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1 Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
2 Division of Neuroscience, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia
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Abstract |
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(Received 10 May 2005;
accepted after revision 23 May 2005;
first published online 26 May 2005)
Corresponding author J. Jarvinen: Division of Neuroscience, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia. Email: jaakko.jarvinen{at}anu.edu.au
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Introduction |
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Although the bulk of the outer segment current is carried by Na+ ions, a substantial fraction (10–25% in different cells) is carried by Ca2+ ions. In the steady state, this influx of calcium is matched by an efflux of calcium via the Na+–Ca2+,K+ exchanger (NCKX). Upon illumination the lowered influx of calcium through the channels causes a drop in Ca2+ concentration due to the continued efflux via the exchanger (Yau & Nakatani, 1985). The reduction in Ca2+ concentration triggers a powerful negative feedback loop that contributes to response recovery and to photoreceptor light adaptation. The drop in calcium activates guanylyl cyclase activating proteins (GCAPs: Dizhoor et al. 1994; Gorczyca et al. 1994), increasing the synthesis of cGMP by guanylyl cyclase (GC), raising the cGMP concentration and re-opening plasma membrane ion channels.
It has generally been taken for granted in the literature that illumination at different positions along the outer segment leads simply to a suppression of outer segment circulating current (e.g. Lamb et al. 1981; see also Schnapf, 1983). Lamb et al. (1981) measured the spatial profile of outer segment responsiveness when a stimulus was moved to different positions along the outer segment, with the outer segment drawn either fully or part-way in to the suction pipette; they reported roughly constant sensitivity wherever the ‘recorded’ region was stimulated, and a declining sensitivity as the stimulus moved onto the ‘unrecorded’ region. Schnapf (1983) found a gradation of sensitivity along the outer segment.
In contrast we report here that stimulation of the ‘unrecorded’ region of the outer segment (outside the suction pipette) leads to a prominent response of inverted polarity, i.e. an increase in circulating current. We investigate the role of intracellular voltage in giving rise to these inverted responses, and we present a model that is able to account for them; this model invokes the known voltage dependence of the Na+–Ca2+,K+ exchanger (Yau & Nakatani, 1984; Lagnado et al. 1988; Cervetto et al. 1989). Our interpretation is that the reduced current in the local region where the photon was absorbed is flanked by increased current over the remaining length of outer segment.
We are aware of anecdotal reports of inverted responses, and such events have been examined briefly by Dr C. S. Leibrock (unpublished observations) in our Cambridge laboratory. In addition, inverted responses are reported in a few publications. Baylor & Lamb (1982; their Fig. 12B) and Donner et al. (1987; their Fig. 6B) reported the occurrence of a negative-going component in the current recorded from the tip of a dark-adapted rod, in response to flash stimulation at the base or over the entire outer segment, respectively. Both studies attributed the inverted responses to changes in intracellular voltage causing changes in current through the ohmic resistance of the outer segment membrane. Here we have designed experiments to investigate the cellular mechanism of these inverted responses, and we present an alternative model that we think is consistent both with our own experiments and with previous reports.
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Methods |
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Animals
We began using cane toads, Bufo marinus, obtained from Peter Douch (Queensland, Australia), but when we experienced difficulty in achieving whole-cell recordings we instead used clawed frogs, Xenopus laevis, obtained from Blades Biological (Kent, UK). Animals were dark adapted overnight, and then killed humanely, under the provisions of Schedule 1 of the Animals (Scientific Procedures) Act, by stunning with a purpose-built captive-bolt gun followed immediately by pithing. Retinal rods were obtained as described by Murnick & Lamb (1996).
Experimental procedures and light stimulation
In most experiments the outer segment of a single rod was drawn (either fully or partly) into the suction pipette, though in some experiments the inner segment (and possibly part of the outer segment) was drawn in. Stimuli were brief (20 ms) flashes of green light (500 nm, circularly polarized; Fain et al. 1989) delivered either diffusely, or else to a spatially restricted region using an adjustable slit. The length of the slit was always greater than the diameter of the cell (usually three or more times greater). To improve image quality, the microscope condenser was replaced by an objective lens (10 x Zeiss Planachromat; Lamb et al. 1981). We presume that the spatial profile of light scatter was as measured in that study.
The stimuli are specified in terms of the incident flash intensity (in photons µm–2) and the nominal slit width (in µm) measured in each experiment. In order to calculate the number of photoisomerizations elicited when this stimulus fell on the outer segment, we used the calculation of effective collecting area given in eqn (14) of Baylor et al. (1979), but with the nominal slit width rather than the full length of the outer segment. For the nominal slit width of 3 µm used in Fig. 1, and the measured outer segment diameter of 6 µm, the effective collecting area for the slit and outer segment was 
1 µm2, so that a flash intensity of 3.7 photons µm–2 would be expected to have elicited 3.7 photoisomerizations (R*). From the spatial profile of light scattering given by Lamb et al. (1981), we estimate that very little of the incident light would have been scattered beyond the recorded region of outer segment, when the stimulus was more than 10 µm from either end.
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Solutions
The ionic composition of the Ringer solution was (mM): Na+ 113, K+ 2.5, Ca2+ 1.0, Mg2+ 1.6, Cl– 119, EDTA 0.01, Hepes 3.0, D-glucose 10, with pH buffered to 7.7–7.8. Bovine serum albumin (0.1 mg ml–1, Sigma) was added to solutions used for dissection or short-term storage, and glucose was omitted from the solution used for filling suction pipettes. The patch pipette solution contained (mM): K+ 101, Na+ 16, Mg2+ 6, Asp– 91, Cl– 12, ATP2– 5, GTP3– 1, Hepes 5, EDTA 0.01, with pH buffered to 7.4. The osmolality of the intracellular solution was measured using a vapour pressure osmometer (Wescor 5500, Germany) as 215 mosmol kg–1, 8% lower than for the extracellular solution (234 mosmol kg–1). For the solutions above, used in our patch-clamp experiments, we estimated the liquid junction potential to be 16.2 mV, from the generalized Henderson equation (see e.g. Ng & Barry, 1995); this value was subtracted from all raw voltages.
Data acquisition
Suction pipette current was recorded with a custom electrometer, and patch pipette current (or voltage) with a List EPC-7 (Darmstadt, Germany). Signals were filtered DC-20 Hz (6-pole Bessel), digitized at 100 Hz, recorded continuously to computer disc, and analysed offline; the custom recording and analysis programs ran under Matlab (The MathWorks, Inc.). During analysis, signals were further low-pass filtered digitally, by a Gaussian filter (usually with a cut-off frequency of 5 Hz); this introduced no additional delay.
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Results |
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We studied spatial properties of the photocurrent by taking advantage of a feature of the suction pipette technique that allows one to record the current flowing through just part of the length of the outer segment, rather than its entire length. When the rod outer segment was drawn part way into the suction pipette and the unrecorded part (the base of the outer segment) was stimulated with spatially restricted flashes, we observed current responses of inverted polarity (i.e. increased circulating current). The phenomenon was observed in rods of both Bufo marinus (n = 41 cells) and Xenopus laevis (n = 32 cells).
Figure 1 illustrates one experiment (out of five similar experiments on different cells) in which the outer segment was first drawn part way into the suction pipette, then drawn fully in; in both configurations, the length of the cell was scanned several times with a dim slit-shaped flash stimulus as described in previous publications (Lamb et al. 1981; Schnapf, 1983). Figure 1A demonstrates that stimulation of the base of the outer segment generated an inverted response when only the tip of the outer segment was recorded, but that the overall current response from the entire outer segment, recorded afterwards, was entirely normal. Thus, inverted responses could not be attributed to damage to cells. Inverted responses were characterized by a slow time course (time to peak up to 4–5 s in Bufo) relative to the conventional light response from the same cell. The time to peak of the inverted response was shorter (1–2 s) in Xenopus rods, and the conventional response was both faster and less sensitive than in Bufo rods (see also Solessio et al. 2004).
The spatial profile of sensitivity (Fig. 1C) was broadly consistent with earlier work (Lamb et al. 1981), except that when unrecorded regions of the cells were stimulated, the responses were inverted. Note that these profiles have been plotted at measurement times near the time-to-peak of the conventional and inverted responses, 1.8–2.3 s and 4.5–5.0 s, respectively, as indicated by the bars in Fig. 1A. As can be seen in Fig. 1B and C, a small response was elicited by a stimulus positioned on the inner segment, and we think that this can be accounted for by light scattered to the region of the outer segment within the suction pipette.
To eliminate the possibility that the inverted responses might somehow have arisen from damage to the tip of the outer segment by the suction pipette, we drew a rod in the other way round, i.e. we drew the inner segment and part of the outer segment into the pipette. In this configuration, illumination of the whole outer segment generated conventional responses (suppression of circulating current), while localized illumination of the base of the outer segment, now inside the pipette, generated a response of opposite polarity (an increase of current through the tip of the outer segment). We interpret this result to indicate that inverted responses (measured at the tip, in response to flashes at the base) are physiological, and are not dependent on which end of the outer segment is drawn in.
Effect of local bleaches
We studied the effect of localized bleaches on inverted responses by drawing a Bufo marinus rod part way into the suction pipette, stimulating the unrecorded part with flashes and bleaching the region inside the suction pipette (the tip) with brief intense light exposures (n = 8 cells). The purpose of bleaching was to close all ion channels in the recorded region. Before any bleaching took place (in the dark-adapted state), responses of inverted polarity were observed (grey trace, Fig. 2A). We attribute the positive-going ‘hump’ at around 1 s to have been elicited by scattered light stimulating the recorded part of the cell directly. After local bleaching of the tip, only the fast negative-going transient remained (continuous black trace, Fig. 2B). The slow inverted component and the positive-going hump were abolished by local bleaches. Bleaching did not change the size or the time course of the fast transient for any flash intensity tested (Fig. 2B; responses to two flash intensities shown), providing further evidence to support the finding of Baylor & Lamb (1982) that this transient represents a capacitive current due to intracellular voltage change.
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We made simultaneous recordings of suction pipette current and patch pipette voltage (or current) from isolated Xenopus laevis rods (Fig. 3A). To our knowledge, these are the first such recordings from this species to be published (for recordings from salamander rods, see Lamb et al. 1986). In our best experiments we were able to record for 10–15 min before excessive run-down of light responses occurred. Our success rate in achieving an adequate whole-cell patch-clamp recording from these cells was low, and we attribute this to the fragility of these cells and to the exposed recording configuration where a large part of the cell protruded from the suction pipette.
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We take the elimination of inverted responses by voltage clamping as evidence that both the fast and slow components of the inverted response are dependent on changes in membrane voltage. However, because the peak of the slow component of the inverted response was always substantially delayed from the peak of the simultaneously measured intracellular voltage response (e.g. Fig. 7B; n = 5 cells), we can rule out the possibility that the inverted response was caused by an ohmic current flowing through the resistance of the cell membrane.
Origin of the fast initial transient
If the origin of the fast initial transient were a capacitive current due to the intracellular voltage change, as suggested by Baylor & Lamb (1982), then the peak of the fast transient should coincide with the steepest gradient of the voltage response. To test this prediction, we took the time derivative of the voltage response and compared it with the simultaneously recorded suction pipette current (Fig. 5). Figure 5B shows that the time course of dV/dt (dashed trace) is very similar to the time course of the initial transient in the suction pipette current (grey trace) and we take this as further evidence, in addition to our bleaching experiments, for the capacitive origin of this component. We can extract an estimate for the membrane capacitance from these recordings as follows. If fin represents the fraction of outer segment drawn into the suction pipette and feff the collecting efficiency, then the recorded capacitive current JC,rec can be written as:
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| (1) |
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We developed a biophysical model (Appendix) to predict the slow component of the inverted response from the measured voltage response. This model is based on the known reactions occurring in the outer segment, as indicated schematically in Fig. 6, and in particular depends on the known relationship between membrane voltage and the activity of the Na+–Ca2+,K+ exchanger. Thus, hyperpolarization of the plasma membrane leads to increased extrusion of calcium by the NCKX, a reduction in [Ca2+]i, increased production of cGMP by guanylyl cyclase due to the enzyme's calcium sensitivity, and opening of cyclic nucleotide gated channels due to the increased [cGMP]. The opening of CNGCs leads to an increased circulating current, which accounts for the polarity of the inverted response. The slow time course of the inverted response can be explained by the two integrating steps involved in changing the concentrations of Ca2+ and cGMP (eqns (A2) and (A4)).
Where possible, the parameter values used in modelling were estimated for the cells that we simulated, as indicated by ‘This study’ in Table 1. The great majority of the remaining parameter values were taken from published estimates for salamander rods, as that is the only amphibian species for which values have been determined. For the exponents ncyc and ncG, values have widely been reported for amphibia, but we have taken the most recently revised estimates, which were in fact obtained in experiments on mouse rods. Thus, the parameters listed in Table 1 are intended to be applicable to amphibian rods generally, and to Xenopus, Bufo and Ambystoma rods in particular.
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We tested whether the fit of the model to experiment could be made even closer by taking into account the effect of scattered light, which we think was masking some of the inverted response (especially its rising phase). We took the suction pipette response recorded under voltage clamp as the best available estimate of the response due to scattered light, and we assumed that this component was also adding in to the current-clamped response. In this approach we are assuming that the current recorded under voltage clamp arises solely from direct stimulation of the recorded region by stray photons, because any components of current due to voltage change will have been eliminated. Figure 7A shows currents recorded from the tip during current clamp (black) and voltage clamp (grey). Figure 7C compares the difference of the two traces from Fig. 7A (black trace, representing the inverted response corrected for the effect of scattered light) with the theoretical prediction. Inspection shows that the fit of the theory to the rising phase is considerably better in Fig. 7C than Fig. 7B, as would be expected if scattered light had been contributing significantly to the suction pipette currents. (As explained in the legend, we used slightly different parameters in Fig. 7C compared with those used in Fig. 7B).
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Discussion |
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One way to compromise this loop would be to incorporate a calcium buffer such as BAPTA into the outer segment. We have simulated the expected effects using our computational model, and as might be expected intuitively, we find that in the absence of any other changes an increase in calcium buffering power is predicted to reduce the amplitude of the slow component and to further retard its time course. However, this preliminary calculation takes no account of complicating factors such as the likely slowing of the intracellular voltage response in the presence of BAPTA. We plan to examine the effects of increased calcium buffering power in future experiments.
For illumination that is spatially restricted along the outer segment (as occurs with a single photoisomerization), the resulting local conductance change elicits a hyperpolarization that is distributed along the length of the outer segment, and which causes a distributed increase in circulating current. The net result is a localized region of reduced current at the point of illumination, flanked by an increase in current over the remainder of the outer segment. The integrated response over the entire length is of course a decrease in current, but the response exhibits characteristics similar to both a spatial centre-surround and a temporal high-pass. In the future it will be interesting to examine the extent to which the normal intracellular voltage response affects the time course of recovery of the single-photon response, by comparing current-clamp and voltage-clamp conditions. However, to accomplish this, it is likely to be necessary to employ the perforated patch technique, to lower the rate of run-down and thereby permit whole-cell recording durations considerably longer than those of 10–15 min that we have been able to achieve so far.
A model of the kind described here is able to account for other related phenomena. Thus, anecdotal reports of spontaneous events of inverted polarity (e.g. from our laboratory) would be explicable naturally in terms of spontaneous photoisomerizations in the region of outer segment that is not ‘electrically’ within the suction pipette. In response to hyperpolarizing voltage steps in voltage-clamp mode, Baylor et al. (1984) and Baylor & Nunn (1986) reported that the outer segment current exhibited a delayed increase; in qualitative terms, this is exactly as predicted by our analysis, though a quantitative test would probably require new experiments. On the other hand, our model has not been formulated to apply to depolarizing voltage steps like those used by Miller & Korenbrot (1993, 1994); those authors reported an increased circulating current in response to depolarizing steps from the resting potential to voltages above +20 mV. As we have presented it, our model is restricted to the range of voltages negative to rest, where the outer segment behaves approximately as a current source, i.e. where current is only weakly dependent on membrane voltage (Bader et al. 1979; Baylor & Nunn, 1986; Cobbs & Pugh, 1987). With large depolarizing steps, one would need to take into account the effect of membrane voltage on the driving force for Ca2+ through the cGMP-gated channels (Miller & Korenbrot, 1993, 1994).
A previous model of inverted and biphasic responses (Donner et al. 1987) assumed that the membrane of the region of outer segment drawn into the suction pipette behaved ohmically, giving a component of current flow directly proportional to the change in membrane voltage. However, a number of previous studies (Bader et al. 1979; Baylor & Nunn, 1986; Cobbs & Pugh, 1987) have shown the slope conductance to be very small, so that a mechanism of this kind could make only a small contribution to the inverted responses.
The delayed increase in circulating current that is elicited by the voltage sensitivity of the calcium extrusion mechanism not only generates inverted responses in our recording configuration, but, more generally, it provides a mechanism to speed the recovery phase of the electrical response to illumination. Thus, it may be that the importance of the cellular mechanism that we have described here is to bring about an increase in the temporal resolution of the photoreceptor.
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Appendix |
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We make the assumption that the cytoplasm of the outer segment behaves as a well-stirred compartment to avoid consideration of the effects of diffusion. Our model takes into account the effect of calcium feedback upon the Na+–Ca2+,K+ exchange current and the activity of guanylyl cyclase, and the feedback effect of cyclic GMP upon the calcium concentration.
The basis of our theoretical analysis is the voltage sensitivity of the Na+–Ca2+,K+ exchange. Lagnado et al. (1988) reported that in the salamander rod, the exchange current Jex increases exponentially with hyperpolarization of the membrane voltage V, i.e.:
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Here Jex,rest and Vrest are the resting exchange current and the membrane voltage (in darkness), and Vex is the e-fold voltage sensitivity of the exchange current, reported as Vex = –70 mV (Lagnado et al. 1988).
The exchange current is also dependent on the intracellular free calcium concentration (written as Ca for brevity). The dependence is described by
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| (A1) |
The second equation of our model is the differential equation for the calcium concentration, which can be written as
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| (A2) |
is Faraday's constant, Vcyt is the volume of cytoplasm in the outer segment, and BCa is the cytoplasmic buffering power for calcium, which is defined as BCa
= dCatot/dCa, where Catot is the total
[Ca2+] in the outer segment, as opposed to the free
[Ca2+]. In our calculations we assumed for simplicity that the buffering power was a constant, BCa
= 75 (Lagnado et al. 1992).
The calcium dependence of the guanylyl cyclase activity 
is given by the third equation of our model (Forti et al. 1989, eqn (16); Koutalos et al. 1995; Nikonov et al. 2000, eqn (A10)):
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| (A3) |
min is the residual GC activity (in the presence of high [Ca2+]i), max is the maximal GC activity (in very low [Ca2+]i), Kcyc is the [Ca2+]i for half-maximal GC activation and ncyc the cooperativity of GC activation by calcium.
The concentration of cyclic GMP (abbreviated as cG) is determined by the rates of its synthesis and hydrolysis, as described by the following differential equation (Kawamura & Murakami, 1986, eqn (4)):
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| (A4) |
The current mediated by the cGMP gated channels (JcG) is given by the fifth equation (Fesenko et al. 1985; Hodgkin & Nunn, 1988, eqn (5); Yau & Baylor, 1989):
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| (A5) |
Predictions for JcG and Jex are obtained by numerically solving eqns (A1)–(A5) for a measured voltage response, V. We coded the numerical solution in Matlab, using the in-built solver function ‘ode45’, and also using Simpson's rule as a check.
The capacitive current JC across the entire outer segment membrane is given by:
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| (A6) |
The final equation of our model sums the three separate currents caused by the voltage change (capacitive, Ca2+ exchange and CNGC-mediated currents), and takes into account the current collecting efficiency of the suction pipette (feff) and the fraction of outer segment drawn in (fin), to give a prediction for the recorded current Jrec as:
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| (A7) |
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References |
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and bandwidth of 5 Hz is 0.1 pA; averaged from 10 traces, the expected peak-to-peak noise would be 
