Axonal differentiation of retinal bipolar cells has largely been studied by comparing the morphology of these interneurons in fixed tissue at different ages. To better understand how bipolar axonal terminals develop in vivo, we imaged fluorescently labeled cells in the zebrafish retina using time-lapse confocal and two photon microscopy. Using the upstream regulatory sequences from the nyx gene that encodes nyctalopin, we constructed a transgenic fish in which a subset of retinal bipolar cells express membrane targeted yellow fluorescent protein (MYFP). Axonal terminals of these YFP-labeled bipolar cells laminated primarily in the inner half of the inner plexiform layer, suggesting that they are likely to be ON-bipolar cells. Transient expression of MYFP in isolated bipolar cells indicates that two or more subsets of bipolar cells, with one or two terminal boutons, are labeled. Live imaging of YFP-expressing bipolar cells in the nyx::MYFP transgenic fish at different ages showed that initially, filopodial-like structures extend and retract from their primary axonal process throughout the inner plexiform layer (IPL). Over time, filopodial exploration becomes concentrated at discrete foci prior to the establishment of large terminal boutons, characteristic of the mature form. This sequence of axonal differentiation suggests that synaptic targeting by bipolar cell axons may involve an early process of trial and error, rather than a process of directed outgrowth and contact. Our observations represent the first in vivo visualization of axonal development of bipolar cells in a vertebrate retina.

Congenital achromatopsia is a stationary retinal disorder with autosomal recessive inheritance that is characterized by loss of color discrimination, low visual acuity, photophobia, and nystagmus. This disorder has been shown to be associated with CNGA3, CNGB3, and GNAT2 mutations, and the frequency of mutations in the CNGA3 gene (encoding α subunit of the cone-specific cGMP-gated cation channel) was 23–33% in European populations. The aim of this study was to test the hypothesis that CNGA3 mutations are also responsible for congenital achromatopsia in Japanese patients. DNA from venous blood samples from a total of 14 patients from 13 Japanese pedigrees was prepared. Mutation screening of the CNGA3 gene was performed using direct sequencing and PCR-single-strand conformation polymorphism analysis. Compound heterozygous missense mutations (p.R436W and p.L633P, the latter of which was novel) were identified in one patient only, a 22-year-old female. Neither of these two mutations was found in 150 Japanese control individuals. The patient's parents and sister carried one of these mutations each but were not affected. No mutations in the CNGB3 or GNAT2 genes were identified in the patient. Clinically, best-corrected visual acuity was 0.1 in both eyes. No specific findings were obtained in funduscopy. Optical coherence topography revealed a normal foveal thickness but a 20% decrease in parafoveal thickness. Ganzfeld full-field electroretinograms (ERGs) showed normal responses in rod and mixed rod-plus-cone ERGs but no response in cone or 30-Hz flicker ERGs. Spectral sensitivity on a white background revealed a curve with only one peak at around 500 nm, which fits the absorption spectrum of human rhodopsin. L633, conserved among vertebrate orthologs of human CNGA3, is a hydrophobic residue forming part of the carboxy-terminal leucine zipper (CLZ) domain, which is functionally important in the mediation of intracellular interactions. To our knowledge, this is the first report of a Japanese complete achromat with CNGA3 mutations, and of any patient with a missense mutation within the CLZ domain. The outcome suggests low frequency (7%, 1/14) of CNGA3 mutations in Japanese patients.

Inherited tritan color vision deficiency is caused by defects in the function of the short-wavelength-sensitive (S) cones. This heterozygous group of disorders has an autosomal dominant pattern of inheritance. Amino acid variations of the S cone opsin are rare and all that have been identified thus far are associated with inherited tritan color vision defects. Here we report the identification of a 30-year-old male who made errors on standard color vision tests consistent with the presence of a mild tritan color vision deficiency. We tested the hypothesis that his color vision impairment was due to a mutation in the S cone photopigment gene. He was found to be heterozygous for a mutation that caused the amino acid proline to be substituted in place of a highly conserved leucine at amino acid position 56 in the S cone opsin. This mutation was absent in 564 S cone photopigment genes from 282 subjects who did not make tritan errors. Thus, we conclude that this mutation disrupts the normal function of S cones.

In normal trichromats, the long- (L) and middle-wavelength-sensitive (M) pigment genes are arranged in a head-to-tandem array on the X chromosome. Two amino acids at positions 277 and 285, encoded by exon 5 of the L and M genes, respectively, are essential for the spectral difference between L and M pigments whose spectral peaks are at approximately 560 and 530 nm. Intragenic or intergenic unequal crossing-over commonly occurs between the highly homologous L and M genes, resulting in red-green color vision deficiencies. The dichromacy is usually associated with a single L gene for deuteranopia or a single 5′ L-M 3′ hybrid gene with M-gene exon 5 for protanopia. We clinically diagnosed a total of 88 male dichromats using a Nagel model I anomaloscope, which included one unclassified subject in addition to 31 protanopes and 56 deuteranopes. The objective of this study was to characterize the phenotype of the subject and to determine the genotype of his X-linked pigment genes. The subject accepted not only any red-green mixture but also an extended yellow-scale range at each matching point (i.e. 20 to 32 scale units at the green primary and 3.5 to 6 scale units at the red primary). The slopes of regression lines were in the range of −0.34 to −0.23, while the mean slopes for the protanopes and deuteranopes were −0.38 and −0.01, respectively. Spectral sensitivity tests showed that the subject's curve was shifted between the protanope and deuteranope curves. Molecular analysis revealed a novel form of a single pigment gene with a unique arrangement of exon 5 (Y277 from the L gene and A285 from the M gene). The predicted λmax (541 to 546 nm) of the unique pigment was closer to the M than to the L pigment. Our outcome suggests that intragenic unequal crossing-over may have occurred between amino acid positions 279 and 283.

Shift in the wavelength of peak sensitivity of the cone photo pigments is a major cause of inter-individual variations in the Rayleigh match. Normal color observers performed multiple Rayleigh matches (i.e., a series of seven Rayleigh-type color matches using various sets of test and primary lights) in order to derive estimates of the M- and L-photo pigment wavelengths of maximum absorbance. We predicted the ratio of incident radiances Pr/Pg for a range of plausible values in terms of wavelength of the peak sensitivity of the M-cone and L-cone. An algorithm allowed us to adjust the values of the peak wavelength of the cone photo pigments in order to minimize the sum of squared differences between predicted and real results of matches. To create candidate M-cone and L-cone photo pigments that best predict the set of equation values, we used the low density spectral absorbance curves of the M-cone and L-cone photo pigments respectively, as tabulated by Stockman and Sharpe (2000), and shifted each template along a logarithmic wavelength scale. For all observers, the system of seven Rayleigh-like equations converges to a unique solution. M- and L-cone photo pigments are estimated separately. The distribution of the wavelength of maximum sensitivity of the photo pigments includes a cluster. Only a few women's results lie outside the cluster. The choice of the template has a considerable influence on the convergence of the algorithm.

In mammals, cone photoreceptor subtypes are thought to establish topographies that reflect the species-relevant properties of the visual environment. Middle- to long-wavelength-sensitive (M) cones are the dominant population and in most species they form an area centralis at the visual axis. Short-wavelength-sensitive (S) cone topographies do not always match this pattern. We here correlate the interrelationship of S and M cone topographies in representatives of several mammalian orders with different visual ecology, including man, cheetah, cat, Eurasian lynx, African lion, wild hog, roe deer, and red deer. Retinas were labeled with opsin antisera and S and M cone distributions as well as S/M cone ratios were mapped. We find that species inhabiting open environments show M cone horizontal streaks (cheetah, pig, deer). Species living in structured habitats (tiger, lynx, red deer) have increased S cone densities along the retinal margin. In species with active vision (cheetah, bear, tiger, man), S cone distributions are more likely to follow the centripetal M cone gradients. Small species show a ventral bias of peak S cone density which either matches the peak of M cone density in a temporal area centralis (diurnal sciurid rodents, tree shrews) or not (cat, manul, roe deer).

Thus, in addition to habitat structure, physical size and specific lifestyle patterns (e.g. food acquisition) appear to underlie the independent variations of M and S cone topographies.

The objective of the present work was to determine the interaction of cone inputs in the response of horizontal cells using heterochromatic flicker photometry (HFP). Intracellular electrophysiological recordings were made in horizontal cells of isolated retinae of carp maintained in physiological solution, with the receptor side up. Sharp glass microelectrodes filled with 3 M KCl solution with resistances between 100 and 120 MΩ were used. Stimuli comprised six cycles of two 6-Hz sinusoidal light waves in counterphase adjusted for the same number of quanta: a green light (550 nm) from a monochromator with a Xenon lamp and an LED red light (628 nm). The stimulation program consisted of 10 steps with the 550-nm wave at constant amplitude, while the 628-nm wave varied in increments of 10% up to 100%, followed by another 10 steps with the 628-nm wave at constant amplitude while the 550-nm wave varied in increments of 10% up to 100%. We recorded responses from four different horizontal cell classes: H1 (monophasic, broadband, n = 37), H2 (biphasic, red-green color-opponent, n = 13), and H3 (biphasic, blue-yellow color-opponent, n = 2) cone horizontal cells; and RH (monophasic, broadband, n = 3) rod horizontal cells. H1 and RH horizontal cells showed a similar cancellation point at a heterochromatic mixture consistent with mixed inputs from 630- and 550-nm cones. No cancellation point was found for the H2 cell class. Fish H1 cells add cone inputs and signal “luminance” in light levels appropriate for cone stimulation. The same occurs with RH cells, which also signal “luminance,” but in light levels appropriate for rod work. For both cell classes there is an HFP cancellation point occurring at a combination of 628-nm and 550-nm lights in opposing phase that leads to the cancellation of the cell's response. No cancellation was found for H2 and H3 cells, which are the chromatically opponent horizontal cells in lower vertebrates.

We recently developed a new technique to measure cone inputs to visual neurons and used this technique to seek short-wavelength-sensitive (S) cone inputs to parasol, magnocellular (MC) and midget, parvocellular (PC) ganglion cells. Here, we compare our physiological measurements of S-cone weights to those predicted by a random wiring model that assumes cells' receptive fields receive input from mixed cone types. The random wiring model predicts the average weights of S-cone input to be similar to the total percentage of S-cones but with considerable scatter, and the S-cone input polarity to be consistent with that of PC cells' surround and of MC cells' center. This is not consistent with our physiological measurements. We suggest that the ganglion cells' receptive fields may have a mechanism to avoid S-cone inputs, as is the case in the H1 horizontal cells. Previous reports of S-cone inputs, in particular substantial input to MC cells, are likely to reflect variation in prereceptoral filtering and/or the failure to correct for variation in macular pigment.

The aim is to study chromatic visual evoked potentials (VEP) to isoluminant red-green (R-G) stimulus in schoolchildren. Sixty children (7–19 years) with normal color vision were examined, 30 binocularly and 30 monocularly. The isoluminant point was determined for each child subjectively by using heterochromatic flicker photometry, and objectively from recordings. The stimulus was a 7° circle composed of horizontal sinusoidal gratings, with spatial frequency 2 cycles/degrees and 90% contrast, presented in onset-offset mode. VEP were recorded from Oz (mid-occipital) position. Age-dependent waveform changes and changes of the positive and negative wave were studied to both binocular and monocular R-G stimulation. Age-dependent waveform changes were observed to binocular and monocular R-G stimulation. In younger children the positive wave was prominent, whereas in older children also the negative wave became more evident. The latency of the positive wave decreased linearly with age to R-G binocular stimulation. To monocular stimulation no significant changes of the latency were observed. The amplitude of the positive wave dropped exponentially with age to binocular and monocular stimulation. The latency of the negative wave increased linearly with age to binocular and monocular stimulation, whereas the amplitude did not show age-dependent changes. These findings suggest that the chromatic VEP response undergoes evident age-dependent changes during the school-age period.

In the reverse Stroop task, observers are instructed to ignore the ink color in which a color word is printed (the distractor color) and to respond to the meaning of the color word (the target). Reaction times (RTs) are faster with congruent combinations when the ink color matches the word than with incongruent combinations when the ink color does not match the word. We manipulated the distracting ink color from congruent to incongruent and measured the transition from facilitation to interference. In Experiment 1, we confirmed that this transition could be assessed independently from the contextual influence of particular sets of stimuli and responses, implying that the color space in which interference and facilitation occurs is generalizable. In Experiment 2, we obtained reverse Stroop data for transitions between red and yellow, yellow and green, green and blue, and blue and red, and compared them with independent estimates of color appearance obtained by hue scaling for the same chromaticity samples. We find that the magnitude of the reverse Stroop effect can provide a reliable index of the similarity of color appearance between the distracting chromaticity and the color category represented by the target color word. Moreover, it will allow us to quantify the mapping between the chromaticity space defined at the cone photoreceptors and a cognitive color space defined at an advanced level of neural processing.

We evaluated binocular summation, eye dominance, and learning in the Trivector and Ellipses procedures of the Cambridge Colour Test (CCT). Subjects (n = 36, 18–30 years old) were recruited among students and staff from the University of São Paulo. Inclusion criteria were absence of ophthalmological complaints and best-corrected Snellen VA 20/20 or better. The subjects were tested in three randomly selected eye conditions: binocular, monocular dominant eye, and nondominant eye. Results obtained in the binocular and monocular conditions did not differ statistically for thresholds measured along the protan, deutan, and tritan confusion axes (ANOVA, P > 0.05). No statistical difference was detected among discrimination ellipses obtained in binocular or monocular conditions (ANOVA, P > 0.05), suggesting absence of binocular summation or of an effect of eye dominance. Possible effects of learning were examined by comparing successive thresholds obtained in the three testing conditions. There was no evidence of improvement as a function of testing order (ANCOVA, P > 0.05). We conclude that CCT thresholds are not affected by binocularity, eye dominance, or learning. Our results differ from those found by Verriest et al. (1982) using the Farnsworth-Munsell 100 Hue test and Hovis et al. (2004) using the Farnsworth-Munsell panel D-15 test.

Paradoxical shifts in human color (spectral) sensitivity occur on deep-red (658 nm) background fields. As the radiance of the deep-red background is increased from low to moderate levels, the spectral sensitivity for detecting 15-Hz flicker shifts toward shorter wavelengths, although by more than is predicted by selective chromatic adaptation (e.g., Eisner & MacLeod, 1981; Stromeyer et al., 1987; Stockman et al., 1993). Remarkably, though, at higher background radiances, the spectral sensitivity then shifts precipitously back towards longer wavelengths. Here, we show that both effects are due in large part to destructive and constructive interference between signals generated by the same cone type. Contrary to the conventional model of the human visual system, the M- and L-cone types contribute not just the customary fast signals to the achromatic or luminance pathway, but also slower signals of the same or opposite sign. The predominant signs of the slow M- and L-cone signals change with background radiance, but always remain spectrally opposed (M-L or L-M). Consequently, when the slow and fast signals from one cone type destructively interfere, as they do near 15 Hz, those from the other cone type constructively interfere, causing the paradoxical shifts in spectral sensitivity. The shift in spectral sensitivity towards longer wavelengths is accentuated at higher temporal frequencies by a suppression of fast M-cone signals by deep-red fields.

Temporally varying chromatic-inducing light was used to infer receptive-field organization. Time-varying shifts in color appearance within a test field were induced by a surrounding chromatic pattern; the shifts were then nulled by adding a time-varying stimulus to the test area so the observer perceived a steady test. This method measured chromatic induction without requiring an observer to judge the color appearance of the test. The induced color shifts were consistent with a +s/−s spatially antagonistic neural receptive field, which also accounts for color shifts induced by static chromatic patterns (Monnier & Shevell, 2003, Monnier & Shevell, 2004). The response of this type of receptive-field, which is found only in the visual cortex, increases with S-cone stimulation at its center and decreases with S-cone stimulation within its surround. The measurements also showed a negligible influence of temporal inducing frequency in the range 0.5–4 Hz.

The color appearance of a physically steady central region can appear to vary over time if a surrounding chromatic light varies in time. The induced temporal variation, however, is strongly attenuated at surround temporal frequencies above ∼3 Hz. At these higher temporal frequencies, the central region appears steady (De Valois et al., 1986). The posited explanation is a cortical low-pass temporal filter. Here, we investigate whether higher temporal-frequency surrounds induce color shifts in the steady appearance of the central test. Surrounds modulated in time along the l or s chromatic direction of MacLeod-Boynton color space were symmetric around equal-energy white (EEW). The temporal frequency of the surround was varied. If observers perceived the central test to be temporally modulating between two points in time, they set two separate matches to the extreme points of this modulation. If the central test appeared steady in time, then color matches were made to this steady appearance. Corroborating previous reports, measurements showed that surround temporal frequencies below ∼3 Hz induced temporal modulation. At higher temporal frequencies, however, the surround induced steady color shifts, compared to a steady surround at its time average (EEW). The measurements imply that a nonlinear neural process affects chromatic induction from time-varying context.

Inhibitory effects have been reported when a target is preceded by a cue of the same color and location. Color-based inhibition was found using red and blue nonisoluminant stimuli (Law et al., 1995). Here we investigate whether this phenomenon depends on the chromatic subsystem involved by employing isoluminant colors varying along either the violet-yellow or purple-turquoise cardinal axis. Experiment 1 replicated Law et al.'s study: After fixating magenta, either a red or blue cue was presented, followed by a magenta “neutral attractor,” and, finally, by a red or blue target. In Experiment 2, violet and yellow, cue or target, varied along a tritan confusion line in the CIE 1976 chromaticity diagram. In Experiment 3, purple and turquoise, cue or target, varied along a deutan confusion line in the CIE 1976 chromaticity diagram. Normal trichromats (n = 19) participated in all three experiments. In Experiment 1, color repetition indeed resulted in longer reaction times (RTs) (4.7 ms, P = 0.038). In Experiment 2, however, no significant color repetition effect was found; RTs to violet and yellow were not significantly different, though tending toward slower responses (2 ms) for violet repetition but faster (5 ms) for yellow. Experiment 3 also showed no color repetition effect (P = 0.58); notably, RTs were overall faster for purple than for turquoise (22 ms, P < 0.0001). Furthermore, responses tended to be slower for purple repetition (4 ms, P > 0.05), but faster for turquoise (7 ms, P > 0.05). These findings demonstrate that color repetition is not always inhibitory but may turn facilitatory depending on the colors employed. The results indicate that disengagement of attention is an unlikely mechanism to be the sole explanation of previously reported color-based inhibition of return. We suggest a complementary, perceptual explanation: response (dis)advantage depends on whether the stimuli are isoluminant and on the opponent chromatic subsystem involved. The choice of the colors employed and the cue-attractor-target constellation also may be of significance.

Discrimination for equiluminant chromatic stimuli that vary in L- and M-cone excitation depends on the chromaticity difference between the test field and the surrounding area. The current study investigated the effect of the proximity in space and time of a surround to the test field on chromatic contrast discrimination. The experimental paradigm isolated spatial, temporal, and spatial-and-temporal chromatic contrast effects on discrimination. Chromatic contrast discrimination thresholds were assessed by a four-alternative spatial forced-choice procedure. Stimuli were either metameric to the equal energy spectrum, or varied in L-cone activation along a line of constant S-cone activation. A model based on primate parvocellular pathway physiology described the data. Spatial and temporal contrast produced equivalent reductions in chromatic discriminability as the chromatic difference between the test and surround increased. For all test chromaticities, discrimination was best in the absence of chromatic contrast. Chromatic contrast discrimination is determined by either the spatial or temporal contrast component of the signal.

Discrimination for equiluminant chromatic stimuli that vary in L- and M-cone excitation depends on the chromaticity difference between the test field and the surrounding area. The current study investigated the effect of the proximity in space and time of a surround to the test field on chromatic contrast discrimination. The experimental paradigm isolated spatial, temporal, and spatial-and-temporal chromatic contrast effects on discrimination. Chromatic contrast discrimination thresholds were assessed by a four-alternative spatial forced-choice procedure. Stimuli were either metameric to the equal energy spectrum, or varied in L-cone activation along a line of constant S-cone activation. A model based on primate parvocellular pathway physiology described the data. Spatial and temporal contrast produced equivalent reductions in chromatic discriminability as the chromatic difference between the test and surround increased. For all test chromaticities, discrimination was best in the absence of chromatic contrast. Chromatic contrast discrimination is determined by either the spatial or temporal contrast component of the signal.

Changes of color perception in the peripheral field are measured using an asymmetric simultaneous matching paradigm. The data confirm previous observations in that saturation changes can be neutralized if the test target is increased in size. However, this compensation does not apply to hue shifts. We show that some hues remain unchanged with eccentricity whereas others exhibit substantial changes. Here the color shifts are plotted in terms of a second-stage cone opponent model. The data suggest that the S-L+M channel is more robust to increasing eccentricity than the L-M channel. Observations are interpreted in terms of the known underlying morphological and physiological differences in these channels.

In central vision, the discrimination of colors lying on a tritan line is improved if a small gap is introduced between the two stimulus fields. Boynton et al. (1977) called this a “positive gap effect.” They found that the effect was weak or absent for discriminations based on the ratio of the signals of long-wave and middle-wave cones; and even for tritan stimuli, the gap effect was weakened when forced choice or brief durations were used. We here describe measurements of the gap effect in the parafovea. The stimuli were 1 deg of visual angle in width and were centered on an imaginary circle of radius 5 deg. They were brief (100 ms), and thresholds were measured with a spatial two-alternative forced choice. Under these conditions we find a clear gap effect, which is of similar magnitude for both the cardinal chromatic axes. It may be a chromatic analog of the crowding effect observed for parafoveal perception of form.

To understand the generality and mechanisms of previously reported rod hue biases, we examined whether they are present for small foveal stimuli by comparing the wavelengths of the three spectral unique hues under dark-adapted and flash-bleached conditions. Rod green bias (shift of unique yellow) and rod blue bias (shift of unique green) were found for some observers with 1°-diameter foveal stimuli, the size most likely to stimulate rods. Smaller stimuli (0.2° and 0.6° diameter), which were least likely to stimulate rods, produced no large or consistent differences between dark-adapted and bleached conditions. This suggests that rod hue biases result from the local stimulation of rods by light, not from remote suppression by dark-adapted, unstimulated rods, and not from bleaching light artifacts.