Visual opsin expression and morphological characterization of retinal photoreceptors in the pouched lamprey (Geotria australis, Gray).

Lampreys are extant members of the agnathan (jawless) vertebrates that diverged ~500 million years ago, during a critical stage of vertebrate evolution when image-forming eyes first emerged. Among lamprey species assessed thus far, the retina of the southern hemisphere pouched lamprey, Geotria australis, is unique, in that it possesses morphologically distinct photoreceptors and expresses five visual photopigments. This study focused on determining the number of different photoreceptors present in the retina of G. australis and whether each cell type expresses a single opsin class. Five photoreceptor subtypes were identified based on ultrastructure and differential expression of one of each of the five different visual opsin classes (lws, sws1, sws2, rh1, and rh2) known to be expressed in the retina. This suggests, therefore, that the retina of G. australis possesses five spectrally and morphologically distinct photoreceptors, with the potential for complex color vision. Each photoreceptor subtype was shown to have a specific spatial distribution in the retina, which is potentially associated with changes in spectral radiance across different lines of sight. These results suggest that there have been strong selection pressures for G. australis to maintain broad spectral sensitivity for the brightly lit surface waters that this species inhabits during its marine phase. These findings provide important insights into the functional anatomy of the early vertebrate retina and the selection pressures that may have led to the evolution of complex color vision.

A role for melanopsin in alpha retinal ganglion cells and contrast detection.

Distinct subclasses of retinal ganglion cells (RGCs) mediate vision and nonimage-forming functions such as circadian photoentrainment. This distinction stems from studies that ablated melanopsin-expressing intrinsically photosensitive RGCs (ipRGCs) and showed deficits in nonimage-forming behaviors, but not image vision. However, we show that the ON alpha RGC, a conventional RGC type, is intrinsically photosensitive in mammals. In addition to their classical response to fast changes in contrast through rod/cone signaling, melanopsin expression allows ON alpha RGCs to signal prior light exposure and environmental luminance over long periods of time. Consistent with the high contrast sensitivity of ON alpha RGCs, mice lacking either melanopsin or ON alpha RGCs have behavioral deficits in contrast sensitivity. These findings indicate a surprising role for melanopsin and ipRGCs in vision.

The topography of cone photoreceptors in the retina of a diurnal rodent, the agouti (Dasyprocta aguti).

The presence, density distribution, and mosaic regularity of cone types were studied in the retina of the diurnal agouti, Dasyprocta aguti. Longwave-sensitive (L-) and shortwave-sensitive (S-) cones were detected by antibodies against the respective cone opsins. L- and S-cones were found to represent around 90 and 10% of the cone population, respectively. There was no evidence for L- and S-opsin coexpression in agouti cones. L-cone densities were highest, up to 14,000/mm2, along a horizontal visual streak located about 2-3 mm dorsal to the optic nerve, and the L-cone distribution showed a dorsoventral asymmetry with higher densities in ventral (about 10,000/mm2) than in dorsal (about 4000/mm2) retinal regions. This L-cone topography parallels the agouti's ganglion cell topography. S-cones had a peak density of 1500-2000/mm2 in the central retinal region but did not form a visual streak. Their distribution also showed a dorsoventral asymmetry with densities around 600/mm2 in dorsal and around 1000/mm2 in ventral retinal regions. The patterning of cone arrays was assessed by the density recovery profile analysis. At all eccentricities evaluated, the S-cone mosaic less efficiently packed than the L-cone mosaic. Rod densities ranged from 47,000/mm2 in peripheral to 64,000/mm2 in central retina, and rod:cone ratios were 4:1-9:1. The comparatively low rod density and high cone proportion appear well adapted to the diurnal lifestyle of the agouti.

Mechanisms of opsin activation.

Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu113 and cytoplasmic acidic residues, including Glu134, lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff base-derivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.

Topography of opsin within disk and plasma membranes revealed by a rapid-freeze deep-etch technique.

Rod outer segments in fresh rat retinas were examined by a rapid-freeze, deep-etch technique to explore how membrane proteins are organized at the macromolecular level. Cross-fractures revealed that intradiscal membranes are adherent to each other except at the rim. When an isolated fresh retina was incubated in a hypotonic solution for a few minutes, the interdiscal space was expanded and the cytoplasmic surface of the disk membrane was found to be covered with protrusions except at the rim. A few particles were scattered among the protrusions and were attached to the cytoplasmic surface. Since the distribution density of the cytoplasmic surface protrusions was similar to that of the P-face particles, which are known to reflect opsins, the protrusions were considered to be portions of opsins extending into the cytoplasm. The intradiscal surfaces in chemically-fixed retinas were rather smooth and were labelled with anti-opsin antibodies and wheat germ agglutinin. The true surfaces of the plasma membrane were found to be similar in fine structure to those of the disk. A model of the macromolecular organization of rod outer segments is proposed on the basis of these observations. The model shows apposed opsins within a disk membrane adhering to one another except at the rim. These opsins, as well as those in the plasma membrane, are minimally exposed to the extracellular surface, but protrude deeply into the cytoplasm.