Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses.

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.

Prenatal development of central optic pathways in albino rats.

The development of the central optic projections in albino rat fetuses has been studied using light and electron microscopic degeneration techniques and the horseradish peroxidase method for demonstrating axonal projections of neurons. The first optic axons to reach the region of the optic chiasm arrive at day 15. By day 16, a substantial optic chiasm is seen and the optic tract can be traced into the epithalamus, having first passed through the ventral lateral geniculate nucleus and a thin lamina of cells which is thought to correspond to part of the future dorsal geniculate nucleus. A growth rate of 80-100 mum per hour is estimated for the fastest growing axons. By day 16-1/3 the first axons have entered the anterior border of the superior colliculus and in the next day have grown across the entire rostrocaudal extent with the exception of the medial and lateral edges. The optic axons are recognized at day 17 as bundles lying just below the surface, but in older animals they come to lie deeper, as the whole layer of optic innervation broadens. The first synapses to be formed in the superior colliculus (some of them of optic origin) appear on day 17. Subsequently, there is a gradual increase in the number of contacts, the great majority being formed by optic axons. Compared with previous studies on Xenopus and chick, one of the most striking features of the development of the central visual connections in the rat is the relatively long time before the first optic axons reach the brain and the speed with which they innervate the central structures once they have arrived.

Demonstration of bilateral projection of the central retina of the monkey with horseradish peroxidase neuronography.

In the primate, ganglion cells of the temporal retina project ipsilaterally and those of the nasal retina, contralaterally into the optic tract. The vertical meridian passing through the fovea defines the border between these two populations of ganglion cells and has been demonstrated in four Macaque monkeys after unilateral injection of horseradish peroxidase into the dorsal lateral geniculate nucleus and examination of the pattern of retrograde labeling of those ganglion cells projecting to the injected side. A median 1 degree vertical strip in which ipsi- and contralaterally projecting ganglion cells intermingle was found, confirming the report by Stone et al. ('73). In addition, occasional extrafoveal labeled ganglion cells were found as far as 2 degrees from the vertical midline in the otherwise unlabeled hemiretinae. These ganglion cells were not numerous and had somata of all sizes, suggesting that they do not constitute a separate class of ganglion cells as found in the temporal retina of the cat. In contrast to the description by Stone et al. ('73), the strip of vertical overlap did not show a constant width through the fovea, since mixing of labeled and unlabeled ganglion cells was found in a band approximately 1/2 degree wide along both the nasal and temporal rims of the foveal pit which is 500 mum (2 degrees) in diameter. Beyond these 1/2 degree arcs, the appropriate hemiretina was either completely unlabeled, or contained virtually every ganglion cell labeled on the side projecting to the injected dorsal lateral geniculate nucleus. The scattered labeled ganglion cells rimming an otherwise unlabeled hemifovea represent a possible anatomical basis for the phenomenon of "macular" or "foveal sparing" in which unilateral damage to the occipital cortex produces homonymous hemianopsia with sparing of a small island of centralmost vision extending about 1 degree from the foveal center. From this study, it is not possible to define the receptive fields or specific photoreceptor connections of the ganglion cells labeled with horseradish peroxidase, so that at the present time quantitative correlations cannot be made between the numbers of ganglion cells remaining on the affected side of the fovea and the extent of preservation of visual function in the spared zone. The presence of labeled ganglion cells rimming the fovea in its entirety is compatible with the sequence of foveal development in late prenatal life. After lateral displacement both nasally and temporally of ganglion cells which initially lay in the median vertical overlap strip of 1 degree, in the adult retina a strip approximately 1/2 degree wide around the perimeter of the foveola should contain a mixture of ipsi- and contralaterally projecting ganglion cells. The total population of ganglion cells beyond this 1/2 degree band should be completely ipsi- or contralateral in their projection patterns, as is observed...

The origin of efferent pathways from the primary visual cortex, area 17, of the macaque monkey as shown by retrograde transport of horseradish peroxidase.

The retrograde transport of horseradish peroxidase has been used to identify efferent cells in area 17 of the macaque. Cells projecting to the lateral geniculate nucleus are small to medium sized pyramidal neurons with somata in lamina 6 and the adjacent white matter. The projection to the parvocellular division arises preferentially from the upper half of lamina 6, while that to the magnocellular division arises preferentially from the lower part of the lamina. The projection to both superior colliculus and inferior pulvinar arises from all sizes of pyramidal neurons lying in lamina 58 (Lund and Boothe, '75); at least pyramidal neurons of lamina 5B send collateral axon branches to both destinations. Injections with extensive spread of horseradish peroxidase show that many cells of lamina 4B and the large pyramidal neurons of upper lamina 6 also project extrinsically but their terminal sites have not been identified. Other studies have indicated that cells of laminae 2 and 3 project to areas 18 and 19. Therefore every lamina of the visual cortex, with the exception of those receiving a direct thalamic input, contains cells projecting extrinsically. Further, each lamina projects to a different destination and from Golgi studies can be shown to contain cells with specific patterns of dendritic branching which relate to the distribution of thalamic afferents and to the patterns of intracortical connections. These findings emphasise the significance of the horizontal organisation of the cortex with relation to the flow of information through it and contrast with the current concept of columnar organisation shown in physiological studies.

Monkey retinal ganglion cells: morphometric analysis and tracing of axonal projections, with a consideration of the peroxidase technique.

This paper presents evidence on the retinal distribution and central projections of retinal ganglion cells of various cell body sizes in the adult macaque monkey. The ganglion cell sizes have been determined by computer assisted measurement of camera lucida drawings at various eccentricities of both flat mounted and sectioned retinae. The pattern of projections of individual ganglion cells to the dorsal lateral geniculate nucleus and superior colliculus has been studied using retrograde axonal transport of horseradish peroxidase. Following peroxidase injections into the parvocellular laminae of the geniculate, virtually every ganglion cell was labeled within a circumscribed zone of the retina known to project to the region of the geniculate immediately surrounding the injection needle tip. After magnocellular injections, only the largest cells of the peripheral retina and approximately 26% of the ganglion cells of the parafovea were labeled. Peroxidase injections into the superior colliculus produced labeling of scattered ganglion cells of all sizes in the retina, although no labeled cells were found within the centralmost 10 degrees eccentricity. From these observations it is concluded that all ganglion cells of the macaque retina project to the parvocellular layers of the dorsal lateral geniculate, but that only the largest ganglion cells of the more peripheral retina and not all cells of the parafovea project to the magnocellular laminae. In contrast, only scattered ganglion cells, although these are of all sizes, appear to project to the superior colliculus. Two major problems with the peroxidase tracing technique are described: 1. The extent of stainable peroxidase activity around the injection site appears to be larger than the area of injected tracer actually available for uptake by axons to produce labeled cells. 2. Cut or damaged axons appear to incorporate peroxidase sufficiently to produce labeling of the cell body.

Fine structure and radioautography of rabbit photoreceptor cells.

The retina of the adult rabbit contains two types of photoreceptors. Type I predominates and is a typical rod. The identity of the type II cell has been uncertain until now. This study demonstrates that the type II cell has certain conelike morphologic features including a pale nucleus, complex synaptic pedicel, and multiple wrappings of the outer segment by microvilli of the pigment epithelium. The type II outer segment is also conelike in that some intradisc spaces are patent to the extracellular space, that the pattern of renewal is diffuse as demonstrated radioautographically with sugar and amino acids as precursors, and that the few examples of distal tip shedding found in this study occur mainly in the early part of the dark cycle. The interior of the type II outer segment is labeled selectively with 3H-fucose, whereas the interior of the type I outer segment remains unlabeled with this sugar at all time intervals examined. The label probably reflects the presence of a fucose-containing glycoprotein in the conelike outer segment which is lacking in the rod outer segment and may represent a visual pigment or other intrinsic molecule(s). An amorphous material which forms a symmetric sleeve around the photoreceptor connecting cilium has been demonstrated after intravitreal injections of horeseradish peroxidase. This cilium-associated material is labeled specifically by 4 hr following intravitreal injection of 3H-fucose, before the interphotoreceptor matrix shows significant labeling, suggesting that it is composed of a sugar-containing material. Possible functions of the cilium-associated material, including structural support and ionic regulation, are discussed.

Displaced ganglion cells in the retina of the monkey.

Horseradish peroxidase has been injected into the dorsal lateral geniculate nucleus of macaque monkeys to label ganglion cells of the retina by retrograde axoplasmic transport. Displaced ganglion cells, with somata in the inner nuclear layer and dendrites in the inner plexiform layer, were detected by virtue of their filling with peroxidase-positive granules. These cells were numerous in the peripapillary region, but relatively uncommon elsewhere in the retina.

Fine structure and radioautography of retinal cone outer segments in goldfish and carp.

Cone but not rod outer segments (OSs) in the retinas of goldfish and carp are diffusely labeled after intravitreal injection of 3H-fucose. Among the different cone types, the OSs are heavily labeled in the long double, short single, and some long single cones. The OSs of short double and the remaining long single cones are consistently more lightly labeled. A correlation thus exists between heavy labeling of OSs maximally sensitive to red and blue light and lighter labeling of OSs with maximum sensitivity to green light, and this may reflect different carbohydrate compositions of the cone visual pigments. New information is presented on OS fine structure of rods and cones, and on differential marking of cone OSs with the extracellular tracers horseradish peroxidase (only green-sensitive cone OSs "stained") and microperoxidase (red- and blue- but not green-sensitive cones OSs "stained").

Localization of cellular retinal-binding protein in bovine retina and retinal pigment epithelium, with a consideration of the pigment epithelium isolation technique.

Bovine RPE was isolated by commonly used brushout procedures and analyzed by light and electron microscopy. The preparation was found to consist almost entirely of cells with retained organelles (mitochondria, pigment, and other granules) but with broken surface membranes and extracted cytoplasm. In keeping with this, the wash obtained by sedimenting these broken cells contained approximately 97 percent of the cellular retinol-binding protein present in the suspension. Cellular retinoic acid-binding protein, present in bovine retinal extracts, was found in low amounts in the wash from RPE. The cellular retinol-binding protein present in the RPE wash was of high specific activity and similar in properties to that obtained from bovine retina. Supernatant obtained from sonicated rod outer segments contained approximately 10 percent of the retinol-binding protein of the retina. No retinoic acid-binding protein was found. The relatively large amount of cellular retinol-binding protein present in the RPE (more than is found in the retina) is consistent with a functional role of this protein in uptake and transport of retinol by the RPE.

Orthograde and retrograde axoplasmic transport during acute ocular hypertension in the monkey.

Orthograde and retrograde axoplasmic transport have been studied in the optic nerve heads of 37 Macaca fascicularis eyes with normal or elevated intraocular pressure (IOP) produced by cannulation of the anterior chamber. Orthograde transport was labeled by 3H-amino acids injected intravitreally and incorporated into retinal ganglion cell proteins. Retrograde transport was studied in the same eyes by injecting horseradish peroxidase (HRP) into one or both optic tracts and dorsal lateral geniculate nuclei (dLGN). Both tracers accumulated in the lamina scleralis (LS) of eyes maintained at pressures of 25 to 150 mm. Hg for 12 to 28 hours (pressure in normal controls = 10 to 14 mm. Hg) but the HRP technique was markedly more sensitive. The degree of retrograde transport obstruction in the LS appeared to be directly proportional to both the height and the duration of elevated IOP. In one experiment, the blockades of orthograde and retrograde transport induced at 50 mm. Hg were demonstrated to be reversible. Serial reconstructions of radioautographs and peroxidase-reacted sections of the optic nerve heads demonstrated that the orthograde and retrograde transport obstructions were coincidental anatomically by light microscopy in the LS and occurred most prominently in the temporal quadrants of the nerve head. These transport obstructions occurred at moderate elevations of IOP (25 TO 50 mm. Hg) despite (1) elevated arterial PO2 levels during inhalation of 100 percent oxygen and (2) intact nerve head capillary circulation, as demonstrated by perfusion with nucleated avian erythrocytes.

Foveal sparing. New anatomical evidence for bilateral representation of the central retina.

The pattern of retinal ganglion cell projections to the dorsal lateral geniculate nuclei has been demonstrated with horseradish peroxidase neuronography. A 1 degree-wide strip centered on the vertical meridian has been found in which ipsilaterally and contralaterally projecting ganglion cells intermingle. This strip expands to a width of 3 degrees at the fovea, since mixing of horseradish peroxidase-labeled and unlabeled ganglion cells was found in a band approximately 0.5 degrees wide along both the nasal and temporal rims of the foveola. These labeled ganglion cells rimming the foveal pit in its entirety represent a possible anatomical basis for "foveal sparing".

Axoplasmic transport in ocular hypotony and papilledema in the monkey.

Orthograde and retrograde axoplasmic transport were studied in optic nerve heads of seven hypotensive Macaca fascicularis eyes. Orthograde transport was studied by radioautography after intravitreal radioisotope injections. Retrograde transport was studied in the same eyes by horseradish peroxidase injection into the dorsal lateral geniculate nuclei or optic tracts. Three eyes had developed marked papilledema before injections. Orthograde axoplasmic transport was blocked in swollen axons of the optic disc anterior to Bruch membrane and in the lamina scleralis. Retrograde transport was blocked in axons within the lamina scleralis along the posterior edges of transverse scleral beams and in axons in the choroidal portion of the nerve head posterior to Bruch membrane. These results support the general concept that axoplasmic transport in the optic nerve head is sensitive to alterations in intraocular pressure, either increases or decreases. The edges of Bruch membrane and the openings in the lamina scleralis may constrict axon bundles in ocular hypotony.

Feulgen-positive deposits in retinoblastoma. Incidence, composition, and ultrastructure.

The incidence of hematoxyphilic extracellular deposits in 159 cases of retinoblastoma was found to be 18%. Six other features of these tumors also were examined. In the majority of eyes with these deposits, the tumors were composed solely of anaplastic cells. None was well differentiated with preponderantly rosette formation. The hematoxyphilic deposits were Feulgen-positive, and this staining was diminished but not abolished by pretreatment with purified deoxyribonuclease (DNase), which did abolish nuclear staining. Feulgen staining of the deposits was totally blocked by pronase--DNase pretreatment--suggesting that the deposits may represent DNA-protein complexes. Ultrastructurally, these deposits were electron dense and finely vacuolar. The deposits may originate from degenerating tumor cell nuclei, and the possibility that they may represent immune complexes warrants future study in view of occasional spontaneous regression of this tumor.

Ultrastructural and histochemical studies of conjunctival concretions.

Ultrastructural and histochemical studies were performed on conjunctival concretions (yellow spots in the tarsal conjunctiva) from two patients who had associated atopic keratoconjunctivitis and posttrachomatous degeneration. By electron microscopy, these concretions were composed of finely granular material and membranous debris accumulating in the conjunctival subepithelium. Histochemically, concretions stained strongly positive for phospholipid and elastin, weakly positive for neutral polysaccharides and lipid, and were negative for amyloid, collagen, glycogen, iron, mucopolysaccharides, RNA, and DNA. In view of the absence of both calcium and phosphate in the accumulated material, the use of the term "lithiasis" is misleading and should be avoided. These findings support a hypothesis that concretions that follow chronic conjunctivitis represent products of cellular degeneration.

Multifocal choroidal melanoma in ocular melanocytosis.

A 29-year-old white women had ocular melanocytosis with scleral pigmentation and a diffuse choroidal nevus. The eye was enucleated because of two choroidal tumors. Light and electron microscopy showed two separate choroidal melanomas with differential cytologic characteristics. The extreme rarity of multicentric choroidal melanomas support the belief that ocular melanocytosis predisposes to malignancy.

Ramification patterns of ganglion cell dendrites in the retina of the albino rat.

Ganglion cell morphology has been analyzed in the retina of the adult, albino rat by the Golgi technique and evidence has been obtained that the ganglion cells of this species show a far greater variety and complexity of organization than described previously. Both diffuse ganglion cells, whose dendrites ramify throughout the inner plexiform layer, and stratified ganglion cells, whose dendritic branching is restricted to one or two planes, have been identified. Evidence is also presented for distinct stratification of the inner plexiform layer in this species, based on radioautographic analysis following intravitreal injection of tritiated amino acids.