D-[3H]-galactose incorporation in the bovine retina: specific uptake and transport of the radiolabel in cones

When bovine neural retinas are incubated in Krebs-Henseleit buffer with D-[3H]-galactose, autoradiography reveals that there is a rapid uptake of the tritium label into the inner segments of cones, but not of rods. Pulse--chase studies show that the label is first associated with the Golgi apparatus in the cones, then appears to travel around the nucleus and along the cone fibre (homologous to an axon) to the synaptic pedicle. The cone-specific label travels along the fibre at a rate of about 0.5-1.0 mm per day. Label is also found in endothelial cells and Müller cells, but does not persist in the Müller cells as long as in the cones. The striking difference between rod and cone labelling may reflect fundamental differences in the neurochemistry of these two photoreceptor cell types.     

Characterization of molecules bound by the cone photoreceptor-specific monoclonal antibody CSA-1

The cone-specific monoclonal antibody, CSA-1, selectively labels cone photoreceptors in the neural retina. Immunofluorescence assays reveal that CSA-1 binds cone outer and inner segments, cell bodies, axons and synaptic pedicles. The binding of the antibody is membrane-associated, not cytoplasmic. Several lines of evidence, including sensitivity to mild alkaline hydrolysis and galactosidic enzymes, suggest that the CSA-1 detected determinant is a galactose-containing carbohydrate moiety. Immunoblotting analyses show CSA-1 immunoreactivity with at least two molecules of 54,000 daltons that differ slightly in isoelectric point, and an additional, higher molecular weight species. CSA-1 should prove to be of value in studies of cone photoreceptor biology, in analyses of the development and differentiation of these cells, and in assessing their fate in various retinal pathologies.     

Green-sensitive cone photoreceptors are selectively labeled by Procion yellow dye in goldfish retina

Selective labeling of intravitreal Procion yellow dye by presumed blue-sensitive cone photoreceptors has been demonstrated in primate retina. To determine whether Procion yellow is selective for this cone type in an unrelated vertebrate species, labeling by this dye was studied in goldfish retina, where cone pigment type can be directly inferred from photoreceptor morphology. At low vitreal concentrations of the dye (less than 0.4%), only cone outer segments were labeled. At vitreal concentrations of 0.4-0.5%, the inner segments of short-double cones and a subset of long single cones (presumed green-sensitive cones) were selectively stained. At still higher vitreal concentrations (0.6-0.7%), the inner segments of short-single cones and miniature short-single cones (presumed blue-sensitive cones) showed evidence of Procion label, but were not as heavily labeled. The inner segments of long-double cones and a subset of long-single cones (presumed red-sensitive cones) did not label at any of these concentrations. These results show that Procion yellow is not a selective marker for blue-sensitive cones in the goldfish retina. In addition, stained rod and cone nuclei were observed at each dye concentration, including those concentrations at which no inner segments were labeled.     

Interphotoreceptor retinoid-binding protein in the cone matrix sheath. Electron microscopic immunocytochemical localization.

Using a monoclonal antibody to chondroitin 6-sulfate, a major constituent of the cone matrix sheath, the location of the cone matrix sheath and the relative distribution of interphotoreceptor retinoid-binding protein (IRBP) was examined by light and electron microscopic immunocytochemistry. Chondroitin 6-sulfate immunoreactivity was localized to regions surrounding cone outer and inner segments, extending from the pigment epithelium to the outer limiting membrane. Only a background level of chondroitin 6-sulfate immunoreactivity was found around rods. Ultrathin sections reacted with antibodies to IRBP and chondroitin 6-sulfate showed the presence of both in the matrix surrounding the cones. Chondroitin 6-sulfate immunoreactivity extended from cone inner and outer segments to the adjoining interphotoreceptor matrix (IPM) surrounding the rods, but only background labeling was observed around the rods. The label for IRBP was found in the region containing chondroitin 6-sulfate and the IPM surrounding the rod photoreceptors. The label for IRBP and chondroitin 6-sulfate appeared to be associated with the filamentous reticulum of the IPM. These findings not only show that the cone matrix sheath has not collapsed and that IRBP is present in the cone matrix sheath, but they also demonstrate retention of structural components of the IPM.     

Localization of iodopsin in the chick retina during in vivo and in vitro cone differentiation

Using highly specific antibodies against a chick red-sensitive cone pigment, iodopsin, we investigated the localization of iodopsin in the developing and mature chick retina. The chick retina contains several different photoreceptor types, including a rod, a double cone with a principal and accessory cone, and four different types of single cones. Immunocytochemical observations revealed that outer segments (OS) of one of the single cones (type 1) and both cells of the double cone were strongly immunoreactive to anti-iodopsin antibodies. The Golgi regions and small vesicular structures in the inner segments (IS) of these cells also were intensely stained, indicating a continuous synthesis of iodopsin and its addition to the newly formed cone OS. In the differentiating cones of the developing but immature chick retina, iodopsin immunoreactivity was found at the plasma membranes of both the IS and the terminals (pedicles). This suggests that unidirectional transport of iodopsin to the outer segment may be established during cone differentiation. Immunostaining in the outer plexiform layer (OPL) produced two bands, suggesting that the pedicles of the double cones and type 1 single cones terminate at different positions in this layer. Application of the antibodies to a cell culture system of the chick retina revealed that cells immunoreactive to anti-iodopsin differ slightly in morphology from those reactive with anti-rhodopsin. Since antibodies to iodopsin and rhodopsin stained different types of photoreceptors in the intact chick retina, it will be possible to analyze cell lineage of rods and cones in vitro by use of these antibodies.     

Differential distribution of interphotoreceptor retinoid-binding protein (IRBP) around retinal rod and cone photoreceptors

Light microscopic immunogold cytochemistry was used to examine the distribution of interphotoreceptor retinoid-binding protein (IRBP) in the interphotoreceptor matrix (IPM) surrounding rod and cone photoreceptors. Silver enhancement of retinas reacted with anti-IRBP antibodies using the two stage labeling procedure showed dense staining of the IPM around rod photoreceptor outer segments and the apical region of the RPE. However, the IPM around cone photoreceptors was lightly labeled for IRBP. This region of light labeling extended from the RPE to the distal one-fourth to one-third of the cone inner segment. Although most of the area surrounding cone outer segments was lightly labeled, a dense band of label was seen along the margins of cone outer segments. This study confirms that heterogeneity exists in the distribution of IRBP in the IPM and provides new evidence that IRBP is not homogeneously distributed around rod and cone photoreceptors.     

Ultrastructure of adult rd mouse retina

PURPOSE: To examine the ultrastructure of the adult rd mouse retina in order to determine what structures are altered or lost and thus to better interpret changes produced by photoreceptor and/or retinal transplantation in this model of retinal degeneration. METHODS: rd mutant mice expressing a LacZ reporter gene in rod bipolars were used in order to identify these cells and their processes. Mice of age 6 weeks to 5 months were studied by electron microscopy, concentrating on the posterior pole where retinal transplants are usually placed. RESULTS: The adult rd mouse retina contains degenerating cones, cone outer segments, cone synaptic pedicles with synaptic vesicles and post-synaptic contacts. The major abnormalities occur in the subretinal space where all traces of rods are gone and the main structures are inner segments of cones. These inner segments are smaller than normal, contain fewer and smaller mitochondria, have organized arrays of microtubules, resembling those in cone axonal processes, and are completely engulfed by massive proliferation of apical processes of the retinal epithelium. The subretinal space is well defined by the external limiting membrane vitreally and the retinal epithelium choroidally. Muller cells extend globular rather than filamentous processes into the subretinal space which contact the apical processes of the epithelium. Rod bipolar cells survive and retain processes in the external plexiform layer. CONCLUSIONS: The adult rd mouse retains structural elements necessary for phototransduction and transmission of signals to the inner layers of the retina by the cone system. The major deficits are located in the subretinal space where all rods are lost and cone inner segments undergo a slow degeneration. Rod bipolar cells survive but appear to be de-afferented; there was no evidence that they contact residual cone processes in the external plexiform layer. The rd mouse is a logical model to study the effects of transplantation of photoreceptors because second- and third-order retinal neurons as well as degenerating cones survive in the adult retina.     

Abnormal distribution of red/green cone opsins in a patient with an autosomal dominant cone dystrophy

PURPOSE: To define the distribution of the red/green and blue opsins in cones from donor eyes from an affected member of a clinically well-characterized family with an autosomal dominant form of cone dystrophy. METHODS: Tissue was fixed and processed for immunohistochemistry. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodies to cone cytoplasm, rhodopsin, and cone opsins. The cone-associated matrix was also labeled with the lectin PNA. The affected donor eyes were compared to a postmortem matched normal eye. RESULTS: Electroretinogram (ERG) testing three years prior to the affected member's death showed normal rod function, while the cone b-wave amplitude was reduced 40% below the lower limit of normal. Fundus exam showed only isolated drusen within the macula. Either a normal-appearing or only nonspecific macular findings were noted in the other affected family members who were examined. Immunofluorescence studies showed that blue cone opsin was restricted to the outer segments of blue cones in the affected retina. Red/green opsins were distributed along the entire plasma membrane of these cone types, from the tip of the outer segment to the synaptic base. Cone-associated matrix displayed a heterogeneous distribution. These patterns were observed both in the macula and in the periphery of the affected retina. Cone pedicles appeared larger than normal. In contrast, rhodopsin staining appeared normal. CONCLUSIONS: The immunocytochemical data obtained suggest that the clinical manifestation of this dystrophy is associated with an abnormal distribution of cone red/green opsins. Additionally, changes in the cone pedicles could have contributed to the abnormal cone ERG in this patient.     

Idiopathic retinal degeneration in the dog: differential patterns of [3H]uridine incorporation and HIOMT-like immunoreactivity in surviving photoreceptors

Photoreceptor cell pathology was investigated in an 8-yr-old mixed-breed dog which had displayed visual symptoms of 1 month duration. An electroretinogram detected no light-evoked responses. Light and electron microscopic features showed marked thinning and atrophy of the outer both the tapetal and non-tapetal retina appeared to be involved. In the non-tapetal region, a majority of the rod inner segments were missing, while scattered mitochondria-filled stubby inner segments of cones were readily identified. Inner segments of both rods and cones were observed in the tapetal region. Photoreceptor outer segments were completely absent from the affected retina, and no outer segment debris was observed between the photoreceptor layer and the retinal pigmented epithelium (RPE). Autoradiographic analysis of 3-mm retinal disks from the degenerate retina following incubation with [3H]uridine indicated that only 61% +/- 13 S.D. of the remaining nuclei of rod photoreceptors were undergoing RNA synthesis, whereas more than 99% of cone nuclei incorporated the label. Normal and degenerate retina were also analysed for localization of hydroxyindole-O-methyltransferase (HIOMT)-like immunoreactivity. While the normal retina showed immunoreactivity in both rod and cone photoreceptors with more intense immunoreactivity present in cones, the degenerate retina showed HIOMT-like immunoreactivity only in the remaining cone photoreceptors. The results of this study of idiopathic photoreceptor degeneration of the canine retina suggest that although both photoreceptor types are involved, rods are more severely affected than cones.     

DNA repair synthesis in the rat retina following in vivo exposure to 300-nm radiation

Quantitative autoradiography was used to study the incorporation of 3H-thymidine into the retina of albino rats following in vivo exposure to 300-nm radiation. Relative to background labeling in unexposed eyes, there was 8-20 times as much label per unit area in the outer nuclear layer, inner nuclear layer, and ganglion cells of 300-nm exposed retinas. The photoreceptor inner segments also showed thymidine labeling in both control and exposed retinas.     

Synthesis and secretion of interstitial retinol-binding protein by the human retina

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein present between the retina and pigmented epithelium, which may function to shuttle vitamin A derivatives between these tissues. While previous studies have shown that the retina is solely responsible for IRBP synthesis, the specific retinal cell(s) in which this occurs has not been established. Since the carbohydrate moiety of IRBP contains fucose, the authors have analyzed the sites of incorporation of 3H-fucose in the human retina in vitro, using autoradiography. Following a 30-min pulse incubation, all retinal layers exhibited incorporation of label; however, the rod photoreceptor inner segments contained one- to two-fold more radioactivity than was present in any other retinal compartment. In autoradiographs of retinas recovered following a 4 hr chase incubation, all retinal layers retained similar levels of radioactivity with the exception of the rod photoreceptors, cone photoreceptors and cells in the inner nuclear layer, which lost 75, 11, and 14 percent, respectively of the radioactivity present immediately following the 30-min pulse. Proteins present in the chase incubation medium were analyzed by polyacrylamide gel electrophoresis and fluorography. The principal labeled component in the chase medium was identified as IRBP by immunoprecipitation with antibovine-IRBP immunoglobulins. Thus, the major loss of 3H-fucose radioactivity from rod photoreceptors coupled with the appearance of 3H-labeled IRBP in the incubation media suggests that the rod photoreceptors are primarily responsible for the synthesis and secretion of IRBP.     

Retinal degenerations in the dog II. Development of the retina in rod-cone dysplasia

The kinetics of rod outer segment production and turnover were studied in Irish setter dogs with a recessively inherited photoreceptor degeneration (rod-cone dysplasia). Affected and littermate control (heterozygote) dogs, 16–50 days of age, were injected intravitreally with [³H]leucine after electroretinographic studies. Normal age-matched mongrel dogs were also used as controls. Between 6 hr and 8 days following intravitreal injection, eyes were removed and processed for microscopic and radioautographic studies.In affected dogs, the rod components of the electroretinogram were not recordable. Cone components were recordable but failed to develop normally. Morphologically, rod inner segments were diminutive and the outer segments short and contained disorganized and disoriented disc material. Cone inner segments became broad and club-shaped. Radio-autography showed label accumulation within inner segments, but only sporadic and randomly distributed label in the narrow outer segment layer. These results suggest that in Irish setters with rod-cone dysplasia, there is a defect in the morphogenesis of the rod outer segments during postnatal retinal differentiation.     

Intravitreal injection of fluorochrome-conjugated peanut agglutinin results in specific and reversible labeling of mammalian cones in vivo

PURPOSE: To investigate whether intravitreally injected peanut agglutinin (PNA) conjugated with a fluorochrome can specifically label retinal cones in vivo and to evaluate its clinical potential. METHODS: Fluorescein- or rhodamine-conjugated PNA (0.005%-0.5%) was intravitreally injected into anesthetized mouse, guinea pig, or monkey and retinas were removed at various intervals for fluorescence microscopy. Immunofluorescence and TUNEL assay were carried out to investigate whether PNA injection adversely affected other retinal neurons. Gross visual function was studied in a visual cliff test. The retina of an N-methyl, N-nitrosourea (MNU)-induced mouse model of retinal degeneration was stained with PNA to evaluate how spatiotemporal pattern of the staining would reflect the progression of degeneration. RESULTS: Intravitreally injected PNA resulted in specific labeling of cone outer and inner segments and cone pedicles within 30 minutes over the entire retina and in all tested species. The labeling was reversible; cones did not show any labeling 3 weeks after the injection but could be restained with PNA. TUNEL signal and expression pattern of several retinal proteins in PNA-injected mouse retina were indistinguishable from normal. Similarly, visual behavior of mouse 10 hours after the injection was normal. The pattern of PNA labeling in mice with MNU-induced retinal degeneration showed progressive disappearance of cones from the center to the periphery. CONCLUSIONS: Intravitreal injection of fluorochrome-conjugated PNA results in specific and reversible labeling of mammalian cones in vivo without causing any gross adverse effects. This novel method may eventually provide a clinical tool to examine diseased retina.     

Endocannabinoids in the intact retina: 3 H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide

There is much evidence for an endocannabinoid system in the retina. However, neither the distribution of endocannabinoid uptake, the regulation of endocannabinoid levels, nor the role of endocannabinoid metabolism have been investigated in the retina. Here we focused on one endocannabinoid, anandamide (AEA), and its major hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), in the goldfish retina. Immunoblots of FAAH immunoreactivity (IR) in goldfish retina, brain and rat retina, and brain homogenates showed a single band at 61 kDa that was blocked by preadsorption with peptide antigen. Specific FAAH IR (blocked by preadsorption) was most prominent over Müller cells and cone inner segments. Weaker label was observed over some amacrine cells, rare cell bodies in the ganglion cell layer, and in four lamina in the inner plexiform layer. FAAH activity assays showed that goldfish-retinal and brain homogenates hydrolyzed AEA at rates comparable to rat brain homogenate, and the hydrolysis was inhibited by methyl arachidonyl fluorophosphonate (MAFP) and N-(4 hydroxyphenyl)-arachidonamide (AM404), with IC(50)s of 21 nM and 1.5 microM, respectively. Cellular 3H-AEA uptake in the intact retina was determined by in vitro autoradiography. Silver-grain accumulation at 20 degrees C was most prominent over cone photoreceptors and Müller cells. Uptake was significantly reduced when retinas were incubated at 4 degrees C, or preincubated with 100 nM MAFP or 10 microM AM404. There was no differential effect of blocking conditions on the distribution of silver grains over cones or Müller cells. The codistribution of FAAH IR and 3H-AEA uptake in cones and Müller cells suggests that the bulk clearance of AEA in the retina occurs as a consequence of a concentration gradient created by FAAH activity. We conclude that endocannabinoids are present in the goldfish retina and underlay the electrophysiological effects of cannabinoid ligands previously shown on goldfish cones and bipolar cells.     

Comparison of photoreceptor-specific matrix domains in the cat and monkey retinas

Two lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), were used to compare domains within the interphotoreceptor matrices (IPM) of the cat and monkey, two species where the morphological relationship between the retinal pigment epithelium (RPE) and photoreceptors is distinctly different. In the monkey, PNA labeling was heaviest over the cone outer segments and a discrete region of the interphotoreceptor matrix bordering the cone inner and outer segments--a region which has been termed the cone matrix sheath. Near the apical border of the RPE, the outer margin of the PNA-labeled matrix is surrounded by a circular array of apical microvilli. In the cat retina, PNA labeling was highest over a region of the IPM lying between the outer margin of the cone sheath processes and surrounding rod matrix. In contrast, intracellular labeling of cone inner and outer segments was sparse. The RPE apical processes forming the cone sheath were not labeled. In the monkey retina, WGA preferentially labeled rod outer segments and the region of the IPM around rod inner and outer segments. The cone matrix sheath was not preferentially labeled using this lectin. Rod inner segments and cone inner and outer segments were labeled moderately. In the cat retina, WGA labeling was dense over both rod outer segments and cone outer segments as well as the cone sheath. Rod and cone inner segments, as well as the IPM around both rods and cones, were moderately labeled. These observations suggest that the specialized processes arising from the apical surface of retinal pigment epithelial cells, together with photoreceptor-specific extracellular matrix domains, contribute to the formation of specific microenvironments around rod and cone photoreceptor cells.     

Distribution of membrane proteins in mechanically dissociated retinal rods

Solitary rods were isolated from frog retinas by mechanical dissociation. Typically, the rods cleave sclerad to the nucleus and consist of outer segments with attached partial inner segments with either tapered or rounded profiles. Light and electron microscopy reveal that the outer and inner segments of rods with tapered inner segments, like rods in the intact retina, are joined by a single connecting cilium. In contrast, the outer and inner segments of rods with rounded inner segments are fused, with no extracellular cleft between the two segments. Opsin distribution was studied in both unfused and fused rods by light and electron microscopic immunocytochemistry. Extensive surface labeling is restricted to the outer segments of tapered rods, as observed in vivo. In contrast, both inner and outer segments of rods with rounded inner segments (fused) label heavily with anti-opsin. Thus opsin, a mobile membrane protein, diffuses from the outer to the inner segment of fused rods. Segregated distribution of opsin in unfused rods suggests that the connecting cilium and/or its associated structures may normally act as a diffusion barrier between the outer and inner segments to mobile membrane proteins such as opsin. Immunofluorescence studies demonstrate that Na+/K+ ATPase is restricted in distribution to the inner segment and calycal processes of both fused and unfused isolated rods, as observed in vivo. Maintenance of its restricted distribution in fused cells indicates that Na+/K+ ATPase is not mobile and may be tethered in the surface membrane of the inner segment.     

Signal integration at the pedicle of turtle cone photoreceptors: an anatomical and electrophysiological study

The morphology of the axon which connects the cell body and pedicle of turtle cone photoreceptors was studied by light and electron microscopy. The axon which contains numerous synaptic vesicles, some endoplasmic reticulum, and a few cisternae is basically filled with cytoplasm. The length of the axon is related to the class of cone and varies slightly with retinal location, with axons as short as 3-6 microns found in red cones, and as long as 60 microns in cones containing colorless oil droplets. By simultaneously voltage clamping the cell body and pedicle regions of single isolated cones, we measured the longitudinal axonal resistance and the cell body and pedicle membrane resistances. For each cell studied, the axonal resistance of cones with short axons was lower than the cell and pedicle membrane resistances. Thus, the cell can be considered to be an isopotential structure. However, in some cones with long axons, the axonal, cell body, and pedicle resistances were comparable. The pedicles of these cones, therefore, could act like summing points and may provide a locus for spatial signal integration. Electrical coupling between the principal and accessory members of double cones was also studied. Electron-microscopic observation of the membrane junction between the apposed inner segments of the double cones in the intact retina show narrow segments which resemble gap junctions. However, in every double cone studied in culture, passing currents into one member of the double cone did not result in measurable current flow in the adjacent cell. Thus, the two members of the double cone, isolated from the turtle retina, are not electrically coupled.     

Interphotoreceptor matrix domains ensheath vertebrate cone photoreceptor cells

The retinal interphotoreceptor matrix (IPM) occupies the space between the neural retina and the retinal pigmented epithelium (RPE), two neuroectoderm-derived epithelia. While the IPM appears to be a major route by which photoreceptor cells receive vital metabolic factors, relatively little is known concerning its structure and function. The studies reported here describe the presence of specialized domains of the IPM that ensheath cone, but not rod, inner and outer segments in pig, monkey, and human retinae. These cone extracellular matrix sheaths are chemically and structurally distinct from the remainder of the IPM as revealed by their specific binding of the lectin peanut agglutinin (PNA) and their structural stability during physical dissociation of the retina. Biochemical studies suggest that the PNA-binding components of the cone matrix sheaths are trypsin-sensitive glycoproteins. These structures may play a role in establishing a specialized microenvironment for cone photoreceptors, maintaining proper orientation of cone outer segments, and/or facilitating cone-RPE interactions.     

Binding pattern of anti-rhodopsin monoclonal antibodies to photoreceptor cells: an immunocytochemical study

A panel of anti-rhodopsin monoclonal antibodies (MAbs) of defined epitope specificity has been evaluated by immunocytochemistry. Most of the IgG class MAbs (23/27) gave positive results, but only a few of the IgM class MAbs (2/21) were useful for this application. MAbs specific to the N-terminal region stained rod outer segments most strongly, with progressively less staining in the Golgi, perikarya, plasma membrane of the inner segment, and synaptic region. Phagosomes located basally in the pigment epithelium were stained; cone cells were consistently negative. Antibodies to the C-terminus of rhodopsin labeled the same cell structures (except for phagosomes) but showed diversity in their binding pattern. Many of these MAbs bound to cone outer segments in addition to rods, and showed different patterns of binding to red/green and blue cones. Antibodies specific for rhodopsin sequence 340-348 labeled different types of cone cells, indicating differences in their binding sites. Two MAbs were found to label hydrophilic loop sequences which connect rhodopsin's transmembrane segment: MAb K42-41 which binds loop 5-6, and MAb A1-55 which binds loop 2-3. At least these two regions of the rhodopsin sequence in addition to the C- and N-termini, are available for antibody reaction in fixed retina.     

The cone matrix sheath in the normal and diseased retina: cytochemical and biochemical studies of peanut agglutinin-binding proteins in cone and rod-cone degeneration.

The fate of the cone-associated extracellular domain, or cone matrix sheath (CMS), was examined in two canine models of hereditary retinal degeneration. The diseases, which affect cones selectively (cd = cone degeneration), or rods and cones temporally (prcd = progressive rod-cone degeneration), were examined biochemically (SDS-PAGE/lectin blots) and cytochemically (light microscopy) using peanut agglutinin lectin (PNA) to selectively label this domain and associated structures. Most of the cones had disappeared in the adult cd retina. In the remaining cones, PNA labeled the ectopically located somata and the CMSs that were present around severely diseased ones. Loss of cones resulted in background label in the IPM and the loss of the pedicle-associated label in the OPL. SDS-PAGE of retinal extracts showed that all the major classes of the lower molecular weight PNA-binding proteins were present, but only the 40- and 60-kD bands remained prominent. Because of the selectivity of the cd mutation, this suggests considerable heterogeneity within the various size classifications of the retinal PNA-binding glycoproteins. In prcd, CMSs were normal at a time when cones were structurally normal and disease was limited to the rod outer segments. The CMSs remained intact during the degenerative phase of the disease, and only became compressed in association with the collapse and narrowing of the photoreceptor layer; CMS labeling was lost with disappearance of the cone inner segment. The lectin biochemical results were normal until 1.7 years of age; thereafter, there was a decreased prominence of all major bands. Because of spatial heterogeneity in disease severity, it was not possible to correlate the lectin biochemical and cytochemical results in prcd.