Freeze‐fracture study of filipin binding in photoreceptor outer segments and pigment epithelium of dystrophic and normal retinas

We have studied sterol distribution in the retinal pigment epithelial (RPE) microvillous and outer segment disc membranes of rats with inherited retinal degeneration (RCS; RCS‐p/+) and of normal genetic controls (RCS‐rdy⁺, RCS‐rdy⁺‐p/+) by using the polyene antibiotic filipin, which binds specifically to 3‐B‐hydroxy‐sterols, and freeze‐fracture techniques. Retinas were perfusion‐fixed, incubated with filipin in the same fixative, and prepared routinely for freeze‐fracture electron microscopy. In the normal retina, the distribution of filipin binding sites on both RPE microvillous and outer segment disc membranes changes during development. Prior to outer segment elongation and the onset of phagocytosis (10 days postnatal), filipin sterol complexes are homogeneously distributed in both microvillous and outer segment membranes. With the onset of phagocytosis (2 weeks postnatal and later) filipin binding in both tissues forms a proximal‐to‐distal gradient, and binding sites decrease as distance from the cell body increases. In the normal RPE microvillous membranes, binding sites are numerous proximally and sparse on the distal tips. In the normal outer segment disc membranes, binding sites are often present on the basal discs, but are sparse on the intact apical discs prior to shedding. As the discs are cast off and engulfed by the RPE, however, filipin binding increases on both disc and phagosome membranes. In the dystrophic retina, the distribution of filipin binding sites differs from the normal. First, in the microvillous membranes, the proximal‐to‐distal gradient in filipin binding is rarely present at 2 weeks postnatal and becomes prominent only after the buildup of membranous debris has begun (3–6 weeks postnatal). Second, as the photoreceptors degenerate and the membrane debris disappears (4 months postnatal), filipin binding on the microvillous membranes becomes relatively sparse and homogeneous. Third, filipin binding on the intact disc membranes does not change with outer segment elongation, and numerous filipin binding sites are present on both apical and basal outer segment disc membranes. Fourth, large aggregates of filipin binding sites occupy the vast expanses of particlefree areas of debris membranes which accumulate between the photoreceptors and the RPE. These changes in the amount and distribution of filipin binding sites in the dystrophic retina add to the evidence that the disease process involves outer segment as well as RPE membranes and suggest that alterations in cholesterol distribution could contribute to the phagocytic defect.