Julie L. Schnapf

The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fascicularis.

Visual transduction in rods of the cynomolgus monkey, Macaca fascicularis, was studied by recording membrane current from single outer segments projecting from small pieces of retina. Light flashes evoked transient outward‐going photocurrents with saturating amplitudes of up to 34 pA. A flash causing twenty to fifty photoisomerizations gave a response of half the saturating amplitude. The response‐stimulus relation was of the form 1‐e‐x where x is flash strength. The response to a dim flash usually had a time to peak of 150‐250 ms and resembled the impulse response of a series of six low‐pass filters. From the average spectral sensitivity of ten rods the rhodopsin was estimated to have a peak absorption near 491 nm. The spectral sensitivity of the rods was in good agreement with the average human scotopic visibility curve determined by Crawford (1949), when the human curve was corrected for lens absorption and self‐screening of rhodopsin. Fluctuations in the photocurrent evoked by dim lights were consistent with a quantal event about 0.7 pA in peak amplitude. A steady light causing about 100 photoisomerizations s‐1 reduced the flash sensitivity to half the dark‐adapted value. At higher background levels the rod rapidly saturated. These results support the idea that dim background light desensitizes human scotopic vision by a mechanism central to the rod outer segments while scotopic saturation may occur within the outer segments. Recovery of the photocurrent after bright flashes was marked by quantized step‐like events. The events had the properties expected if bleached rhodopsin in the disks occasionally caused an abrupt blockage of the dark current over about one‐twentieth of the length of the outer segment. It is suggested that superposition of these events after bleaching may contribute to the threshold elevation measured psychophysically. The current in darkness showed random fluctuations which disappeared in bright light. The continuous component of the noise had a variance of about 0.03 pA2 and a power spectrum that fell to half near 3 Hz. A second component, consisting of discrete events resembling single‐photon responses, was estimated to occur at a rate of 0.006 s‐1. It is suggested that the continuous component of the noise may be removed from scotopic vision by a thresholding operation near the rod output.

The Photovoltage of Macaque Cone Photoreceptors: Adaptation, Noise, and Kinetics

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−1 per cone. Coupling between rods and cones was not modulated by either dim backgrounds or dopamine. Voltage noise in dark-adapted cones was dominated by elementary events other than photopigment isomerizations. The dark noise was equivalent in magnitude to a steady light eliciting ∼3800 photoisomerizations sec−1 per cone, a value significantly higher than the psychophysical estimates of cone “dark light.”

Visual transduction in human rod photoreceptors

1. Photocurrents were recorded with suction electrodes from rod photoreceptors of seven humans. 2. Brief flashes of light evoked transient outward currents of up to 20 pA. With increasing light intensity the peak response amplitude increased along an exponential saturation function. A half‐saturating peak response was evoked by approximately sixty‐five photoisomerizations. 3. Responses to brief dim flashes rose to a peak in about 200 ms. The waveform was roughly like the impulse response of a series of four to five low‐pass filters. 4. The rising phases of the responses to flashes of increasing strength were found to fit with a biochemical model of phototransduction with an ‘effective delay time’ and ‘characteristic time’ of about 2 and 800 ms, respectively. 5. Spectral sensitivities were obtained over a wavelength range from 380 to 760 nm. The action spectrum, which peaked at 495 nm, followed the template described for photoreceptors in the macaque retina. Variation between rods in the position of the spectrum on the wavelength axis was small. 6. The scotopic luminosity function derived from human psychophysical experiments was found to agree well with the measured rod action spectrum after adjustments were made for lens absorption and photopigment self‐screening in the intact eye. 7. Responses to steps of light rose monotonically to a maintained level, showing little or no relaxation. Nevertheless, the relationship between light intensity and steady‐state response amplitude was shallower than that expected from simple response saturation. This is consistent with an adaptation mechanism acting on a rapid time scale. 8. Flash sensitivity fell with increasing intensities of background light according to Webers law. Sensitivity was reduced twofold by lights evoking about 120 photoisomerizations per second. Background lights decreased the time to peak and the integration time of the flash response by up to 20%.

Gap-Junctional Coupling and Absolute Sensitivity of Photoreceptors in Macaque Retina

We investigated gap-junctional coupling of rods and cones in macaque retina. Cone voltage responses evoked by light absorption in neighboring rods were briefer and smaller than responses recorded in the rods themselves. Rod detection thresholds, calculated from noise and response amplitude histograms, closely matched the threshold for an ideal detector limited by quantal fluctuations in the stimulus. Surprisingly, cone thresholds were only approximately two times higher. Amplitude fluctuations in cones could be explained by a Poisson distribution of photoisomerizations within a pool of seven or more coupled rods. Neurobiotin coupling between rods and cones was consistent with our electrical recordings, with approximately six rods labeled per injected cone. The spatial distribution of tracer-coupled rods matched the light-evoked cone receptive field. The gap junction inhibitor carbenoxolone abolished both electrical and tracer coupling. Amplitude fluctuations in most rods were accounted for by the expected rate of light absorption in their outer segments. The fluctuations in some rods, however, were consistent with a summation pool of up to six rods. When single rods were injected with Neurobiotin, up to 10 rods were labeled. Rod-rod and rod-cone electrical coupling is expected to extend the range of scotopic vision by circumventing saturation at the rod to rod-bipolar cell synapse; however, because coupling also renders the rod synapse less effective at separating out photon signals from dark noise, coupling is expected to elevate the absolute threshold of dark-adapted observers.

Spectral sensitivity of primate photoreceptors

The spectral sensitivities of rods and cones in macaque and human retinas were determined by recording the membrane current from single outer segments. In the macaque retina, the wavelengths of maximum sensitivity were at about 430, 530, and 561 nm for the blue, green, and red cones, respectively, and at 491 nm for the rods. The shapes of the spectra of the three cones were similar when plotted on a log wavenumber scale; the rod spectrum was slightly broader. Spectral sensitivities of the red and green cones from a human retina were virtually identical to those of macaque cones. For comparison with human psychophysical measurements, the rod and cone spectra were adjusted to give the sensitivities expected for light incident on the cornea of the human eye. These functions satisfactorily predicted the scotopic and photopic luminosity functions as well as results from human color-matching experiments. The adjusted spectra of the red and green cones also agreed well with the pi-mechanism of Stiles (1953, 1959).

Blue-Yellow Opponency in Primate S Cone Photoreceptors

The neural coding of human color vision begins in the retina. The outputs of long (L)-, middle (M)-, and short (S)-wavelength-sensitive cone photoreceptors combine antagonistically to produce “red-green” and “blue-yellow” spectrally opponent signals (Hering, 1878; Hurvich and Jameson, 1957). Spectral opponency is well established in primate retinal ganglion cells (Reid and Shapley, 1992; Dacey and Lee, 1994; Dacey et al., 1996), but the retinal circuitry creating the opponency remains uncertain. Here we find, from whole-cell recordings of photoreceptors in macaque monkey, that “blue-yellow” opponency is already present in the center-surround receptive fields of S cones. The inward current evoked by blue light derives from phototransduction within the outer segment of the S cone. The outward current evoked by yellow light is caused by feedback from horizontal cells that are driven by surrounding L and M cones. Stimulation of the surround modulates calcium conductance in the center S cone.

Noise and light adaptation in rods of the macaque monkey.

Membrane voltage was recorded in rod photoreceptors in retina isolated from macaque monkey. The size of the single photon response and the magnitude of membrane voltage fluctuations were assessed in dark- and light-adapted retina. The dark light rate I(D), defined as the rate of spontaneous photopigment isomerizations that would produce a variance equivalent to that of the noise measured in the dark, was calculated after matched filtering. The average value of 0.08 s(-1) fell at the higher end of psychophysical estimates of dark light in human observers. In light-adapted rods the photon response decreased in amplitude and duration, and the magnitude of the voltage fluctuations increased with increasing background light intensity. The signal-to-noise ratio (SNR) for single rods was defined as the ratio of the peak amplitude of the photon response to the standard deviation of the noise fluctuations. The signal-to-noise ratio for dark-adapted rods SNR(D) was about 7. With increasing background intensity I, the SNR fell as SNR(D)(1 + I/I(D))(-1/2). This function may account for the increment thresholds measured with small brief test flashes in human psychophysical experiments.

Gap-Junctional Coupling of Mammalian Rod Photoreceptors and Its Effect on Visual Detection

The presence of gap junctions between rods in mammalian retina suggests a role for rod-rod coupling in human vision. Rod coupling is known to reduce response variability, but because junctional conductances are not known, the downstream effects on visual performance are uncertain. Here we assessed rod coupling in guinea pig retina by measuring: (1) the variability in responses to dim flashes, (2) Neurobiotin tracer coupling, and (3) junctional conductances. Results were consolidated into an electrical network model and a model of human psychophysical detection. Guinea pig rods form tracer pools of 1 to ∼20 rods, with junctional conductances averaging ∼350 pS. We calculate that coupling will reduce human dark-adapted sensitivity ∼10% by impairing the noise filtering of the synapse between rods and rod bipolar cells. However, coupling also mitigates synaptic saturation and is thus calculated to improve sensitivity when stimuli are spatially restricted or are superimposed over background illumination.

Aberrant photon responses in rods of the macaque monkey

Recovery from bright light was studied in macaque rods by measuring the membrane current of single outer segments. The recovery phase of some responses displayed a plateau current of about one picoampere lasting for several seconds. The following evidence suggests these steps are single photon responses of abnormally long duration. (1) Over a limited range of intensities, step amplitude remained constant and summed linearly with intensity. The collecting area for step generation was about 2.6 x 10(-3) microns2. (2) Step duration varied exponentially with a mean duration of about 6.5 s. (3) Fluctuation analysis of the tail currents was consistent with the idea that a step is evoked by isomerization of a single rhodopsin molecule, and that only 1 in 400 isomerizations leads to a responses with a step-like waveform. (4) With only the distal portion of the outer segment in the electrode, the polarity of the step response reversed when the proximal portion of the outer segment was illuminated, indicating that step generation results from a local change in outer segment conductance near the site of photon absorption. (5) The probability of eliciting a step varied with the wavelength of light in the manner expected from the absorption spectrum of rhodopsin.