Cone Photoresponses Research Articles

Diagnosis and classification of macular degenerations: an approach based on retinal function testing.

The results from literature concerning some aspects of retinal function in macular degenerations (MDs) were reviewed in order to evaluate whether (a) specific patterns of retinal dysfunction may be linked to different clinical phenotypes, and (b) distinct functional profiles may help in orienting molecular diagnosis of diseases. Examined clinical phenotypes included: Stargardt disease/fundus flavimaculatus (St/FF), age-related maculopathy (ARM) and macular degeneration (AMD), pattern dystrophies (PD), Best vitelliform dystrophy (BVD), Sorsby's fundus dystrophy (SFD), autosomal cone-rod dystrophies (CRD). The following functional tests were evaluated: (1) electroretinogram (ERG) (scotopic and photopic according to ISCEV standards, rod and cone photoresponses, rod and cone b-wave intensity-response function, focal ERGs); (2) dark adaptometry (pre-bleach sensitivity and post-bleach recovery kinetics); (3) fundus reflectometry (pigment density and regeneration kinetics). Specific patterns of retinal dysfunction were identified for St/FF, ARM/AMD, SFD and BVD, whereas partially overlapping profiles were found for PD and CRD. Specific functional patterns were associated with different peripherin/RDS gene mutations, as well as with CRX mutations. Combined analysis of different retinal function tests may help to identify different phenotypes of MD, and to orient molecular diagnosis for selected genotypes.

Time course and Ca(2+) dependence of sensitivity modulation in cyclic GMP-gated currents of intact cone photoreceptors.

We determined the Ca(2+) dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca(2+) levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K(1/2), the ligand concentration that half-saturates current, that, in turn, depend on Ca(2+). K(1/2) increases as Ca(2+) rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca(2+) with a single site without apparent cooperativity. (Ca)K(m), the Michaelis-Menten constant for Ca(2+) concentration is 857 +/- 68 nM for cGMP and 863 +/- 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca(2+) caused by sudden Ca(2+) sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 micro, Ca(2+) concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 +/- 0.95-fold with a mean time to peak of 1.25 +/- 0.23 s. We developed a model that assumes channel modulation is a pseudo-first-order process kinetically limited by free Ca(2+). Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s. Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.

UV-sensitive input to horizontal cells in the turtle retina.

Microspectrophotometry, electroretinography and behavioural studies have indicated that ultraviolet (UV) light contributes to functional vision in various vertebrate species. Based on behavioural evidence, this was also suggested for turtle vision. In order to reveal the interactions underlying detection of UV light in the distal retina, we recorded intracellularly the photoresponses of cones and horizontal cells in retinas of Pseudemys scripta elegans and Mauremys caspica and calculated the action spectra of these cells under different conditions of adaptation. In the dark-adapted retina, all three types of horizontal cells; luminosity-type, red/green chromaticity-type and yellow/blue chromaticity-type exhibited increased sensitivity in the UV region of the spectrum. However, chromatic adaptation indicated that only the yellow/blue chromaticity-type horizontal cells received excitatory input from UV-sensitive cones with peak sensitivity approximately 360 nm. The enhanced UV sensitivity of luminosity-type horizontal cells probably reflected the beta-band of the long-wavelength sensitive visual pigment as indicated by the action spectra of dark-adapted L-cones. It is suggested that the enhanced UV sensitivity of red/green chromaticity-type horizontal cells reflects the beta-band of the medium-wavelength sensitive visual pigment. Transmission measurements of the optical media (cornea, lens and vitreous) indicated that UV vision can be functional under normal circumstances.

Neural interactions between cone photoreceptors and horizontal cells in the turtle (Mauremys caspica) retina.

Horizontal cells and cone photoreceptors in the vertebrate retina are interconnected by a complex network of synapses leading to the generation of color-coded responses in chromaticity horizontal cells. A simple cascade model of excitatory feedforward and inhibitory feedback synapses had been suggested to underlie these observations. In this study, the photoresponses of cones and horizontal cells were recorded intracellularly from the turtle eyecup. Three different approaches were adopted in order to test quantitatively the cascade model. Comparing linearity functions between these neurons indicated multiple excitatory inputs to each type of horizontal cells. The depolarizing photoresponses of R/G C-type horizontal cells were considerably faster than those of L-type horizontal cells but slower than those recorded from L-cones. This observation disagrees with the basic assumption of the cascade model that assign the depolarizing photoresponses of R/G C-type horizontal cells to a negative feedback pathway from L-type horizontal cells onto M-cones. Finally, the action spectra of each of the three types of horizontal cells could not be solely accounted for by input from one spectral type of cones. Only by assuming excitatory and inhibitory inputs from all spectral types of cones, the action spectra of all types of horizontal cells could be reconstructed. These findings suggest that the negative feedback pathways from horizontal cells onto cones in the turtle retina cannot solely account for the chromatic properties of the horizontal cells and support a direct inhibitory inputs from cones to turtle horizontal cells.

Light adaptation of cone photoresponses studied at the photoreceptor and ganglion cell levels in the frog retina

The sensitivity and time scale of the dominant (562 nm) cone system of the frog, Rana temporaria, were studied as functions of steady adapting illuminance (IB). Photoreceptor responses to brief flashes of light were recorded as aspartate-isolated ERG mass potentials from the isolated retina. The characteristics of the cone signal after transmission through the retina were derived from response thresholds and stimulus--intensity-response--latency functions for extracellularly recorded spike discharges of single ganglion cells in the eyecup. At 14 degrees C, the single-photon response of dark-adapted cones, extrapolated from ERG intensity-response functions, had an amplitude of 0.5% of the saturated response (Umax) and peaked at tp approximately 0.4 sec. Steady background illumination decreased both tp and flash sensitivity (SF), starting from apparent "dark lights" of, respectively, less than 10 (for time scale) and about 100 (for sensitivity) photoisomerisations per cone per second [P*sec-1]. From there upwards, two distinct ranges of background adaptation were apparent. Under moderate backgrounds (up to IB approximately 10(4) - 10(5) P*sec-1), sensitivity fell according to the relation SF alpha IB-0.64 and time scale shortened according to tp alpha IB-0.16. Under brighter backgrounds, from approx. 10(5) P*sec-1 up to the limit of our light source at 10(7) P*sec-1, the decrease in SF was significantly stronger than predicted by the Weber relation (SF alpha IB-1), while the decrease in tp levelled out and even tended to reverse. All these changes were virtually identical at the photoreceptor and ganglion cell levels, although the absolute time scale of cone signals apparent at the latter level was 2-fold longer. Our general conclusion is that photoreceptors have several distinct regimes for light adaptation, and traditional descriptions of functional changes (in sensitivity and kinetics) relevant to vision need to be restated with higher resolution, in view also of recent insights into the diversity of underlying mechanisms.

The action spectra of cone photoreceptors in the turtle (Mauremys caspica) retina.

Cone photoreceptors in the turtle retina are involved in intricate neuronal interactions with other retinal neurons that modify the responses of the cones to photons absorbed in their outer segments. Therefore, the action spectra of cones strongly depend upon the conditions of measurements. This study describes an attempt to derive the action spectra of turtle cones which are the least distorted by neuronal interactions. To achieve this goal, the photoresponses of cones and horizontal cells were recorded from the turtle retina under different conditions of adaptation using different patterns of the stimulating test flashes. The sensitivity action spectra, derived from small-amplitude (<1 mV) photoresponses, were strongly affected by the recording conditions indicating the contributions of multiple neuronal inputs. Action spectra, constructed from large criterion photoresponses, were less distorted by neuronal interactions and better described the spectral properties of the "isolated" cones. The action spectra of the hyperpolarizing inputs to chromaticity-type horizontal cells were derived by stimulating these cells with mixtures of a saturating red light and a monochromatic light of different wavelength and intensity. The action spectra were constructed from the intensity of the addend component needed to "pull down" the depolarizing response to the red component by a fixed criterion. These spectra, measured in red/green and yellow/blue C-type horizontal cells, are suggested to best represent the "isolated" M-cones and S-cones, respectively.

PH regulation in frog cones studied by mass receptor photoresponses from the isolated retina

Mass cone photoresponses were recorded across the aspartate-treated frog retina under treatments chosen to affect putative pH-regulating mechanisms. The saturated response amplitude (Umax) was found to be a monotonically increasing function of perfusion pH in the range 7-8, and thus presumably of intracellular pH (pHi). Accepting that Umax can be used as an index of pHi changes, two results indicate the importance of bicarbonate transport for preventing intracellular acidification: (1) bicarbonate-buffered (6 mM HCO3- + 6 mM HEPES) perfusate increased Umax compared with nominally bicarbonate-free perfusate (12 mM HEPES); (2) the anion transport blocker DIDS (0.1 mM) caused a strong decrease in the amplitude of photoresponses. Substitution of 95 mM chloride by gluconate in the perfusing fluid boosted photoresponses indicating that at least part of the bicarbonate transport involves HCO3-/Cl- exchange. Amiloride (2 mM) also caused a decrease of photoresponse amplitude, which suggests that Na+/H+ exchange contributes to pHi regulation. In all these respects, cones behaved similarly to rods. Cones differed from rods (in the intact retina) in that addition of 0.5 mM of the carbonic anhydrase inhibitor acetazolamide reduced (never augmented) photoresponses. The difference is considered in relation to the presence of carbonic anhydrase in cone, as opposed to rod, outer segments.

Visual latency and brightness: An interpretation based on the responses of rods and ganglion cells in the frog retina

Rod and cone photoresponses in a variety of species have been accurately described with linear multistage filter models. In this study, the response latency and initial coding of intensity at two higher levels of visual processing are related to such photoreceptor responses. One level is the retinal output (spiking discharges from frog ganglion cells, based on experimental data reported here), the other is the perceptual level in humans (psychophysical latency and brightness functions, based on data from the literature). Photoreceptor responses are described with the "independent activation" model of Baylor et al. (1974). The intensity dependence of the early ganglion cell discharge, its latency and initial impulse frequency, is shown to follow from such a waveform, assuming that 1) latency L = l + D, where l is the time it takes for the rod response linearly summed over the ganglion cell's receptive field to reach a criterion amplitude, and D is a constant delay; and 2) the initial frequency (below saturation) is proportional to the steepness of rise of the summed rod response at time l. It is shown that the intensity dependences of 1) human visual latency and 2) brightness sensation, including effects of stimulus area and duration, are accounted for by the same model. The predicted functions are not power functions of intensity, but approximate such over wide ranges. Thus, a large body of psychophysical data is explained simply by the waveform of photoreceptor responses.

Mixing of color signals by turtle cone photoreceptors.

The direct mixing of red and green cone signals in the outer plexiform layer of the turtle retina was studied by using intracellular recordings from red cone photoreceptors. Cone photoresponses were a function of the wavelength of the photons that stimulated them, even when small-diameter stimuli were used. The intensity response curves measured with red and green test flashes had different shapes. The kinetics of approximately equal amplitude red and green responses also differed. To quantify the short wavelength input onto red cones, differential chromatic adaptation was used. The relative sensitivity of the red cone to red and green test flashes was a function of the color and intensity of the background illumination; red backgrounds decreased relative red sensitivity, and green backgrounds increased relative red sensitivity. The spectral sensitivity of the additional short wavelength input onto red cones was determined by using differential chromatic adaptation, and was found to peak approximately 550 nm. We conclude that red cones receive an additional excitatory input from green cones (and possibly blue cones). A model of the cone mosaic suggests that approximately 50% of the red cone response (linear range) to a dim green test flash arises from neighboring green cones.

Direct excitatory interactions between cones of different spectral types in the turtle retina.

Cone linear sensitivities to red and green stimuli were measured intracellularly in the dark- and light-adapted turtle retina. Test flashes of small diameter were used to minimize horizontal cell feedback. Light adaptation was achieved with either green or red background illumination. The ratio of cone sensitivities to red and green light depended on the color of the background light and differed from the ratio measured in the dark. Electron microscope studies of Golgi-stained turtle cones revealed direct synaptic connections between red and green cones mediated by cone telodendria. These data indicate that the red cone photoresponse is not univariant as has been previously supposed and suggest that mixing of signals from different spectral classes of cones can occur via direct excitatory connections between cones.

The effects of background illumination on the photoresponses of red and green cones.

1. The photoresponses of light- and dark-adapted red and green cone photoreceptors were recorded intracellularly in the retina of the turtle, Pseduemys scripta elegans. Background illumination produced similar effects on both types of cones. 2. In response to the onset of a prolonged, steady background illumination the cone initially hyperpolarized to a peak which then sagged back to a steady-state polarization that was typically about one half the initial peak amplitude. This sag was observed for all backgrounds studied (dim as well as bright). 3. A resensitization was observed concomitantly with this sag; both the maximum increment and decrement responses grew in amplitude as light-adaptation proceeded. After about 2--3 min of background illumination, the amplitudes of these responses stabilized. 4. The dark-adapted cone produced graded responses to test pulses over a range of intensities spanning about 3.5 log units. The amplitudes of these responses were well fit by the relationship V = I.Vm/(I + sigma). 5. After 2--3 min of background illumination, 500 msec test pulses either brighter or dimmer than the background intensity were substituted for the background. The light-adapted intensity-response curves constructed from this data were similar to the dark-adapted curve but were shifted horizontally and slightly vertically, so that they still spanned about 3.5 log units of intensity. Thus, in the light-adapted cone, graded responses were elicited by a range of bright test pulses which would have produced saturated responses when delivered to the dark-adapted cone. 6. The 'off response' observed at the offset of the background became faster as the background intensity was increased. It also became faster with time following the onset of any particular background intensity. 7. It was concluded that cone sensitivity during any state of light-adaptation is determined by two mechanisms; response compression resulting from the instantaneous non-linearity between 'internal transmitter' concentration and membrane potential and a more active 'cellular adaptation' mechanism which is manifest as a shift in the intensity-response curve. In the steady-state condition of light-adaptation, most of the sensitivity changes are a result of the cellular adaptation mechanism. 8. Photopigment bleaching caused by the backgrounds, negative feed-back from horizontal cells and voltage dependent mechanisms in the cones could not account for this cellular adaptation. These effects of background illumination were interpreted in terms of the 'internal transmitter' hypothesis of phototransduction.

Signal transmission from red cones to horizontal cells in the turtle retina.

1. Intracellular recordings were made from L-type horizontal cells in the retina of the turtle Pseudemys scripta elegans. The responses were evoked by 500 msec pulses of 'white' light. 2. L-type horizontal cells were classified as either, 'small receptive field' s.r.f. or 'large receptive field' l.r.f. based upon (1) receptive field size and (2) kinetics of responses to test flashes covering small and big spots. 3. Constant illumination of the entire receptive field, with any intensity studied, evoked a response that reached a peak and then slowly sagged back to a steady-state level that was about half the peak response. 4. Termination of backgrounds resulted in a very fast recovery of the membrane potential that overshot the dark-adapted potential. This 'off' response had faster kinetics in horizontal cells than in red cones. 5. The intensity-response curve measured around any background intensity was shifted along the log intensity axis toward higher test intensities. The curves obtained under light-adapted conditions were sharper than the curve measured in the dark-adapted state. 6. The photoresponses of red cones and s.r.f. horizontal cells were compared under similar states of adaptation. In the dark-adapted state of the gain, expressed as the millivolt change in the horizontal cell per millivolt change in the cone, was not linearly related to cone potential, and was highest for dim stimuli. Light-adaption modified the synaptic transmission to make the horizontal cells most sensitive to light modulation around the background illumination. 7. The mechanisms by which signal transmission can be modified by light-adaptation are discussed in terms of transmitter release by the presynaptic terminals and its binding to post-synaptic sites.

Changes in pigeon cone photocurrent caused by reduction in extracellular calcium activity.

1. Photocurrents have been recorded from the red spot of the isolated superfused pigeon retina. The technique used was to record photovoltage gradients and extracellular fluid resistivity in a direction parallel with the long axes of the receptors. 2. Cone and rod responses were identified, and experiments designed so that only the former were elicited. 3. In the outer portion of the receptor layer, the wave form of the cone photoresponse lacks the initial transient (the 'nose') seen in the portions of the receptor layer nearer the synapses. It is argued that this observation permits the use of a simple equivalent circuit for the generation of the extracellular photocurrent, to infer membrane properties from extracellular recordings. 4. When the superfusing Ringer is changed to one which has a very low calcium activity (2 X 10(-7)M) the first result is that photoresponses increase in magnitude (X 7.7) but the relationship between light intensity and response amplitude and the light intensity (sigma) required to produce a half maximal response remains unchanged. 5. This increase in photocurrent in low calcium also occurs if the superfusing fluid is cooled to 10 degrees. 6. After 2--3 min, the photoresponses in low calcium begin to decrease in amplitude, and the value of sigma is progressively reduced, tenfold in 10 min. 7. During this time, the wave form of the photocurrent alters, the rate of increase and decrease of the responses being slowed. 8. The relationship between peak photocurrent and duration of light flash is modified. 9. The response to a step of light is not well maintained in higher calcium, but is well maintained in low calcium. 10. In higher calcium, the current overshoots during recovery from a flash to below the previous dark level. This does not happen in low calcium. 11. In low calcium, a light adapting background illumination desensitizes the cones. All changes in wave form of the response can be accounted for in terms of the membrane non-linearities. The calculated time course of the change in concentration of the 'internal transmitter' is unaffected. The same is true of desensitization, in the dark, following exposure to intense illumination.

Light-induced resistance changes in retinal rods and cones of the tiger salamander.

1. The electrical properties of retinal rods and cones of the larval tiger salamander were investigated with intracellular electrodes, and the cells identified by means of dye injections.2. Both types of photoreceptors are hyperpolarized by illumination. Following stimulation with brief flashes of dim light, rod responses show a slower time course than cone responses; with bright flashes, rod responses can be recognized because of their long recovery time.3. Values of input resistance were derived from the voltage displacement induced by constant current pulses in darkness or at the peak of the photoresponse. The input resistance following illumination was also calculated from the effect of steady polarizing currents on the amplitude of the photoresponse.4. In darkness, the input resistance of the rod cells is time- and voltage-dependent, but the voltage-current relations of most cells have a linear region which includes the physiological limits of membrane potential. At the peak of the photoresponse, the input resistance (slope of the linear region of the v-i relations) is decreased.5. Cone cells show approximately linear v-i relations. As reported by previous authors, illumination increases the input resistance.6. These results support the current view that the cone photoresponse is the consequence of a reduction in the permeability of channels which in darkness shunt the membrane. In rods, however, it appears that the main effect of illumination is to increase the permeability of the membrane to ions for which the equilibrium potential is more negative than the membrane potential in darkness.

The effect of ions on the photoresponses of pigeon cones.

1. Pigeon cone receptor potentials have been identified in a preparation of isolated, incubated retina.2. There is an approximately linear relationship between response amplitude and sodium concentration of the bathing medium.3. A decrease of chloride ions produces results equivalent to a decrease of sodium ions. The response amplitude is a power function of the product of the sodium and chloride concentrations. For most experiments, the exponent is ca. 1.4. After treatment of the retina with ouabain, responses vanish, but can be temporarily restored, in normal, or inverted polarity, by appropriate changes in sodium or chloride concentration.5. Ammonium and potassium ions can substitute quantitatively for sodium ions. Lithium ions initially carry response currents, but later block all responses. Bromide, nitrate and thiocyanate can substitute for chloride ions.6. The response wave form, and the amplitude/light intensity relationship are independent of ions.7. Models of the photoreceptor are discussed. It is concluded that the membrane potential of the cone in light is chiefly determined by the distribution of anions across the membrane, and in darkness there is an increase in outer limb cation permeability.