark [Ca2+]i between wild-type and Trα–/– mice for any of the dyes tested (5 % level, unpaired Student's t test).

Table 2.  Rod outer segment calcium concentration(nm)
Mouse typeDyeRoads (n)Dark-adaptedSaturating light
  1. Errors are given as s.e.m.

WildFluo-320260 ± 3020 ± 3
 Fluo-46250 ± 9024 ± 8
 Fluo-5F14230 ± 3026 ± 2
 Combined40250 ± 2023 ± 2
Trα–/–Fluo-5F12225 ± 24 

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

The principal conclusion of this work is that the outer segments of rods from wild-type mice show a light-dependent decrease in [Ca2+]i similar to that previously described for gecko (Gray-Keller & Detwiler, 1994) and salamander (Sampath et al. 1998a), and that this decrease must result from activation of transducin since it is absent in mice lacking a functional gene for the α subunit of this protein. The dark resting value of [Ca2+]i is lower in mouse rods than in the rods of lower vertebrates, and the dynamic range for Ca2+-dependent modulation of transduction appears also to be diminished.

Measurement of free Ca2+ concentration

Our ability to measure [Ca2+]i in the small outer segments of mouse rods depended critically upon the high sensitivity of the spot-confocal method we originally devised for recording from salamander (Sampath et al. 1998a, 1999; Matthews & Fain, 2000, 2002). In adapting this method for mouse, however, the concentration of dye in the incubation medium had to be decreased to prevent an accumulation of dye large enough to alter the normal physiological properties of the photoreceptor (Fig. 2). The reason for this probably lies with the greater surface-to-volume ratio of the outer segments of mouse than of salamander, enabling a higher rate of entry of the AM ester across the outer segment membrane per unit intracellular volume, and therefore a more efficient loading of the dye.

To estimate the absolute level of [Ca2+]i, we determined the dissociation constants of fluo-3, fluo-4 and fluo-5F as a function of temperature and Mg2+. All of the dyes showed a pronounced dependence of Kd on temperature (see also Lattanzio, 1990; Lattanzio & Bartschat, 1991; Shuttleworth & Thompson, 1991; Paltauf-Doburzynska & Graier, 1997; Larsson et al. 1999), the common value of ΔH for all three dyes (+12 to +13 kcal mol−1) reflecting the similarity of the Ca2+ binding site of these chemically-related molecules. A Mg2+ concentration of 1 mm produced an increase in the Kd for all three dyes at all temperatures of about 100–200 nm, as would be expected if Mg2+ were simply competing with Ca2+ for the ion binding site of the dye.

Our measurements emphasise the importance of calibrating dye Kd under conditions as close as possible to those actually used in the physiological experiments, since had we used the room temperature Kd values quoted for these dyes by the manufacturer, our estimates of dark resting [Ca2+]i would have been much larger and dramatically different for each of the three dyes. In contrast, our own determinations of Kd at the temperature used in our experiments (37 °C) gave values for [Ca2+]i that are in substantial agreement (see Table 2). This seems to us to be of some importance, since fluo and other fluorescent calcium dyes are being used increasingly in studies of mammalian cells at elevated temperature, and the temperature dependence of the Kd is rarely taken into consideration.

Dark resting free Ca2+ concentration

We estimate the dark resting [Ca2+]i in mouse rods to be of the order of 250 nm and to decrease to a steady-state value of about 23 nm after exposure to bright light and the closure of the cyclic nucleotide-gated channels. These estimates are in excellent agreement with recent measurements of the activation of mouse guanylyl cyclase, which spans nearly its complete range between the calcium concentrations we have measured in darkness and after bright light (Calvert et al. 2001). They are however subject to several qualifications. It is difficult to be certain that, in the determination of Fmin and Fmax, the calibration procedure actually brought [Ca2+]i to minimal and maximal values. In a few cells, we tested for saturation of the dye in high Ca2+/ionomycin solution by further exposing the rod to 0.01 % saponin, and for most cells this produced little additional fluorescence. Nevertheless, the ratios of Fmax/Fmin calculated from measurements from outer segments were consistently smaller than the values obtained for the dyes in the in vitro measurements (fluo-3, 9.5 vs. 176; fluo-4, 20.5 vs. 100; fluo-5F, 31 vs. 150). In addition, we have used values for dye Kd from in vitro calibrations, but a number of studies have shown that dye Kd is influenced by ionic strength and viscosity and may be larger in the cytoplasm of a living cell (see for example Bassani et al. 1995; Du et al. 2001). However, our measurements are probably not greatly affected by binding of dye to protein, which is known in other tissues to produce a large change in dye Kd (Baylor & Hollingworth, 2000). This is because measurements of the diffusion constant of fluo dyes in salamander rod outer segments (Nakatani et al. 2002) indicate that most of the dye is freely diffusible and unbound to protein or other cytosolic components.

Our estimates of [Ca2+]i are also complicated by an initial rise in fluorescence with a time constant of a few milliseconds, which precedes the light-dependent fall caused by the decline in [Ca2+]i. This initial rise seems not to be produced by the normal transduction cascade, since it is of similar time course and amplitude in wild-type and Trα–/– rods. As in salamander (Matthews & Fain, 2002), it appears to be triggered by rhodopsin bleaching, since it can largely be suppressed by pre-exposure to bright bleaching light, whether from the laser or the optical stimulator. Nevertheless, it is unaffected by incorporation of the Ca2+ chelator BAPTA, either in salamander (Matthews & Fain, 2002) or in mouse (see Fig. 5D), suggesting that it does not reflect an actual increase in [Ca2+]i but rather some interaction between rhodopsin and the fluorescent dye. We have therefore used the fluorescence level 15 ms after the beginning of the laser flash in our estimate of dark [Ca2+]i. If a part of the rapid fluorescence increase in fact reflects a change in [Ca2+]i, a possibility we cannot entirely exclude (see for example Matthews & Fain, 2002), our estimates of [Ca2+]i in darkness will be too high. On the other hand, some decrease in [Ca2+]i must certainly occur during the first 15 ms as the cyclic-nucleotide-gated channels close, and this would cause our estimates to be too low. Back extrapolation, however, suggests that this latter effect is rather small (< 5 %), since the time constants of the light-dependent decrease are too slow to produce much change in [Ca2+]i during the first 15 ms.

Calcium and light adaptation

Calcium plays an important role as a second messenger in adaptation (Pugh et al. 1999; Fain et al. 2001): changes in sensitivity in background light (Matthews et al. 1988; Nakatani & Yau, 1988) and after bleaching (Matthews et al. 1996) are largely suppressed when the changes in [Ca2+]i are prevented. Calcium modulates guanylyl cyclase (Koch & Stryer, 1988) and has been proposed also to affect several other components of the transduction cascade (see Fain et al. 2001). It is therefore of some importance to determine the range within which [Ca2+]i varies during adaptation (see for example Gray-Keller & Detwiler, 1996), since this directly affects the extent to which transduction will be modulated and rod sensitivity will change in backgrounds and after bleaches (see Koutalos & Yau, 1996).

Previous measurements in salamander rods estimated that [Ca2+]i changes from 600–700 nm in darkness to about 30 nm in saturating light (Sampath et al. 1998a), giving a dark-to-light ratio of about 30–35 (see also Gray-Keller & Detwiler, 1994). More extensive determinations with all three fluo dyes have more recently provided values of 500 nm in darkness and only 8 nm in the light, giving an even larger dark-to-light ratio of around 60 (Matthews & Fain, unpublished data). In mouse, on the other hand, our estimate of this ratio (which is approximately the same for all three dyes and independent of the value of dye Kd) is considerably smaller, of the order of 10. This suggests that Ca2+ may vary over a smaller range in mammalian rods than in salamander.

Several studies have been made of light adaptation in mammalian species (Tamura et al. 1989; Matthews, 1991; Nakatani et al. 1991; Tamura et al. 1991), including mouse (Dodd, 1998). These studies all show that rods alter their sensitivity in background light, and there is evidence that Ca2+ plays a role in mammals (Matthews, 1991; Tamura et al. 1991) similar to that in amphibians (Matthews et al. 1988; Nakatani & Yau, 1988). One of the principal functions of the change in [Ca2+]i is to modulate guanylyl cyclase to prevent the rod photocurrent from saturating in constant background illumination. Since the outer segments of mammalian rods have a smaller diameter than amphibian rods, each mammalian rod will absorb photons at a lower rate for any given light intensity. It seems possible, therefore, that the smaller range of modulation of [Ca2+]i in mammals has the useful consequence that mammalian rods will reach increment saturation at about the same ambient light intensity as in other species despite their lower collecting area (see Fain, 1976).

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  5. Discussion
  6. References
  7. Acknowledgements
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Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

We thank Professor Roger Thomas of the Physiological Laboratory, University of Cambridge, UK for assistance with Ca2+ electrode measurements, and for support we acknowledge grants from the NIH (EY01844 to G.L.F. and EY12008 to J.L.), from the Wellcome Trust (to H.R.M.), and from Lion's of Massachusetts (to J.L.).