MOUSE CONE PIGMENTS

long-wavelength sensitive) yields cone pigments with peaks distributed over the range from about 493 to 565 nm. Mouse cone pigments are drawn from these two respective families. As is the mammalian norm, the mouse M-cone opsin gene is X chromosome linked, while the UV opsin gene is derived from an autosome, chromosome 7. The SWS1 family includes genes that specify both UV and short-wavelength-sensitive (S) cones. Comparative examinations of opsin gene sequences suggested that the ancestral pigments in the SWS1 group were actually UV pigments, and that in the course of the evolution of different lineages, some of these were converted to become S pigments (and occasionally reconverted to UV pigments) [21]. Mammals that have retained the ancestral UV pigments are mostly rodents, but not all rodents follow this pattern; for example, the retinas of most sciurids contain S pigments. So far, there does not appear to be any consistent correlation between visual lifestyle and short-wavelength sensitivity; for example, there are other nocturnal rodents like the mouse with UV pigments, while still other rodents also classified as nocturnal have S pigments.

Mammalian cone opsins derived from LWS genes typically consist of string of about 365 amino acids folded into seven helical arrays to form a transmembrane protein that is covalently linked to the photopigment chromophore 11-cis retinal. Sequence comparisons of a collection of mammalian cone opsin genes carried out in conjunction with measurements of photopigment absorption spectra suggested that dimorphic substitutions of amino acids at a total of only five sites in the protein can account for all of the shifts in spectral positioning of LWS pigments [22]. According to this account, the ancestral mammalian LWS pigment is inferred to have had a λmax value of about 531 nm. From that starting point, various combinations of changes at the five sites can be used to account for the spectral positions of the entire array of mammalian LWS pigments. In the case of the mouse, shifting the ancestral LWS-derived pigment from a peak at 531 nm to the spectral position of the M pigment of the mouse (510 nm) requires at minimum only two amino acid substitutions from the ancestral arrangement. The evolutionary timing and events surrounding these proposed changes remain unknown.

The spectral tuning of pigments derived from the SWS1 gene family is more complicated. As noted, this partly reflects the fact that SWS1 genes specify photopigments falling into two spectral classes having different ranges of peak spectral sensitivity, UV and S, and that in the course of evolution there have been numerous interconversions between these two groups. As many as 8 amino acid sites (of a total of about 350) have been implicated in the spectral tuning of pigments in this group [23]. Three of these are believed to be most critical, and at those three sites, the amino acids of the mouse UV opsin are identical to the arrangement believed to represent the ancestral condition in pigments specified by the SWS1 gene family [21].

Regional Distribution of Mouse Cone Pigments

Although there are well-documented exceptions (e.g., the absence of S cones from the center of the primate fovea), generally cones containing different photopigment types are rather uniformly intermixed across mammalian retinas. This seems a logical arrangement as it allows local circuits positioned downstream of the photoreceptors to have access to signals originating in the different types of cone, thus allowing various excitatory and inhibitory combinations of the two. To satisfy much the same goals, only a single type

D

T N

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Fig. 2. The distribution of cone pigments in the retina of Mus musculus as derived by the application of opsin-specific antibodies [24]. Depicted is a flat-mount map of the mouse retina. The distribution of cone types is given by the fill as follows: vertical lines exclusively populated by cones containing UV (ultraviolet) pigment; horizontal lines predominantly M cones with a small intermixed population of cones containing UV pigment; vertical and horizontal lines with shading cones coexpressing M and UV pigment. As described in the text, there are competing claims on the distribution of cone pigments in the mouse retina. Orientation of the retina: D dorsal; V ventral; T temporal; N nasal.

of cone pigment is usually expressed in any given cone. Mouse cones violate both of these principles. In 1992, Szel and coworkers [24] used opsin antibody labeling to reveal a striking separation of the two cone types across the retina such that cones containing UV pigment are largely relegated to the ventral retina, while cones containing M cone pigment predominate in the dorsal retina. In most mammalian retinas, cones containing pigments derived from LWS opsin genes greatly outnumber those derived from SWS1 genes (often by ratios of ~10:1), but these labeling experiments revealed the mouse has more UV than M cones. As a third surprise, Szel and coworkers documented the presence of a central transitional zone between these two regions where individual cones routinely expressed both UV and M pigments. This unique pattern of cone distribution in the mouse retina is illustrated in Fig. 2.

Several functional correlates of these unusual cone distributions in the mouse have been reported. First, in accord with the claim that UV pigment is more abundant than M pigment in the mouse retina, electroretinograms (ERGs) recorded in response to large-field test lights show much higher sensitivity to UV test lights than to similar lights drawn from the middle wavelengths [15, 17]. Second, in support of the idea that cones containing UV and M pigment have different retinal distributions, when UV and middle-wavelength stimuli are imaged onto various subregions of the mouse retina, the ERG shows relative higher sensitivity to UV lights directed to the ventral retina then when the dorsal retina

is similarly stimulated [25]. Finally, exposure of the mouse eye to an intense light flash having a wavelength content that is exclusively absorbed by M cones greatly suppresses sensitivity to subsequently presented UV test lights [17]. That result is consistent with what would be expected if some mouse cones coexpress both UV and M pigments.

It has been discovered that regional disparities in distribution of the different types of cone pigment of the sort first documented in the mouse also occur in other mammalian retinas. For instance, a range of different mouse species have retinal subfields that are dominated by cones containing the short-wavelength-sensitive (presumably, UV) pigment, as do a number of other species, such as rabbit, guinea pig, and vole [9]. Coexpression of cone pigments has now also been seen in a range of other mammalian retinas [9]. The patterning of the regions dominated by one cone type varies considerably across species, as does the extent of coexpression. Despite these species variations, it now seems clear that the mouse is not alone in supporting what were originally thought to be very unusual cone distribution arrangements.

The extent of photopigment coexpression in mouse cones remains a somewhat unsettled issue. As noted (Fig. 2), the original observation [24] was that mouse cones coexpressing UV and M pigments were restricted to a narrow strip effectively separating dorsal and ventral retinal subfields, each of which had cones containing a single type of pigment. A subsequent study reached a quite different conclusion. In that case, Northern blotting was used to assess messenger RNA levels for the UV and M opsins in conjunction with opsin antibody labeling to examine the distribution of the cone types across the mouse retina [26]. The essential finding was that, although a small number of cones in the far dorsal retina express only M photopigment, virtually all cones throughout the mouse retina coexpress the two cone types. Further, this study provided evidence that there are gradients of pigment expression across the retina, with M pigment progressively decreasing from the dorsal to the ventral retina, while UV pigment expression shows the inverse pattern. In addition, in accord with earlier ERG results (as described in this section), the retina was judged to contain overall about three times as much UV pigment as it does M pigment [26].

Two more recent studies have bearing on the issue of cone pigment coexpression. One of these used M and UV cone opsin antibodies and searched for double labeling [27]. Counter to the results of the previous study [26], this experiment found that most cones in the dorsal retina express only M pigment, with a small minority (3–5%) expressing only UV pigment. In the ventral retina, most cones express both pigment types, although again a minority of cones (8–20%) contains only UV pigment. Finally, in a recent technical tour de force it proved possible to use suction pipettes to record from the outer segments of individual mouse cones [28]. Although the number of cones examined was somewhat restricted, most of these receptors had sensitivity spectra consistent with the joint presence of UV and M pigments. In addition, results obtained from a majority of the cones (21/29) indicated they contained relatively greater amounts of UV pigment.

Given these contradictory conclusions, consensus about the distribution of cone pigments in the mouse retina is not yet at hand. What presently seems clear is that (1) overall, the mouse retina expresses greater amounts of UV than M pigment, (2) a substantial proportion of mouse cones coexpress the two pigments with reciprocal variations in the relative amounts that roughly follow a dorsal/ventral gradient, and (3) opsin-labeling