M-cone opsin gene number does not correlate with variation in L/M-cone sensitivity.

Molecular genetic studies demonstrate that the human cone opsin gene array on the q-arm of the X-chromosome typically consists of one long-wave-sensitive (L) cone opsin gene and from one to several middle-wave-sensitive (M) cone opsin genes. Although the presence of the single L-cone opsin gene and at least one M-cone opsin gene is essential for normal red-green colour discrimination, the function of the additional M-cone opsin genes is still unclear. To investigate whether any variations in phenotype correlate with differences in the number of M-cone opsin genes, we selected 13 normal trichromat males, for whom four independent molecular techniques have exactly determined their number of M-cone opsin genes, ranging from one to four. Their phenotype was characterized by estimating their foveal L- to M-cone ratio from heterochromatic flicker photometric (HFP) thresholds, by measuring the wavelength corresponding to their 'unique yellow', and by determining their L- and M-cone modulation thresholds (CMTs). No correlation was found between these psychophysical measures and the number of M-cone opsin genes. Although, we found a reasonably good correlation between the L/M-cone ratios based on HFP and on CMT, we did not find any correlation between the estimated L/M-cone ratios and the settings of 'unique yellow'. Our results accord with previous molecular genetic studies that suggest that only the first two genes in the X-linked opsin gene array are expressed.

Thresholds for detecting slowly changing Ganzfeld luminances.

Detection thresholds for luminance increments or decrements are normally related to rapid light changes. The goal of this study was to determine detection thresholds for slowly changing achromatic Ganzfeld luminances before and after adaptation to a constant Ganzfeld illumination, subsequently called Ganzfeld adaptation. During Ganzfeld adaptation, perceived brightness decreased slowly and leveled off(on average after 5-7 min), despite constant illumination of the retina. The state of adaptation was characterized by using magnitude estimation. Comparing detection thresholds for changing light intensities before and after Ganzfeld adaptation showed that the sensitivity for luminance changes is independent of the perceived brightness. A further issue addressed was the time dependence of the luminance change. Is there a limit below which a change of luminance is no longer perceivable? Even for the slowest gradient tested (0.01 log/min), subjects were able to detect the change of luminance, although they were not able to perceive a continuous brightness change. Similar thresholds (ca. 0.24 log unit) for shallow and steep luminance gradients suggest an absolute luminance detection mechanism. Possible underlying mechanisms and neurophysiological substrates are discussed.

L/M cone ratios in human trichromats assessed by psychophysics, electroretinography, and retinal densitometry.

Estimates of the relative numbers of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) cones vary considerably among normal trichromats and depend significantly on the nature of the experimental method employed. Here we estimate L/M cone ratios in a population of normal observers, using three psychophysical tasks-detection thresholds for cone-isolating stimuli at different temporal frequencies, heterochromatic flicker photometry, and cone contrast ratios at minimal flicker perception--as well as flicker electroretinography and retinal densitometry. The psychophysical tasks involving high temporal frequencies, specifically designed to tap into the luminance channel, provide average L/M cone ratios that significantly differ from unity with large interindividual variation. In contrast, the psychophysical tasks involving low temporal frequencies, chosen to tap into the red-green chromatic channel, provide L/M cone ratios that are always close to unity. L/M cone ratios determined from electroretinographic recordings or from retinal densitometry correlate with those determined from the high-temporal-frequency tasks. These findings suggest that the sensitivity of the luminance channel is directly related to the relative densities of the L and the M cones and that the red-green chromatic channel introduces a gain adjustment to compensate for differences in L and M cone signal strength.

Direct visual resolution of gene copy number in the human photopigment gene array.

PURPOSE: To visualize by direct fluorescent in situ hybridization the entire human visual pigment gene array on single X-chromosome fibers and to compare the results with values obtained by other molecular techniques. METHODS: The size of the opsin gene array on the X-chromosome in eight male subjects was investigated by (i) direct visual in situ hybridization (DIRVISH) on elongated DNA fibers: (ii) quantitation of genomic restriction fragments after Southern blot hybridization; (iii) quantitation of restriction fragment length polymorphism after PCR amplification (PCR/RFLP), and (iv) sizing of NotI fragments by pulsed field gel electrophoresis and Southern blot detection. Each male subject's color vision was assessed by Rayleigh matches on a Nagel Type 1 anomaloscope. RESULTS: The number of genes resolved by the DIRVISH protocol, which ranges from 1 to 6, agrees exactly with the gene array sizes obtained in the same male subjects from pulsed field gel electrophoresis, but differs from the estimates derived from the commonly used indirect Southern blot hybridization and PCR/RFLP quantitation methods. In particular, the PCR/RFLP method overestimates the copy number in all but the smallest arrays. CONCLUSIONS: Visualization of the X-chromosome opsin gene array by DIRVISH provides a new, direct method for obtaining exact copy numbers and helps to resolve the controversy about the range and the average visual pigment gene number in the human population in favor of smaller average array sizes.

L, M and L-M hybrid cone photopigments in man: deriving lambda max from flicker photometric spectral sensitivities.

Using heterochromatic flicker photometry, we have measured the corneal spectral sensitivities of the X-chromosome-linked photopigments in 40 dichromats, 37 of whom have a single opsin gene in their tandem array. The photopigments encoded by their genes include: the alanine variant of the normal middle-wavelength sensitive photopigment, M(A180); the alanine and serine variants of the normal long-wavelength sensitive photopigment, L(A180) and L(S180); four different L-M hybrid or anomalous photopigments, L2M3(A180), L3M4(S180), L4M5(A180) and L4M5(S180); and two variants of the L-cone photopigment, encoded by genes with embedded M-cone exon two sequences, L(M2; A180) and L(M2; S180). The peak absorbances (lambda max) of the underlying photopigment spectra associated with each genotype were estimated by correcting the corneal spectral sensitivities back to the retinal level, after removing the effects of the macular and lens pigments and fitting a template of fixed shape to the dilute photopigment spectrum. Details of the genotype-phenotype correlations are summarized elsewhere (Sharpe, L. T., Stockman, A., Jägle, H., Knau, H., Klausen, G., Reitner, A. et al. (1998). J. Neuroscience, 18, 10053-10069). Here, we present the individual corneal spectral sensitivities for the first time as well as details and a comparison of three analyses used to estimate the lambda max values, including one in which the lens and macular pigment densities of each observer were individually measured.

Red, green, and red-green hybrid pigments in the human retina: correlations between deduced protein sequences and psychophysically measured spectral sensitivities.

To analyze the human red, green, and red-green hybrid cone pigments in vivo, we studied 41 male dichromats, each of whose X chromosome carries only a single visual pigment gene (single-gene dichromats). This simplified arrangement avoids the difficulties of complex opsin gene arrays and overlapping cone spectral sensitivities present in trichromats and of multiple genes encoding identical or nearly identical cone pigments in many dichromats. It thus allows for a straightforward correlation between each observer's spectral sensitivity measured at the cornea and the amino acid sequence of his visual pigment. For each of the 41 single-gene dichromats we determined the amino acid sequences of the X-linked cone pigment as deduced from its gene sequence. To correlate these sequences with spectral sensitivities in vivo, we determined the Rayleigh matches to different red/green ratios for 29 single-gene dichromats and measured psychophysically the spectral sensitivity of the remaining green (middle wavelength) or red (long wavelength) cones in 37 single-gene dichromats. Cone spectral sensitivity maxima obtained from subjects with identical visual pigment amino acid sequences show up to a approximately 3 nm variation from subject to subject, presumably because of a combination of inexact (or no) corrections for variation in preretinal absorption, variation in photopigment optical density, optical effects within the photoreceptor, and measurement error. This variation implies that spectral sensitivities must be averaged over multiple subjects with the same genotype to obtain representative values for a given pigment. The principal results of this study are that (1) approximately 54% of the single-gene protanopes (and approximately 19% of all protanopes) possess any one of several 5'red-3'green hybrid genes that encode anomalous pigments and that would be predicted to produce protanomaly if present in anomalous trichromats; (2) the alanine/serine polymorphism at position 180 in the red pigment gene produces a spectral shift of approximately 2.7 nm; (3) for each exon the set of amino acids normally associated with the red pigment produces spectral shifts to longer wavelengths, and the set of amino acids normally associated with the green pigment produces spectral shifts to shorter wavelengths; and (4) changes in exons 2, 3, 4, and 5 from green to red are associated with average spectral shifts to long wavelengths of approximately 1 nm (range, -0.5 to 2.5 nm), approximately 3.3 nm (range, -0.5 to 7 nm), approximately 2.8 nm (range, -0.5 to 6 nm), and approximately 24.9 nm (range, 22.2-27.6 nm).

Numbers and ratios of X-chromosomal-linked opsin genes.

Quantitative Southern blotting and PCR/RFLP analysis were used to determine the number and ratio of long-wave-sensitive (L-) and mid-wave-sensitive (M-) opsin genes in 25 colour-normal caucasian males. The average observed ratio was 1:2.8 +/- 1.2 for Southern blot analysis and 1:3.0 +/- 1.7 for PCR/RFLP analysis. Thus, the two techniques yielded similar results for the ratio of L- to M-opsin genes (Wilcoxon t-test, P < 0.01). PCR/RFLP analysis of a Sma I polymorphism specific for the most proximal opsin gene suggested an average gene number of 6.0 +/- 2.1, with a range from 4 to 12 in individual subjects. In contrast, Southern blot analysis suggested an average number of 3.8 +/- 1.2, with a range from 2 to 7 (on the assumption that only one L-opsin gene is ever present). Differences between the L- to M-opsin gene ratio and the total gene number in some subjects may result from the presence of multiple L-opsin genes and/or hybrid opsin genes in colour-normal males. An exact determination of the total gene number will require employing other molecular techniques.

Macular pigment densities derived from central and peripheral spectral sensitivity differences.

Estimates of the density spectrum of the macular pigment (Wyszecki G, Stiles WS. Color Science: Concepts and Methods. Quantitative Data and Formulas. 1st ed. New York: Wiley, 1967); (Vos JJ. Literature review of human macular absorption in the visible and its consequences for the cone receptor primaries. Institute for Perception. Soesterberg, The Netherlands, 1972) are partially based on the difference between central and peripheral spectral sensitivities, measured under conditions chosen to isolate a single cone class (Stiles WS. Madrid: Union Internationale de Physique Pure et Appliquée, 1953;1:65-103). Such derivations assume that the isolated spectral sensitivity is the same at both retinal locations, save for the intervening macular pigment. If this is true, then the type of cone class mediating detection should not influence the calculated difference spectrum. To test this assumption, we measured central and peripheral spectral sensitivities in a deuteranope, a protanope and a normal trichromat observer: (a) for short-wave sensitive (S-) cone detection; and (b) for long-wave sensitive (L-) cone detection (deuteranope), for middle-wave sensitive (M-) cone detection (protanope) or for both L- and M-cone detection (normal trichromat). The difference spectra determined for L- or M-cone detection deviate significantly from those measured for S-cone detection, at wavelengths below 450 nm. A theoretical analysis suggests that the discrepancies are owing, in part, to regional variation in the optical density of the cone pigments; and that such receptor variation cannot be ignored when deriving the standard density spectrum of the macular pigment.

Brightness fading during Ganzfeld adaptation.

The time course and extent of brightness fading in a Ganzfeld were determined for adapting luminances ranging from 0.01 to 100 cd/m2. Magnitude estimation and interocular brightness matching were used. During Ganzfeld adaptation, perceived brightness decreased slowly and leveled off, on average, after 5-7 min (adapting time increasing with luminance). On average, the total brightness loss was equivalent to a 1.2 log unit reduction in luminance, independent of adapting luminance. The residual brightness perceived at the final plateau was generally higher than the brightness of the Eigengrau, suggesting a partially sustained luminance channel.