ATP8A2 is a P 4 -ATPase that is highly expressed in the retina, brain, spinal cord and testes. In the retina, ATP8A2 is localized in photoreceptors where it uses ATP to transport phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the exoplasmic to the cytoplasmic leaflet of membranes. Although mutations in ATP8A2 have been reported to cause mental retardation in humans and degeneration of spinal motor neurons in mice, the role of ATP8A2 in sensory systems has not been investigated. We have analyzed the retina and cochlea of ATP8A2-deficient mice to determine the role of ATP8A2 in visual and auditory systems. ATP8A2-deficient mice have shortened photoreceptor outer segments, a reduction in photoresponses and decreased photoreceptor viability. The ultrastructure and phagocytosis of the photoreceptor outer segment appeared normal, but the PS and PE compositions were altered and the rhodopsin content was decreased. The auditory brainstem response threshold was significantly higher and degeneration of spiral ganglion cells was apparent. Our studies indicate that ATP8A2 plays a crucial role in photoreceptor and spiral ganglion cell function and survival by maintaining phospholipid composition and contributing to vesicle trafficking.

The evolutionary position of tarsiers with respect to primates is still debated. The type of photoreceptors in the nocturnal Tarsius spectrum retina has been compared with the nocturnal New World monkey Aotus trivulgaris and the Old World monkey Macaca nemestrina by using immunocytochemical labeling for antisera known to be specific for primate cone and rod proteins. In all three species, antisera to long/medium (L/M) -wavelength specific cone opsin and cone-specific alpha-transducin detected a single row of cones. Only Macaca and tarsier retina contained cones labeled by antiserum to short (S) -wavelength specific cone opsin. Tarsier rod cell bodies were 6-12 deep, depending on retinal eccentricity. Tarsier central cones had 2-microm-wide outer (OS) and inner segments, which came straight off the cell body. Cone morphology differed little from rods except OS were shorter. Macaca cones labeled for 7G6 and calbindin, Aotus cones did not label for calbindin, and Tarsius cones did not label for 7G6 or calbindin. In tarsier retinal whole-mounts, peak cone density ranged from 11,600-14,200/cones mm(2). The 11- to 12-mm-wide peak region centered roughly on the optic disc, although foveal counts remain to be completed. Density decreased symmetrically to a far peripheral band of 4,200-7, 000/cones mm(2). In contrast, S cone density was very low in central retina (0-300/mm(2)), rose symmetrically with eccentricity, and peaked at 1,100-1,600/mm(2) in a 2- to 3-mm-wide zone in the far periphery. In this zone, S cones were 9-14% of all cones. L/M cones were regularly spaced, whereas S cones showed no regular distribution pattern. Although the functional characteristics of the tarsier S and L/M cone systems are yet to be determined, tarsier cone proteins and distribution have some similarities to both New and Old World monkey retinas.

RD3 is a 23 kDa protein implicated in the stable expression of guanylate cyclase in photoreceptor cells. Truncation mutations are responsible for photoreceptor degeneration and severe early-onset vision loss in Leber congenital amaurosis 12 (LCA12) patients, the rd3 mouse and the rcd2 collie. To further investigate the role of RD3 in photoreceptors and explore gene therapy as a potential treatment for LCA12, we delivered adeno-associated viral vector (AAV8) with a Y733F capsid mutation and containing the mouse Rd3 complementary DNA (cDNA) under the control of the human rhodopsin kinase promoter to photoreceptors of 14-day-old Rb(11.13)4Bnr/J and In (5)30Rk/J strains of rd3 mice by subretinal injections. Strong RD3 transgene expression led to the translocation of guanylate cyclase from the endoplasmic reticulum (ER) to rod and cone outer segments (OSs) as visualized by immunofluorescence microscopy. Guanylate cyclase expression and localization coincided with the survival of rod and cone photoreceptors for at least 7 months. Rod and cone visual function was restored in the In (5)30Rk/J strain of rd3 mice as measured by electroretinography (ERG), but only rod function was recovered in the Rb(11.13)4Bnr/J strain, suggesting that the latter may have another defect in cone phototransduction. These studies indicate that RD3 plays an essential role in the exit of guanylate cyclase from the ER and its trafficking to photoreceptor OSs and provide a 'proof of concept' for AAV-mediated gene therapy as a potential therapeutic treatment for LCA12.

Marmoset photoreceptor development was studied to determine the expression sequence for synaptic, opsin, and phototransduction proteins. All markers appear first in cones within the incipient foveal center or in rods at the foveal edge. Recoverin appears in cones across 70% of the retina at fetal day (Fd) 88, indicating that it is expressed shortly after photoreceptors are generated. Synaptic markers synaptophysin, SV2, glutamate vesicular transporter 1, and CTBP2 label foveal cones at Fd 88 and cones at the retinal edge around birth. Cones and rods have distinctly different patterns of synaptic protein and opsin expression. Synaptic markers are expressed first in cones, with a considerable delay before they appear in rods at the same eccentricity. Cones express synaptic markers 2-3 weeks before they express opsin, but rods express opsin 2-4 weeks before rod synaptic marker labeling is detected. Medium/long-wavelength-selective (M&L) opsin appears in foveal cones and rod opsin in rods around the fovea at Fd 100. Very few cones expressing short-wavelength-selective (S) opsin are found in the Fd 105 fovea. Across peripheral retina, opsin appears first in rods, followed about 1 week later by M&L cone opsin. S cone opsin appears last, and all opsins reach the retinal edge by 1 week after birth. Cone transducin and rod arrestin are expressed concurrently with opsin, but cone arrestin appears slightly later. Marmoset photoreceptor development differs from that in Macaca and humans. It starts relatively late, at 56% gestation, compared with Macaca at 32% gestation. The marmoset opsin expression sequence is also different from that of either Macaca or human. Indexing termsretina; opsin; cones; rods; arrestin; transducin; synaptophysin; recoverin The New World common marmoset monkey (Callithrix jacchus) has several practical advantages over Old World Macaca monkeys for experimental investigation of primate retinal development. Their smaller size, rapid maturation, and more frequent breeding with multiple births provide significant cost and time savings when studying primate retinal NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript development. In a previous paper , we showed that the marmoset retina and its vasculature have a spatial developmental pattern that is identical to previous morphological descriptions of fetal and infant macaque and human retinal development (Hendrickson and Kupfer, 1976;Provis et al., 1985Provis et al., , 1998Yuodelis and Hendrickson, 1986;Packer et al., 1990;LaVail et al., 1991;Hendrickson and Drucker, 1992;Hendrickson, 1992;Dorn et al., 1995;Bumsted et al., 1997;Georges et al., 1999;Xiao and Hendrickson, 2000;Provis, 2001;Springer and Hendrickson, 2004;Hendrickson and Provis, 2006).The one marked difference between macaque and marmoset is in the temporal development of the fovea, the specialized region responsible for high visual acuity. At birth, the marmoset fovea is relatively immature compared with a macaque neonate, but it then undergoes a rapid postnata...

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Identification and visualization of specific cells and cellular structures in the retina are fundamental for understanding the visual process, retinal development, disease progression, and therapeutic intervention. The increased usage of transgenic and naturally occurring mutant mice has further emphasized the need for retinal cell-specific imaging. Immunofluorescence microscopy of retinal cryosections and whole mount tissue labeled with cell-specific markers has emerged as the method of choice for identifying specific cell populations and mapping their distribution within the retina. In most cases indirect labeling methods are employed in which lightly fixed retinal samples are first labeled with a primary antibody targeted against a cell-specific protein of interest and then labeled with a fluorescent dye-tagged secondary antibody that recognizes the primary antibody. The localization and relative abundance of the protein can readily be imaged under a conventional fluorescent or confocal scanning microscope. Immunofluorescence labeling can be adapted for imaging more than one protein antigen through the use of multiple antibodies and different, nonoverlapping fluorescent dyes. A number of well-characterized immunochemical markers are now available for detecting photoreceptors, bipolar cells, amacrine cells, horizontal cells, Müller cells, and retinal pigment epithelial cells in the retina of mice, and other mammals.