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The Journal of Neuroscience, February 15, 1999, 19(4):1203-1216
The Photovoltage of Macaque Cone Photoreceptors: Adaptation, Noise, and Kinetics
David M. Schneeweis and Julie L. Schnapf Departments of Ophthalmology and Physiology, University of California, San Francisco, California 94143-0730
ABSTRACT
Top
Abstract
Introduction
Whole-cell voltage and current recordings were obtained from red and green cone photoreceptors in isolated retina from macaque monkey. It was demonstrated previously that the cone photovoltage is generated from two sources, phototransduction current in the cone outer segment and photocurrent from neighboring rods. Rod signals are likely transmitted to cones across the gap junctions between rods and cones. In this study, the "pure" cone and rod components of the response were extracted with rod-adapting backgrounds or by subtracting the responses to flashes of different wavelength equated in their excitation of either rods or cones. For dim flashes, the pure cone component was similar in waveform to the cone outer segment current, and the rod component was similar to the photovoltage measured directly in rods. With bright flashes, the high frequencies of the rod signal were filtered out by the rod/cone network. The two components of the cone photovoltage adapted separately to background illumination. The amplitude of the rod component was halved by backgrounds eliciting ~100 photoisomerizations sec
1 per rod; the cone component was halved by backgrounds of 8700 photoisomerizations sec
Key words: rod; cone; photoreceptor; primate; adaptation; coupling
INTRODUCTION
Certain aspects of human vision, such as color sensitivity, are determined by the process of light absorption and phototransduction in cone outer segments (Baylor et al., 1987
; Schnapf et al., 1987
Psychophysicists describe two distinct retinal mechanisms by which rod signals are processed in parallel. One mechanism is slow and sensitive to dim light, and the second is faster and operates at mesopic light levels (Conner and MacLeod, 1977
Under photopic (cone-mediated) conditions, human subjects can reliably detect a flash of light that elicits <50 photoisomerizations per cone (Hood and Finkelstein, 1986
MATERIALS AND METHODS Preparation and solutions. Eyes were obtained from 10 cynomologus monkeys (Macaca fascicularis) and 4 rhesus monkeys (Macaca mulatta). No differences were found in the physiology of the cones obtained from the two species. At least 30 min before enucleation, an opaque black occluder was placed over the cornea of a monkey under general anesthesia. Retinal pieces were isolated and stored in L-15 medium (Life Technologies, Gaithersburg, MD) at 4°C for up to 3 d. Details of the surgery and dissection are given in Schneeweis and Schnapf (1995)
A piece of retina ~3 × 3 mm2 was isolated from the pigment epithelium and placed receptor side up in a recording chamber perfused with bicarbonate-buffered Locke's solution equilibrated with 95% O2/ 5% CO2 and kept at 37°C. The recording solution contained (in mM): NaCl (120); NaHCO3 (20); KCl (3.6); CaCl2 (1.2); MgCl2 (2.4); HEPES buffer (3), pH 7.4; D-glucose (10); EDTA (0.02); and Basal Medium Eagle amino acids and vitamins (Life Technologies).
Photovoltage recordings from red and green cones were obtained by the perforated-patch method (Horn and Marty, 1988
Light stimuli. Cones were stimulated with unpolarized light incident approximately parallel to the long axis of the outer segments. Wavelength was regulated with interference filters of 10 nm nominal half-bandwidth. Intensity was controlled with calibrated neutral density filters. The flash stimulus was a circular spot 180 µm in diameter. Flash duration was 10 msec. In adaptation experiments, the background diameter was 310-800 µm.
The number of photoisomerizations per flash was calculated from the product of the flash photon density and the photoreceptor collecting area. Except where noted, the collecting area was assumed to have peak values of 0.6 µm2 for cones and 1 µm2 for rods (Schneeweis and Schnapf, 1995
Electrical recording. Signals were recorded with an Axopatch 2D amplifier (Axon Instruments, Foster City, CA), filtered by the Axopatch four- pole Bessel analog filter, and in some cases refiltered by a Gaussian digital filter. Phase delays resulting from filtering were taken into account. Membrane potentials were corrected for the electrode junction potential. Unless otherwise stated, voltage signals were recorded with the amplifier in current-clamp mode. In some cases, photocurrent was recorded with the same electrode configuration and with the amplifier in voltage-clamp mode.
Noise analysis. For characterizing the power spectral density of the membrane voltage fluctuations, voltage recordings were low-pass filtered with the Axopatch Bessel filter and with an additional eight-pole Bessel filter (Frequency Devices, Haverhill, MA). The cutoff frequency of both filters was set at 200 Hz, giving an effective cutoff of ~140 Hz. Records were digitized at a sampling rate of 1 kHz.
Power spectra were computed using Fourier transform routines from the software package Igor (WaveMetrics, Lake Oswego, OR). Digitized records of ~12.3 sec were divided into overlapping segments of 2048 points each, and each segment was multiplied by a Hanning window. Power spectra were computed for each segment and then averaged. Spectra from multiple 12.3 sec sweeps were then averaged together to obtain the final spectrum.
Because the noise spectra reported here are not difference spectra, it was important to ascertain whether the electronics of the recording configuration or the experimental conditions introduced artifactual features into the spectra. Voltage noise spectra were obtained under approximate experimental conditions from voltages measured across a 100 M
resistor, from high-resistance electrodes (>100 M
RESULTS The cone inner segment shapes the photovoltage
In cones of cold-blooded vertebrates, the photovoltage is shaped by voltage-dependent conductances residing in the inner segment (Barnes, 1994
The photovoltage response to a dim brief flash was diphasic (Fig. 1) and could be fitted by the same kinetic equations (see Fig. 16) found to describe the outer segment photocurrent [Schnapf et al. (1990)
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Figure 1. Photovoltage and photocurrent responses to 660 nm flashes. A, Photovoltage of a red cone receiving minimal rod input. Traces are averages of 1-11 responses. Flash strength ranged from 3.68 × 103 to 2.96 × 106 photons µm
The overall durations of the current and voltage responses were similar, as quantified by the "integration time" of the cone (the time integral of the normalized dim-flash response). In a collection of 17 cones, the integration time of the photovoltage response was 25 ± 7 msec (n = 17), similar to the mean of 24 msec found for the photocurrent (Schnapf et al., 1990
With brighter flashes, prominent differences in the photocurrent and photovoltage were observed. In contrast to that of the photocurrent, the time to the peak of the photovoltage shortened dramatically with increasing flash strength. For the brightest flashes, a prominent initial voltage transient was seen that was absent in the photocurrent (see Figs. 1, 4). These differences between photovoltage and photocurrent were expected from the activation of voltage-dependent conductances identified in macaque cones (Yagi and MacLeish, 1994
Rods influence the response waveform and spectral sensitivity of cones
Another conspicuous difference between the outer segment photocurrent and the photovoltage was wavelength dependence. The waveform of the photocurrent depended on the quantity but not the wavelength of photons absorbed (Baylor et al., 1987
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Figure 2. Dependence of response waveform on wavelength. A, B, Superimposed responses of a red cone to flashes of increasing strength at wavelengths of 500 nm (A) and 660 nm (B). Vertical lines indicate the time points at which response amplitudes were measured for plots shown below (see Fig. 3, 18 and 400 msec). Flash photon densities increased by a factor of approximately four between traces and ranged from 8 to 3.07 × 104 photons µm
If the fast and slow components of the cone photovoltage represent cone and rod contributions, respectively, then the early part of the response should reflect the spectral absorption of cone photopigment, and the late part should reflect the spectral absorption of rod photopigment. Figure 3 plots the amplitude of the responses in Figure 2 measured at the peak and at fixed early and late times. The smooth curves near the points are Michaelis-Menten functions of the form:
where i is the flash photon density, r is the response amplitude, rmax is the maximal response amplitude, and i0 is the flash photon density that gives a response amplitude of 0.5 rmax.
(1)
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Figure 3. Response amplitude of a red cone plotted as a function of the log of the flash photon density for wavelengths of 500 nm ( ) and 660 nm (
). Some of the flash responses are illustrated in Figure 2. A-C, Amplitudes were measured at the peak of the response (A), 18 msec after the midpoint of the flash (B), and 400 msec after the flash (C). The smooth curves are described in the Results with the following values for rmax (in mV) and i0 (in photons µm
At the late (400 msec) time point (Fig. 3C), the amplitudes at both 500 and 660 nm were reasonably well fitted by Equation 1. The separation of the two curves on the x-axis (3.64 log units) was close to the separation expected (3.58 log units) for light absorption in rods (Baylor et al., 1984
Rod/cone coupling was also evident in cones recorded under whole-cell voltage clamp, as illustrated for a red cone in Figure 4. Flash strengths at 500 and 660 nm were approximately matched for light absorption in the cone, but they differed in their relative effectiveness for light absorption in rods. In response to 500 nm flashes, the photocurrent displayed a long-lasting outward tail that mirrored the after-hyperpolarization of the photovoltage. The presence of this tail current under voltage clamp indicated that the current did not result from the activation of voltage-dependent conductances in the cone. The tail current had not been observed in suction electrode recordings (Baylor et al., 1987
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Figure 4. Rod input visualized in whole-cell voltage clamp. A, B, Photocurrent responses of a red cone to flashes of increasing strength at wavelengths of 500 nm (A) and 660 nm (B). Flash photon densities increased by a factor of approximately two between traces and ranged from 160 to 1380 photons µm
The time-to-peak of the light response under voltage clamp was invariant with increasing light intensity, in contrast to the shortening of the time-to-peak of the photovoltage (Fig. 1A). Thus the intensity dependence of the rise time of the photovoltage was attributable to the activation of voltage-dependent conductances as opposed to synaptically activated currents.
Coupling between red and green cones?
Gap junctions have been described between cones in primate retina (Raviola and Gilula, 1973
Cone sensitivities fell into two groups that clustered near the predicted values for red or green cones (Fig. 5). (No blue cones were encountered.) For both groups the relative sensitivity to 500 nm light was elevated compared with the value expected for pure red and green photopigment (Fig. 5, dashed lines). Measured values of log S500/S660 were 1.15 ± 0.17 and 2.47 ± 0.20 (mean ± SD), as compared with the expected values of 0.88 and 2.13 for pure red and green cones, respectively. Sensitivities for four rods on the other hand [3.60 ± 0.07 (mean ± SD)] were tightly distributed about the predicted value of 3.58 (Fig. 5, dotted line).
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Figure 5. Spectral sensitivity of rods and cones to 500 and 660 nm light. Points plot the log of the relative spectral sensitivity to 500 and 660 nm flashes measured from 16 red cones ( ), and 4 rods (
If red and green cones were electrically coupled, then their symbols would lie in the interval between the two dashed lines of expected cone sensitivities. Instead, the symbols for each group were displaced toward the rod value, indicating that rods added a measurable contribution to the cone photovoltage even at brief times after a flash of light. These measurements do not rule out some mixing of cone signals, but if cone-cone coupling existed, its effect on spectral sensitivity was overwhelmed by a larger rod contribution.
Estimates of the kinetics of rod and cone inputs
In retinas of cold-blooded vertebrates, rods are extensively coupled to other rods, and to a lesser extent to cones, through gap junctions (Fain, 1975
Stimulus-matching method for decomposing the cone photovoltage
The "pure" cone component of the cone photovoltage was isolated by taking the difference of responses to flashes that were equated for excitation of rods but not of cones. Figure 6A shows responses to 500 and 660 nm flashes estimated to produce an equal number of photoisomerizations in rods but an unequal number in the cone. The closeness of the rod match was verified by the near coincidence of the late phase of the responses. Whereas the 660 nm response had clear rod and cone components, the 500 nm response lacked a cone component because the flash was too dim to excite cones effectively (Schnapf et al., 1990r500) yielded an estimate of the cone component of the 660 nm response (Fig. 6B). As expected, it had a diphasic waveform characteristic of the photocurrent (Fig. 1B). Similar results were obtained from five other cones.
In a complimentary experiment, cone-matched responses to 500 and 660 nm flashes were used to isolate the pure rod component of the cone photovoltage (Fig. 7). The response to the 660 nm flash lacked rod input because the intensity was too dim to evoke a sizable rod response at this wavelength; as expected, the slow rod tail was absent (Fig. 7A). The difference response (r500
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Figure 6. Average responses in a red cone to flashes of 500 and 660 nm light, with flash strength matched for rod photon capture. A, Responses to flashes of 500 nm (dashed line) and 660 nm (solid line). B, Difference response (r660
Was the rod signal altered by the network? To answer this, we obtained rod signals at several other flash intensities by lowering the intensity of the flash pairs in parallel and by comparing the difference responses to photovoltage responses recorded directly in rods with comparable stimuli (Fig. 8). For the dimmest flashes, the response waveforms were similar, indicating that small rod signals were not altered by network filtering. For brighter flashes, the prominent initial transient of the rod photovoltage was nearly absent in the cone photovoltage, and the flat plateau of the rod photovoltage was replaced in the cone by a slowly developing hyperpolarization that never reached a steady state. Although the variability of response kinetics made a detailed comparison of separate rod and cone recordings impractical, in all 11 cones analyzed in this way, the rod component of the cone response lacked the sharp transient and flat plateau found in all rod photovoltage recordings.
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Figure 7. Average responses in a red cone to flashes of 500 and 660 nm light of flash strength matched for cone photon capture. A, Responses to flashes of 500 nm (dashed line) and 660 nm (solid line). B. Difference response (r500
Interpretation of the kinetics of the bright flash response is complicated by possible nonlinearities in the summation of the rod and cone components. With large excursions of the cone membrane potential, nonlinearities would result from voltage-activated conductances and alterations in the driving force for the photocurrent. In a few cones, the effect of these nonlinearities was assessed by eliciting cone-matched responses when the cone was voltage-clamped to the resting membrane potential (Fig. 4). The rod components computed from these voltage-clamp responses were similar to those in Figure 8A, indicating that nonlinear summation did not significantly distort the rod component estimates. The mechanism of temporal filtering of the rod signals in the rod/cone network is not known. The nature of this filtering observed in primates is unlike the high-pass characteristic of other rod/cone networks (Attwell et al., 1984
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Figure 8. Comparison of the rod component of the cone photovoltage to the rod photovoltage. A, The rod component of the cone photovoltage was estimated from the difference responses (r500
Adaptation method for decomposing the cone photovoltage
Another method for decomposing the cone photovoltage that was independent of spectral absorption matching took advantage of adaptation differences of rod and cone phototransduction. The two traces in Figure 9A are photovoltage responses of a green cone to 500 nm test flashes in the presence or absence of a dim steady background, also of 500 nm. The background was of an intensity expected to saturate rod phototransduction (Baylor et al., 1984
Although the background itself evoked only a small change in membrane potential (<0.2 mV), it dramatically altered the response to the test flash. The slow component of the hyperpolarization was eliminated as would be predicted from its rod origin. The peak response was also reduced, reflecting an early rod influence on the cone signal as suggested from the peak spectral sensitivity estimates of Figure 5. The light-adapted response resembled the cone component derived from the spectral difference response (Fig. 6B). Similar results were obtained in five other cones. If one assumes that the rod but not the cone component was suppressed by the background, subtraction of the light-adapted from the dark-adapted response would give another estimate of the rod component. The adaptation difference response in Figure 9B is noisy but similar to the spectral difference response estimate in Figure 8A at a similar flash strength. Comparable adaptation difference responses were obtained in a total of six cones. Although the analysis from Figure 9 did not rely on precise intensity matching, a critical assumption was that cone phototransduction was not altered directly by the adapting light. A test of this assumption is shown for another green cone in Figure 10. The three traces plot responses to an identical test flash recorded in the dark and in the presence of two different adapting backgrounds. A dim 500 nm background reduced the peak and removed the slow rod component from the dark-adapted response. But a cone-matched 660 nm background that was too weak to desensitize rods (Baylor et al., 1984
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Figure 9. Background adaptation used to isolate rod and cone components of the photovoltage of a green cone. A, Test flash responses in the presence (light-adapted) and absence (dark-adapted) of a steady background light expected to saturate rod phototransduction. Traces are averages of 28 dark-adapted or 11 light-adapted responses. Test flash, 500 nm, 274 photons µm
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Figure 10. Dim background light selectively suppressed rod input. Flash responses of a green cone to 500 nm test flashes (274 photons µm
Can rod/cone coupling be modulated?
Light modulates gap junctional coupling of several cell types in nonprimate retina (Vaney, 1994
Neither dopamine nor flupenthixol had any effect on the rod component of the photovoltage (Fig. 11A,B). Dopamine was bath applied to a final concentration of 20 µM, a concentration shown to saturate dopamine receptors in other retinal cells (Piccolino et al., 1984
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Figure 11. Effect of pharmacological agents on rod/cone coupling. A, Responses in a red cone to 500 nm test flashes in control medium (solid line) or in 20 µM dopamine (dashed line) 4-7 min after the onset of dopamine application. Flash strength was 625 photons µm
Although classical chemical synapses between rods and cones have not been observed anatomically in macaque retina, it remains possible that coupling occurs via chemical transmission at an unidentified site. Light responses were recorded before and after superfusion with 0.5 mM CoCl2, a blocker of conventional synaptic transmission. Application of cobalt had no effect on the rod photovoltage itself (data not shown) or on the magnitude of rod signals recorded in cones (Fig. 11C), ruling out a role for conventional chemical transmission from rods to cones. Similar results were obtained in a total of three cones.
Can light itself modulate rod/cone coupling? On the basis of anatomical measurements in cat retina and theoretical considerations about signal and noise characteristics in a coupled network, it has been suggested that rods and cones should couple at background intensities exceeding approximately one photoisomerization sec
Response sensitivity and kinetics as a function of background intensity
Are the changes in cone photovoltage with light adaptation comparable with the light adaptive changes of vision in human observers? Do the rod and cone components of the cone photovoltage light-adapt independently, or do adaptive mechanisms act on the combined signals? To address these questions, we measured cone responses to dim flashes in the presence of steady background lights of varying intensity.
Flash sensitivity (the change in membrane potential evoked by a flash divided by flash photon density) diminished with increasing background intensity (Fig. 12A). Background illumination progressively reduced both the rod and cone components but did not substantially alter the kinetics of the cone component. This can be seen clearly by scaling dark- and light-adapted responses to the same peak amplitude (Fig. 12B). Despite a twofold difference in peak sensitivity, the two scaled responses tracked one another closely for >60 msec. At later times the responses diverged because the light-adapted response lacked the rod component. Similar effects were observed in seven other cones. The invariant kinetics of the cone component of the photovoltage with adaptation was reminiscent of the kinetic invariance of the cone outer segment photocurrent (Schnapf et al., 1990
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Figure 12. Changes in sensitivity and kinetics of photon responses with background illumination in a green cone. A, Flash sensitivity (change in membrane potential photon
The intensity dependence of flash sensitivity is illustrated in Figure 13 (for the same green cone shown in Fig. 12). The points in Figure 13, A and B, plot normalized peak sensitivity. The solid curve in A is a Weber-Fechner function of the form:
where SL is the light-adapted sensitivity, SD is the dark-adapted sensitivity, IB is background light intensity, and I0 is the background that reduced sensitivity to 0.5 SD. The value of I0 that gave the best (least-squares) fit of the data to the curve was 10,600 photons µm
(2)
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Figure 13. Intensity dependence of background desensitization in a green cone. Points plot peak flash sensitivity to a 500 nm test flash (SL), normalized to the dark-adapted sensitivity (SD) as a function of the intensity of a 500 nm background light (IB). Some of the responses used for these data are shown in Figure 12. SL and SD were compared in interleaved trials; no significant reduction in SD was observed between trials. A, B, Points ( = 0.75, IR0 = 70.8 photons µm
Peak sensitivity was better described by a function based on the idea that the cone photovoltage was a weighted sum of rod and cone signals and that the amplitudes of both rod and cone inputs were reduced with background illumination according to Weber-Fechner functions with different I0 values (Fig. 13B). Normalized sensitivity would then be given as a weighted sum of Weber-Fechner functions:
where I0R and I0C are the background intensities that reduce the amplitudes of the rod and cone components to one-half their dark values, and
(3) The solid curve in Figure 13B is the best-fitting weighted function (Eq. 3), and the dashed and dotted curves plot the putative rod and cone contributions, respectively. Equation 3 provided a good description of the peak sensitivity data. The half-desensitizing intensities for 500 nm background light were 71 photons µm
Another way to estimate adaptation of the rod input was to measure the background-induced reduction in amplitude of the cone photovoltage at a late time point in the response. The intensity dependence at 300 msec (Fig. 13C) was well described by a single Weber-Fechner function with I0 = 84 photons µm
Collected results of background desensitization from a total of four cones are plotted in Figure 14. For each cell the points were shifted on the y-axis so that the low intensity cone plateau was set to a normalized sensitivity value of 1. The intensity axis was also normalized by the estimated value of I0C for each cone. The solid curve is a Weber-Fechner function fitted to the cone component alone. Values of I0C ranged from 2320 to 14,800 photoisomerizations sec
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Figure 14. Background desensitization measured in four cones. Peak flash sensitivity as a function of background intensity was plotted on normalized axes. Each symbol type is from a different cone. Data from each cone were normalized on the y-axis so that the low-intensity cone plateau was set to a normalized sensitivity value of 1. I0 is the percent background intensity for each cone that halved the sensitivity of the dark-adapted cone component. The values of I0 (in photons µm ). The smooth curve is from Equation 2.
Further evidence that the reduction in flash sensitivity at the lowest background intensities was caused by rod desensitization came from a comparison of desensitization functions obtained with 500 and 660 nm backgrounds (Fig. 15). The desensitization function for this red cone was nonmonotonic when background strength was plotted in units of photoisomerizations sec
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Figure 15. Spectral sensitivity of background desensitization. Peak flash sensitivity was measured as a function of background strength in a red cone. Flash wavelength was 500 nm. Background wavelength was 500 nm (
Membrane voltage fluctuations in the dark
The ability of a dark-adapted human observer to detect dim flashes of light is limited by the rate of spontaneous isomerization of rhodopsin in rods (Baylor et al., 1984
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Figure 16. Dark noise and single photon responses in a green cone (A-C) and a red cone (D-F). A, D, Representative voltage records in the dark. Bandwidth, DC-140 Hz. B, E, Average voltage responses to 660 nm flashes, divided by the expected number of photoisomerizations per flash. Bandwidth, DC-100 Hz. The number of responses averaged and the photoisomerizations per flash are 12 and 205 in B and 11 and 153 in E. Smooth curves are least-squares fits of the responses to Equation 7 from Schnapf et al. (1990) r, 22 and 23 msec;
, 42 and 38°. Flash duration was 10 msec. Flash monitors are shown above the responses. C, F, Power spectral density of the dark voltage noise (
It was not possible to resolve single photopigment isomerizations in cone voltage noise (Fig. 16A,D) because the expected peak amplitude is only ~5 µV (Schneeweis and Schnapf, 1995
If dark noise arose mainly from spontaneous isomerizations, then the noise spectrum would be proportional to |R(f)|2, the magnitude squared of the Fourier transform of the single photon response r(t). r(t) was estimated from a smoothed approximation to the average response to dim 660 nm flashes (Fig. 16B,E). Figure 16, C and F, illustrates dark noise spectra from a green and red cone, along with the spectra predicted for noise attributable solely to random isomerizations at rates of 13,000 sec
Equivalent dark light
Although the dark noise spectrum implied that isomerization-like events were not the sole source of voltage noise, it is nonetheless the case that noise fluctuations in the dark could limit the detectability of photons in much the same way that random photoisomerizations from a background light reduce the sensitivity of the visual system to light increments. The variance of the dark noise
2 can be expressed as the intensity ID of a steady light that would generate voltage fluctuations of equivalent variance (Barlow, 1958
Because of temporal filtering by the cone synapse and subsequent neural elements (Baylor and Fettiplace, 1977
![I<SUB>D</SUB>=<FR><NU>&sfgr;<SUP>2</SUP></NU><DE><LIM><OP>∫</OP><LL>0</LL><UL>∞</UL></LIM>[r(t)]<SUP>2</SUP><UP>d</UP>t</DE></FR>.](/content/vol19/issue4/fulltext/1203/img004.gif)
(4) The above calculation of ID assumed that the visual system discards high frequencies that provide no information about the occurrence of photoisomerizations. This idea can be extended to suppose that the visual system uses an optimal filtering strategy to extract photon signals from noise. If the cone voltage is passed through a linear filter (assuming that a linear systems approach is appropriate for small signals), then ID is given by:
![I<SUB>D</SUB>=<FR><NU>&sfgr;<SUP>2</SUP><SUB>F</SUB></NU><DE><LIM><OP>∫</OP><LL>0</LL><UL>∞</UL></LIM>[r<SUB>F</SUB>(t)]<SUP>2</SUP><UP>d</UP>t</DE></FR>,](/content/vol19/issue4/fulltext/1203/img005.gif)
(5a) where
(5b) One choice of optimal filter is the Wiener filter. This filter is optimal for minimizing the mean squared error between the filter output and some signal that is embedded in noise (Davenport and Root, 1958
|R(f)|2/ND(f). Substituting this expression into Equation 5b yielded a dark light rate estimate of 12,100 and 4190 sec
DISCUSSION Magnitude of rod input in cones
The rod signal seen in cones was quite robust, although its magnitude varied from cone to cone. We do not know the true magnitude in vivo, but it is likely that the extent of rod coupling with cones was underestimated by our experiments. Some rod outer segments were damaged during retinal isolation and dissection, and we had greater success patching onto cones in retinal areas that were more sparsely populated by rod outer segments.
Rod input to cones could not be modulated using dopamine or a D1/D2 antagonist. This suggests that, unlike in Xenopus in which dopamine acts via D2 receptors to modulate rod/cone coupling (Krizaj et al., 1998
Waveform of the photovoltage
The cone component of the cone photovoltage differed from the outer segment photocurrent in two main ways. For very dim flashes the photovoltage peaked earlier than did the photocurrent, and for bright flashes the photovoltage had a pronounced early transient (or "nose") that was absent in the photocurrent. Could inner segment voltage-dependent conductances account for these differences? Although voltage-dependent conductances in primate cones have not been studied extensively, it is possible to identify at least two that might be involved in shaping the primate cone photovoltage (Yagi and MacLeish, 1994
Changes in kinetics with light adaptation
The temporal sensitivity of human photopic vision is altered by light adaptation. Bright background lights reduce the sensitivity of human observers selectively to stimuli of low temporal frequencies (de Lange, 1958
Light adaptation in cones has also been assessed from ERG recordings in human subjects. Hood and Birch (1993)
The psychophysical parameter, critical duration, defines the period of time over which the visual system can integrate photons from a stimulus. For foveal stimulation the critical duration of the photopic visual system is ~100 msec in the dark and declines to ~25 msec as the background is increased to 1000 td (Watson, 1986
Changes in sensitivity with light adaptation
The sensitivity of the cone component of the photovoltage was halved by backgrounds that elicited ~8700 photoisomerizations sec
In contrast, background intensities of only 1-2 log td are needed to halve psychophysical sensitivity (Hood and Finkelstein, 1986
Dark noise
In most of the cones studied here, the dark noise was dominated by events other than spontaneous photopigment isomerizations. Lamb and Simon (1977)
Rieke and Baylor (1996)
Equivalent dark light
Psychophysical estimates of the cone dark light fall in the range 1.4-140 sec
How could the dark light rate in cones be greater than the rate measured psychophysically? One possible resolution to this apparent paradox is that psychophysical dark light is not directly related to noise as is usually assumed. It could be the case, for instance, that psychophysical sensitivity is more closely linked to an adaptation mechanism that is responsive to small changes in the mean of the cone signal rather than to its variance. This idea should be testable by recording from retinal cells proximal to the cones.
FOOTNOTES Received Aug. 18, 1998; revised Nov. 10, 1998; accepted Nov. 25, 1998.
This work was funded by National Eye Institute Grants R01-EY07642 and F32-EY06399. Additional support was obtained from That Man May See and Research to Prevent Blindness. We thank Drs. David Copenhagen and Jonathan Horton for helpful comments on this manuscript and Dr. Sean McCarthy for useful discussion on optimal filtering.
Correspondence should be addressed to Dr. Julie Schnapf, Departments of Ophthalmology and Physiology, University of California, San Francisco, CA 94143-0730.
Dr. Schneeweis's present address: Smith-Kettlewell Eye Research Institute, 2318 Filmore Street, San Francisco, CA 94115.
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