Opsin gene expression during early and late phases of retinal degeneration in rds mice.

Opsin mRNA levels, opsin synthetic rates and localization of opsin were studied throughout the photoreceptor's life span in the rds mice. Mutant mice 11 days to 11 months old were investigated. Opsin mRNA levels were studied by means of northern blot analysis. Opsin synthesis was measured by incorporation of [35S]methionine into newly synthesized opsin in vitro. Distribution of opsin in the retina was determined by immunoelectron microscopy. Opsin mRNA was detected in young as well as old retinas, and opsin synthesis could be detected at early phases of degeneration but not in late phases. The absence of opsin synthesis in older rds mice might be due to translational down-regulation or some other defect in the capacity to synthesize opsin. In young mice, opsin was detected in the subretinal space in opsin-laden vesicular membranes: such membranes were absent from retinas of older mice. This disappearance parallels the cessation of opsin synthesis and the consequent failure to deliver opsin to the subretinal space in retinas from older mice. Immunochemical analysis revealed the presence of small amounts of opsin in all retinas up to 11 months of age. Immunoelectron microscopy localized the residual opsin, mostly to the plasma membrane which envelops the nuclei and synaptic terminals. These opsin molecules might be a consequence of very low levels of opsin synthesis, too low to be detected by our assays, or may have been synthesized at an earlier age and retained in the plasma membrane of the old mutant photoreceptors.

Characterization of vasoactive intestinal peptide receptors in retina.

Vasoactive intestinal peptide (VIP) is a neuropeptide having a wide range of effects on a large number of tissues. To gain insight into the role VIP plays in retinal function, VIP receptors in bovine retinal membranes were analyzed in competition binding assays and by affinity labeling studies and compared to VIP receptors in rat liver membranes. In both membrane preparations, high affinity VIP binding sites (KD approximately 1 nM) were detected. Secretin and glucagon, each having close structural homology to VIP, were found to have negligible effects on [125I]VIP binding in retina. In contrast, secretin (KD = 70 nM) was modestly effective in inhibiting [125I]VIP binding to rat liver membranes. Affinity labeling analysis revealed a VIP binding site of 59 kDa in both bovine retinal and rat liver membranes. Digestion of affinity-labeled receptor proteins with endoglycosidase F generated final cleavage products of approx. 45 kDa for both receptors. These results indicate that the retina expresses a high affinity, highly selective VIP receptor thereby supporting a specific function for VIP in this tissue.

Localization of immunoreactive cholecystokinin precursor to amacrine cells and bipolar cells of the macaque monkey retina.

We used antisera that recognized precursors of the neuropeptide cholecystokinin extended at the carboxyl terminus in an immunocytochemical study of the macaque retina. A subpopulation of bipolar cells with long, obliquely oriented dendrites was labeled. Their axons terminated exclusively in the fifth stratum of the inner plexiform layer, where they contacted processes of amacrine and ganglion cells. Based on their morphology, these cells appeared to be the type that contacts short-wavelength cones selectively. Two types of amacrine cells were also labeled, and processes from both types formed dense plexuses in the second and fourth strata of the inner plexiform layer. The majority of their synaptic connections were with other amacrine cells, but they had more contacts with bipolar cell axons and retinal ganglion cell dendrites than any other peptidergic cells in the macaque retina. We studied extracts of macaque retina with gel-filtration chromatography and radioimmunoassays to confirm our immunohistochemical results. We found cholecystokinin octapeptide and other immunoreactive forms that were amidated at their carboxyl termini and were therefore likely to be biologically active. Unlike most other regions of the CNS, however, the retina had relatively low concentrations of amidated forms, and forms with extended carboxyl termini that are presumably their precursors were far more abundant. These findings suggest that the rate of cholecystokinin synthesis in the retina is quite high, as we would expect if the peptide were found in tonically active neurons.

Analysis of acidic metabolites of biogenic amines in bovine retina and vitreous and aqueous humour by gas chromatography-negative ion chemical ionisation mass spectrometry.

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.

Novel localization of a G protein, Gz-alpha, in neurons of brain and retina.

Recently, a cDNA coding for a novel G protein alpha-subunit, Gz-alpha, was isolated from a human retinal cDNA library and shown by Northern blot analysis to be expressed at high levels in neural tissues. We have prepared affinity-purified antibodies specifically directed against synthetic Gz-alpha peptides and employed immunohistochemical methods to map the localization of Gz-alpha in human, bovine, and murine retina and brain. By light microscopy, Gz-alpha was localized to the cytoplasm of neurons, with predominant reactivity in ganglion cells of the retina, Purkinje cells of the cerebellum, and most neurons of the hippocampus and cerebral cortex. Reactivity was confined to perikaryon, dendrites, and a very short segment of proximal axons, except for the retinal ganglion cells, in which the axons in the nerve fiber layer showed intense Gz-alpha immunoreactivity proximal to the lamina cribrosa. Pre-embedding immunoelectron microscopy demonstrated the presence of focal Gz-alpha immunoreactivity on the nuclear membranes, endoplasmic reticulum, and plasma membranes of Purkinje cell perikarya and in association with microtubules in their proximal dendrites. Subcellular fractionation studies confirmed the association of Gz-alpha with plasma and intracellular membranes. The localization of Gz-alpha and its unique amino acid sequence suggest that it may have a specialized function in neural tissues.

Localization of insulin-like growth factor-1 binding sites in the embryonic chicken eye.

Insulin-like growth factor-1 (IGF-1) binding sites were localized in the embryonic chicken lens, retina, and retinal pigmented epithelium (RPE) with the use of autoradiography. Each of the ocular tissues exhibited specific binding of radiolabeled ligand. Labeling occurred over the entire surface of the lens from 6-day-old embryos, including the epithelium, basal ends of the fiber cells, and the annular pad. There was relatively little labeling over the lens nucleus. A similar pattern was seen in lenses from 19-day-old embryos. In isolated retinas from embryos of this age, the inner and outer plexiform layers were most heavily labeled. In the 19-day-old RPE, only the apical surface of the cells was heavily labeled. Electron microscopic studies revealed an apical layer of membranous material that may represent outer photoreceptor segments that remained attached to the RPE during dissection. It was uncertain, therefore, whether the IGF-1 was binding to sites on the RPE or to these membrane fragments.

Effects of long-term diabetes and galactosaemia upon lens and retinal mRNA levels in the rat.

The levels of mRNAs encoding the alpha 1 chain of collagen IV and the B1 chain of laminin were assayed in the lenses and retinas of long-term (28-week) diabetic and galactosaemic rats in order to gain some insight into the effects on basement membrane (BM) synthesis in these tissues. mRNAs coding for beta-actin, glucose transporter protein and the alpha 2 catalytic subunit of Na+,K(+)-ATPase were also assayed to determine whether any effects on BM-coding mRNA levels were specific. Long-term diabetes had no significant effect on the levels of alpha 1 (IV) collagen mRNA but caused a significant reduction in the laminin B1 message in the lens. In the same samples, the level of the glucose transporter protein mRNA was found to be elevated significantly in the diabetic tissue, whereas the mRNAsen coding beta-actin and alpha 2 Na+,K(+)-ATPase were unaffected in comparison with age-matched controls. Long-term galactosaemia resulted in significant increases in the levels of all mRNAs assayed when expressed per micrograms total RNA used for each analysis. However, this effect appeared to be due to a specific loss of ribosomal RNA from these severely cataractous lenses. When related to the beta-actin mRNA internal control, the levels of mRNA in the galactosaemic lenses were very similar to that found in the diabetics. Laminin B1 mRNA levels were decreased significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Interplexiform cells of the goldfish retina.

Dopaminergic and glycinergic interplexiform cells (IPCs) in the goldfish retina were impregnated by using two new Golgi protocols. The two cell types have markedly different morphological characteristics: Dopaminergic IPCs have primary dendrites that descend into and stratify in the inner plexiform layer, where they give rise to processes that project to the outer plexiform layer. Conversely, glycinergic IPCs have primary dendrites that ascend to the outer plexiform layer and from this dendritic arbor, many processes then project into the inner plexiform layer. The apparent coverage of dopaminergic IPCs is almost four times that of glycinergic IPCs. Even so, the coverage of each glycinergic IPC in the outer plexiform layer allows it to provide an accurate copy of the S-space to the inner plexiform layer. Considering the known GABAergic and glycinergic synaptologies in the inner plexiform layer, the glycinergic IPC must form a major element in the retinal circuitry of the goldfish.

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues.

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.

Variation in epiretinal membrane components with clinical duration of the proliferative tissue.

Immunohistochemical investigations were conducted on surgically excised epiretinal membranes to determine how cellular and extracellular components of proliferative vitreoretinopathy membranes change with time. Specimens of less than four months' duration contained a significantly higher proportion of retinal pigment epithelial cells than later membranes. No association was found between membrane duration and the content of collagen subtypes I to IV and laminin, but 'early' specimens contained significantly more fibronectin than did 'late' membranes. Fibronectin and collagens I, III, and IV showed a variable relationship with glial cells and were most consistently associated with retinal pigment epithelial and fibroblast-like cells. These observations may explain some of the surgical features of epiretinal membranes.

Use of the biotin-avidin system for labelling, isolation and characterization of neural cell-surface proteins.

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions.

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.

Localization of cellular retinoic acid-binding protein to amacrine cells of rat retina.

Monoclonal antibodies to performic acid-oxidized cellular retinoic acid-binding protein (CRABP) from bovine retina were prepared by fusion of spleen cells from immunized mice with mouse myeloma cells. Five antibodies were studied in detail. It was established by ELISA that the antibodies react with CRABP and oxidized CRABP, but not with other oxidized or unmodified retinoid-binding proteins. Competitive ELISA demonstrated that the antibodies react with heat-denatured antigen but not with native protein. Western blotting and immunostaining, following sodium dodecyl sulfate gel electrophoresis, provided evidence for recognition of a single component in retinal supernatants whose staining is prevented by preabsorption of the antibody with heat-denatured CRABP. The insoluble fraction from a retinal homogenate contains residual CRABP and two weakly-reacting components, whose staining is not affected by preabsorption of the antibody with antigen. Each antibody produces the same staining pattern on cryostat sections of rat retina by indirect immunofluorescence. Amacrine somata on both sides of the inner plexiform layer are labeled, as well as processes forming laminae within this layer. These results suggest that retinoic acid may play a functional role in the inner retina.

Neural cell adhesion molecule (NCAM) in adult vertebrate retinas: tissue localization and evidence against its role in retina-pigment epithelium adhesion.

The presence of neural cell adhesion molecule (NCAM) was examined in the neural retina, interphotoreceptor matrix (IPM), and retinal pigment epithelium (RPE) of adult bovine and frog eyes. Using polyclonal antibodies raised against adult isoforms of NCAM. Western blot analyses revealed the presence of NCAM in the neural retina, but not in the IPM or RPE of these species. As a control, Western blot analysis was used to demonstrate the presence of interphotoreceptor retinoid-binding protein (IRBP) in the IPM preparations. NCAM immunoreactivity was detected by light microscopic immunocytochemistry primarily in the plexiform layers and nerve fibre layer of the frog retina. Minor immunoreactivity was also detected in the inner and outer nuclear layers, but there was no detectable NCAM immunoreactivity in the IPM, outer segments, or RPE. These results indicate that NCAM is not a likely participant in the process of retina-RPE adhesion in the adult eye.

Glutamate-like immunoreactivity in the retina of a marine teleost, the dragonet.

The localisation of endogenous glutamate in the dragonet retina was investigated by light microscopic postembedding silver-enhanced immunogold labeling after incubation with an anti-glutamate antiserum. Rod and cone inner segments and synaptic terminals, as well as the inner plexiform layer, are moderately labeled. Bipolar cells and ganglion cell bodies show strong labeling. In the dorsal inner plexiform layer, the levels with square-patterned bipolar synaptic boutons can be identified by their prominent glutamate-immunoreactivity. These results support the idea that the majority of the neurons that constitute the direct, centripetal pathways through the retina use glutamate as their neurotransmitter.

Ion-selective microelectrodes suitable for recording rapid changes in extracellular ion concentration.

A method for fabricating double-barrel, ion-selective microelectrodes with fine tips (0.5-1.5 microns) and rapid response times is described. When made into K(+)-selective microelectrodes, the electrodes respond to changes in [K+]o with a time constant of 70-95 ms. The electrical response of these electrodes to common-mode voltages can be made to have a time constant of less than 2 ms, which minimizes electrical artifacts from field potentials. The application of these microelectrodes to the measurement of rapid, transient changes in retinal [K+]o is presented.

Ultrastructural localization of extracellular matrix components in human retinal vessels and Bruch's membrane.

We have used the electron microscopic immunogold technique to localize precisely various extracellular matrix components in human retinal vessels and Bruch's membrane. Collagen types IV and V, laminin, and heparan sulfate proteoglycan core protein were localized in basement membranes of retinal capillaries. In addition to the capillary basement membrane components, collagen types I and III and fibronectin were found in the basement membranes of retinal arterioles and venules. The basement membranes of choriocapillaries and retinal pigment epithelial cells in Bruch's membrane also showed a similar distribution of the retinal capillary basement membrane components. Both the inner and outer collagenous layers of Bruch's membrane contained collagen types I and III along with fibronectin, whereas collagen type VI was mostly limited to the central elastic lamina. The precise localization and distribution of various extracellular matrix components in human retinal vessels and Bruch's membrane may be important for understanding their normal function as well as their alteration in disease.

Chromatographic characterization of vasoactive intestinal polypeptide in guinea pig and rhesus monkey eyes.

Acid extracts of guinea pig and rhesus monkey anterior uvea, choroid and retina contain immunoreactive VIP. By reversed phase high performance liquid chromatography, the immunoreactive material from these ocular tissues elutes at a position similar to synthetic porcine VIP. Only in the guinea pig anterior uvea is a second smaller peak detected. By size exclusion high performance liquid chromatography, the peptide in the monkey anterior uvea, choroid and retina elutes in the identical position as the synthetic porcine VIP standard with an apparent molecular weight of 3450 daltons. We conclude that a single form of VIP, chromatographically similar to the porcine standard, is the predominant form of the peptide in the eye of guinea pig and rhesus monkey.

Purification and partial characterization of a novel cellular retinol-binding protein, type three, from the piscine eyes.

A novel cellular retinol-binding protein, termed type three (CRBP III), was isolated from eyes of the bigeye of tuna. CRBP III showed a molecular weight of 15,400, an isoelectric point of 4.80, alpha 1-mobility in electrophoresis, and a lambda max of 350 nm. All-trans-retinol, the endogenous ligand, could be competitively displaced by retinoic acid but not by retinal. CRBP III was differentiated from purified piscine and rat cellular retinol-binding proteins (CRBP) and cellular retinoic acid-binding proteins (CRABP) by its amino-acid composition, electrophoretic mobility, fluorescence spectra and ligand-binding specificity.