Electroretinogram analysis of relative spectral sensitivity in genetically identified dichromatic macaques

Abstract

The retinas of macaque monkeys usually contain three types of photopigment, providing them with trichromatic color vision homologous to that of humans. However, we recently used molecular genetic analysis to identify several macaques with a dichromatic genotype. The affected X chromosome of these animals contains a hybrid gene of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) photopigments instead of separate genes encoding L and M photopigments. The product of the hybrid gene exhibits a spectral sensitivity close to that of M photopigment; consequently, male monkeys carrying the hybrid gene are genetic protanopes, effectively lacking L photopigment. In the present study, we assessed retinal expression of L photopigment in monkeys carrying the hybrid gene. The relative sensitivities to middle-wavelength (green) and long-wavelength (red) light were measured by electroretinogram flicker photometry. We found the sensitivity to red light to be extremely low in protanopic male monkeys compared with monkeys with the normal genotype. In female heterozygotes, sensitivity to red light was intermediate between the genetic protanopes and normal monkeys. Decreased sensitivity to long wavelengths was thus consistent with genetic loss of L photopigment. Trichromatic color vision in Old World primates originates from three types of retinal cone photoreceptors possessing differing spectral sensitivities. This difference arises from the selective expression of three genes respectively encoding long-wavelength-sensitive (L), middle-wavelength-sensitive (M), and short-wavelength-sensitive photopigment. In humans, the genes encoding L and M photopigment are located in a head-to-tail tandem array on the X chromosome, and loss of one or the other— caused by unequal chromosome recombination—results in dichromatic color vision (1). Dichromatism in humans has been studied for the purposes of making clinical diagnoses and achieving a better understanding of the mechanisms responsible for trichromatic color vision; the roles of lost and retained photopigments become more apparent in dichromats. Macaque monkeys have trichromatic color vision homologous to that of humans (2) and have served as subjects in a variety of physiological and psychophysical studies of color vision. Therefore, the dichromatic macaque may be a useful animal model of dichromatism with which to conduct clinical studies and investigate the mechanisms underlying color vision. In many species of New World monkeys, both dichromatic and trichromatic animals are mixed within a species because of the alleles whose products exhibit different spectral sensitivities in a single cone photopigment locus on the X chromosome (3–8). On the other hand, dichromatic macaques were not recognized until recently (9, 10). However, our molecular genetic analysis showed the existence of a dichromatic genotype of the crab-eating macaque (11). By using PCR to specify genotype, we found male protanopes and female heterozygotes spread among some troops in Pangandaran National Park, Indonesia. The genome of male protanopes contains a single hybrid gene—consisting of the 5′ part of the L photopigment and the 3′ part of the M photopigment (R4G5, exons 1–4 come from the former, exons 5 and 6 from the latter; see ref. 11)—instead of separate L and M photopigment genes. The absorbance spectrum of R4G5 photopigment, which was characterized by photobleaching analysis, is very close to that of M photopigment. Consequently, males carrying only the R4G5 gene on their X chromosome should exhibit the retinal sensitivity of a protanopic dichromat, effectively lacking L photopigment. In the present study, we examined the retinal chromatic sensitivity of monkeys carrying the R4G5 hybrid gene to confirm the correspondence between the protanopic genotype and the phenotype. We used electroretinogram (ERG) flicker photometry to measure the relative sensitivities of these animals to long- and middle-wavelength light. ERG flicker photometry has been used to measure the spectral sensitivity of macaque monkeys (10) and also has proved to be quite useful to show correlation between the genotype and phenotype in humans (12–15) and New World monkeys (4, 16). We chose this noninvasive approach from among a number of alternatives to minimize the likelihood of injury to the animals, as these carrier monkeys are rare and invaluable. Measurements were made in three genetic groups (normal males and females, heterozygous females, and protanopic males), and our findings show that sensitivity to long wavelengths corresponds to predicted levels of L photopigment.