Robert J. Ulshafer

Excitatory amino acid involvement in retinal degeneration

Amino acid analysis using high-performance liquid chromatography demonstrated high levels of the excitatory amino acids, aspartate and glutamate, in the retinas of congenitally blind chicks at the time of photoreceptor degeneration. Concentrations of aspartate were about 2 times higher in blind chicks than in retinas of age-matched sighted chicks that were carriers for the genetic defect. Glutamate levels were similar in blind chicks and carriers at 1 day of age, but doubled and tripled sighted chick values at 1 week and 2 weeks of age in blind chick retinas. Light microscopic immunocytochemistry using antibodies that recognize aspartate and glutamate revealed increased levels of these two amino acids specifically in the photoreceptor layer of blind chicks. This report is the first to demonstrate high endogenous levels of excitatory amino acids associated with a hereditary degeneration of photoreceptor cells.

Hereditary retinal degeneration in the Rhode Island Red chicken. I. Histology and ERG

Hereditary blindness in Rhode Island Red chickens was analyzed at various post-hatching stages by light microscopy and electrophysiological recordings. At the time of hatching the retina of affected chicks appeared morphologically normal and identical to that of control, non-affected chicks. Whereas the electroretinographic (ERG) response to light stimulus in normal chicks was near the adult level at the time of hatching, no ERG either under light- or dark-adapted conditions was measurable in affected chicks at any stage examined. Photoreceptor cells of affected animals were seen to undergo degenerative changes after about one week post-hatching. Decrease in number of outer segments, spaces between inner segments and large spaces in the outer nuclear layer were apparent by Day 10. By Day 21, most of the photoreceptor inner segments appeared swollen, and the decrease in number of outer segments and photoreceptor nuclei was noteworthy. By the end of the second month no outer segments were seen and the majority of identifiable inner segments were from cones, a larger proportion than normally present being double cones. By six months, very few photoreceptor inner segments and nuclei remained; most inner segments were deformed and diminutive but usually contained a clearstaining oil droplet characteristic of the principal member of the double cone. In all stages after one week of age, pycnotic nuclei and thinning of inner retinal layers accompanied photoreceptor degeneration. In all specimens examined, degeneration of retinal cells was more pronounced in the superior central retina than in the periphery. Pathological changes were frequently also noted in the pigment epithelium overlying degenerating retina. Because the chick retina is well developed at birth, contains a fovea and a significant cone population and because cones (particularly one specific type) survive rods, we believe that this congenitally-blind chicken may be a useful model for studies on human hereditary retinal degenerations.

Early expression and localization of rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) in the developing fetal bovine retina

Differentiation and maturation of the photoreceptor outer segments are key steps in the development of the visual system. Morphological studies presented here show that the cow and human are nearly identical in the timing of outer segment appearance during fetal development, implying that the bovine retina is a good model system for the final stages of human photoreceptor development. To study photoreceptor maturation, rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) were quantified by ELISA in a developmentally staged series of fetal bovine retinas. In addition, their localization within these retinas was determined by immunogold electron microscopy. Rhodopsin, as detected by antibodies directed against either the N- or C-terminal portions of the molecule, is first found at about 5.5 months gestation. It is first detected on the plasma membrane of the immature cilia and on the earliest emergent outer segment membrane, even before organized disk membranes are apparent. In contrast, whereas rhodopsin levels and outer segments are nearly undetectable before 5 months gestation, IRBP accumulates to a significant level (4-5% of the adult) as early as 3 months gestation. Immunogold electron microscopy confirmed this finding, with localization of IRBP predominantly in the subretinal space.

Neurotransmitter-specific identification and characterization of neurons in the all-cone retina of Anolis carolinensis II: Glutamate and aspartate

Immunocytochemical and autoradiographic methods were used to identify neurons in the pure cone retina of the lizard (Anolis carolinensis) that are likely to employ glutamate (GLU) or aspartate (ASP) as a neurotransmitter. GLU immunocytochemistry demonstrated high levels of endogenous GLU in all cone types and numerous bipolar cells. Moderate GLU levels were found in horizontal and ganglion cells. Müller cells and most amacrine cells had very low GLU levels. GLU immunoreactivity (GLU-IR) in the cones was present from the inner segment to the synaptic pedicle. A large spherical cell type with moderate GLU-IR was identified in the proximal inner plexiform layer (IPL). These cells also contain ASP and have been tentatively identified as amacrine cells. Uptake of [3H]-L-GLU labeled all retinal layers. All cone types and Müller cells sequestered [3H]-D-ASP, a substrate specific for the GLU transporter. Anti-ASP labeling was observed in cones, horizontal cells, amacrine cells, and cells in the ganglion cell layer. ASP immunoreactivity (ASP-IR) in the cones was confined to the inner segment. One ASP-containing pyriform amacrine cell subtype ramifying in IPL sublamina b was identified. Analysis of GLU-IR, ASP-IR, and GABA-IR on serial sections indicated that there were two distinct populations of horizontal cells in the Anolis retina: one containing GABA-IR, GLU-IR, and ASP-IR; and another type containing only GLU-IR and ASP-IR. Light GLU-IR was frequently found in GABA-containing amacrine cells but ASP-IR was not. The distinct distributions of GLU and ASP may indicate distinctly different roles for these amino acids. GLU, not ASP, is probably the major neurotransmitter in the cone-bipolar-ganglion cell pathway of the Anolis retina. Both GLU and ASP are present in horizontal cells and specific subpopulations of amacrine cells, but it is unclear if GLU or ASP have a neurotransmitter role in these cells.

Macular disease in related rhesus monkeys.

During (January) 1986–(May) 1988, we examined 272 eyes in 136 rhesus monkeys in the closed Cayo Santiago colony of the Caribbean Primate Research Center of the University of Puerto Rico. Seventy-eight eyes were less than 10 years of age. One hundred and ninety-four were aged 10–28 years. The fundi were examined and photographed. Fluorescein angiography was performed in some eyes. Selected cases were evaluated for ‘acuity’ loss by recording of pattern-evoked retinal and cortical signals. Light and electron microscopy were used to evaluate the pigment epithelium of some animals. Thirty-eight percent of all eyes had posterior pole drusen. Incidence was highly age-related. When late-stage lesions were found, we did not see neovascularization, but late hyperfluorescence was consistent with degenerative scarring and atrophy. Electrophysiology demonstrated moderately reduced acuity in the presence of numerous macular drusen. Electrooculograms were low normal. Histopathology showed changes identical to those reported in human age-related macular degeneration. No eyes less than 10 years of age had confluent drusen or disciform-like lesions. The incidence of drusen in samples of some social groups was much higher than others.

Degenerative Changes in Maculas of Rhesus Monkeys

In the hope of identifying an animal model for age-related macular degeneration (AMD) we undertook a pilot investigation of aged rhesus monkeys. Twenty-nine monkeys from a seminatural colony were examined at the Caribbean Primate Research Center. Macular drusen were found in 74% of the monkey eyes. Alterations of the retinal pigment epithelium within the macula were noted in 45% of the eyes. Fluorescein angiography in selected animals revealed window defects consistent with drusen. None of this sample showed the exudative form of AMD or disciform scarring. One typical monkey underwent special studies including measurement of visual resolution by electrophysiological study of the retinal and visual cortex. Application of human criteria to this animal supported the diagnosis of early AMD. Histopathologic study of one eye by transmission electron microscopy confirmed the presence of drusen with nearly identical ultrastructural features to those found in the human pigment epithelium in AMD.

Macular degeneration in a community of rhesus monkeys. Ultrastructural observations.

The eyes of 29 aged adult, (mean age, 20 years) rhesus monkeys were examined for the presence of age-related macular degeneration (AMD). This sample represented approximately 25% of the aged population in the seminatural colony at the Caribbean Primate Research Center (CPRC) of the University of Puerto Rico. Approximately 75% of the animals examined had drusen in the posterior pole. Ultrastructural analysis was used to determine whether the pathologic alteration of Bruchs membrane and drusen in the colony resembled those noted in aged or AMD-afflicted human retinas. There were abnormalities in all layers of Bruchs membrane. Deposits of heterogeneous material, comprised of membranous, granular, and cellular components, were seen in both the inner collagenous zone (ICZ) and the outer collagenous zone (OCZ). Accumulation of this drusenoid material in the ICZ produced a scalloping of the basal border of the retinal pigment epithelium (RPE). Dense bodies were seen in both Bruchs membrane and RPE cytoplasm near the basal infoldings. Cytoplasmic processes, as well as whole cells, were seen with regularity within the drusenoid material. In one case there was a cell with a basement membrane crossing the middle elastic layer of Bruchs membrane. These changes are consistent with those reported in human aging and AMD. Aged individuals in this colony appear to be predisposed to macular degenerative changes and may prove to be an invaluable animal model for studying AMD in humans.

Ultrastructural changes in the retinal pigment epithelium of congenitally blind chickens

Pathological changes in the retinal pigment epithelium (RPE) in a strain of chickens having hereditary blindness and retinal degeneration were described at the ultrastructural level. Photoreceptors in the retinal degenerate (rd) chicken had previously been noted to degenerate within a week after hatching. Affected chicks have neural retinas that are morphologically comparable to normal animals prior to that time despite an obvious lack of vision. In the present study, no pathological changes were noted in rd RPE prior to the time of photoreceptor degeneration. However, while mitochondria in the normal chicks RPE underwent diurnal changes in morphology within a few days of hatching, pleomorphic or ring mitochondria were not seen with high frequency in the rd chick. After photoreceptors began degenerating, changes were seen in the rd RPE. By 2 weeks of age, we noted a reduction in the depth and number of basal infoldings, an increase in number and size of autophagic vacuoles and large whorls of membranous material within rd RPE cells. Membranous debris and what appeared to be broken off outer segments were seen in the subretinal space at that time. These phenomena became more prominent and prevalent with time. In 3-4 week old specimens, nearly intact outer segments were seen within RPE cytoplasm. At the same time very few intact outer segments were present on photoreceptors. After this time degenerative changes were seen in the RPE: a thinning of cells (apical to basal cell width), spreading out of cells (increased distance between intercellular junctional complexes), hypopigmentation of cells and presence of free cells in the sub-retinal space. Some RPE cells appeared in a rounded up configuration, bulging into the subretinal space and making junctional complexes with remaining photoreceptor inner segments or Mueller cell processes. Many RPE cells did appear to maintain their phagocytic abilities, as evidenced by presence of many microvilli and pinocytotic vacuoles in the apical cytoplasm.

Neurotransmitter-specific identification and characterization of neurons in the all-cone retina of Anolis carolinensis, I: Gamma-aminobutyric acid.

The inhibitory amino-acid neurotransmitter, gamma-aminobutyric acid (GABA), was localized in the pure cone retina of the lizard Anolis carolinensis by autoradiographic and immunocytochemical techniques. Uptake of [3H]-GABA labeled horizontal cells, amacrine cells, numerous cells in the ganglion cell layer, both plexiform layers, and the nerve fiber layer. Label in the inner plexiform layer showed distinct lamination. The pattern of GABA immunoreactivity was similar to the pattern of [3H]-GABA uptake, although some differences, particularly in labeling of amacrine and ganglion cells, were observed. Immunocytochemistry revealed endogenous stores of GABA in a set of horizontal cells, amacrine cells, and cells in the ganglion cell layer. Both plexiform layers were labeled by the GABA antisera. Labeling in the inner plexiform layer (IPL) was highly stratified and GABA-immunoreactive strata were present in both sublaminae a and b. Six subtypes of conventionally placed GABA-immunoreactive amacrine cells and one displaced amacrine cell subtype were identified. Three of the six conventional amacrine cell subtypes were of pyriform morphology and three subtypes were of multipolar morphology. GABA-immunoreactive interstitial cells also were observed. Under certain conditions the GABA antiserum labeled the cones. Etching the resin eliminated cone labeling, suggesting that GABA in the cones is present in a labile pool, unlike GABA in horizontal or amacrine cells, or the observed labeling was not due to endogenous GABA. Cones did not demonstrate [3H]-GABA uptake.

Alligator rhodopsin: Sequence and biochemical properties

We sequenced selected peptides of alligator rhodopsin that accounted for about half of the total protein. These sequences were confirmed when the total primary structure of alligator rhodopsin was deduced from the cDNA sequence. Differences in the amino-terminal region, compared to that of bovine rhodopsin, account for failure of cross-reactivity of several anti-bovine rhodopsin monoclonal antibodies. Differences in the carboxyl-terminal region give rise to limited antibody cross-reactivity and may also account for a slightly reduced ability of alligator rhodopsin to be phosphorylated by bovine rhodopsin kinase. Alligator rhodopsin regenerates much faster than bovine rhodopsin. The pseudo-first-order rate constant for alligator rhodopsin regeneration is approximately 25 times that of bovine. Phylogenetic analysis of 17 rhodopsin sequences indicates that the alligator is more closely related to the chicken than to the other species examined.