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Topographical organization of the dopamine-dependent adenylate cyclase of the chick embryo retina.

Retinas obtained from 9- and 16-day-old chick embryos and 5-day-old post-hatched chicken were analyzed with regard to the topographical distribution of the dopamine-stimulated adenylate cyclase system. The retinas were sectioned into 4 quadrants, namely ventroanterior, dorsoanterior, dorsoposterior and ventroposterior, taking the beak and the choroid fissure as references. Dopamine (0.1 mM) elicited the accumulation of cAMP in all 4 portions of tissue studied. However, when the ratio between the dopamine-stimulated vs the basal level of cAMP of 9-day-old embryo retina were compared in the different tissue portions, we observed that in the dorsoposterior quadrant the ratio was 15.5 while in the ventroanterior quadrant the ratio was approximately 6. The other two portions showed intermediate values. The same pattern was observed in retinas from 16-day-old embryos. However, the ratio between the dopamine-stimulated vs the basal cAMP levels were 4.6 and 3 in the dorsoposterior and ventroanterior quadrants respectively. Further, the retina responsiveness to dopamine from post-hatched chicken was evenly distributed in the tissue, showing a stimulated/basal cAMP ratio of approximately 2 in all 4 quadrants studied. Our results show that the dopamine-dependent cAMP accumulation of the chick retina is unevenly distributed in the embryonic tissue and tends to homogeneity as the tissue differentiates.

Adenylyl Cyclases↗

D1 dopamine receptors in neurite regions of embryonic and differentiated retina are highly coupled to adenylate cyclase in the embryonic but not in the mature tissue.

[3H]SCH 23390 binds stereospecifically and with high affinity to D1 dopaminergic receptors in the developing chick retina. Autoradiographic experiments revealed that in retinas from 3-day-old chicken and embryos with 12, 14 and 16 days of development, specific labeling of [3H]SCH 23390 was mainly observed over the plexiform layers of the tissue, showing that dopaminergic D1 receptors are localized in retina cell neurites since the initial stages of neurite formation. The total number of [3H]SCH 23390 binding sites increased 5-fold during the differentiation of the retina, while the dopamine-dependent cyclic adenosine monophosphate (AMP) accumulation was significantly decreased. Consequently, the ratio between dopamine-dependent cyclic AMP accumulation and [3H]SCH 23390 binding sites decreased 10-fold as retina differentiated, indicating that a significant portion of D1 receptors in retinas from adult chicken are not effectively coupled to adenylate cyclase molecules.

Adenylyl Cyclases↗

Developmental immunoreactivity for GABA and GAD in the avian retina: possible alternative pathway for GABA synthesis.

Although the distribution of GABAergic neurons in chick retina has been previously described by several investigators, the early appearance of these neurons has not been reported. In the present study immunohistochemical methods were used to localize GABAergic neurons with antisera to both GABA and its synthesizing enzyme, glutamate decarboxylase (GAD), in embryonic chick retina at several stages of development and beyond hatching. GABA-positive neuroblast-like cells were clearly detected in retinas as early as embryonic day 6. In contrast, GAD-containing cells were not observed in retinas until embryonic day 10. These findings indicated that immunocytochemically detectable amounts of GAD were not present in young GABAergic cells. Our data on the developmental appearance of GABA and GAD immunoreactivities are consistent with previous biochemical data for the development of GABA concentration and GAD activity in the chick retina. Together, these data suggest that retina cells from the early stages of development may synthesize GABA from an alternative pathway in which the most likely precursor is putrescine.

Animals↗

Tyrosine hydroxylase-immunoreactive elements in the distal retina of Bufo marinus: a light and electron microscopic study.

Tyrosine hydroxylase-immunoreactive elements in the distal retina of Bufo marinus were investigated using light and electron microscopic immunocytochemistry. At the light microscopic level, immunoreactive somas were seen in the proximal part of the inner nuclear layer, and immunoreactive processes projected both to the inner and outer plexiform layers. In some instances stained axon-like processes traveled from the inner plexiform layer, across the inner nuclear layer to the distal retina. Immunolabeled elements formed basket-like structures around the photoreceptor inner segments. At the ultrastructural level immunostained fibers were observed in close contact with the necks, lateral walls, bases and the outer surfaces of rod outer segments. Synaptic specializations were neither observed at rod contacts nor at other possible contact sites such as bipolar dendrites and horizontal cell somata and processes in the outer plexiform layer. In contrast, synaptic specializations between immunolabeled profiles and amacrine, bipolar and ganglion cells were regularly present in the inner plexiform layer. These findings suggest that a population of dopaminergic interplexiform cells is present in the Bufo retina and sends axon-like processes towards the distal retina. It is assumed that dopamine is probably released non-synaptically from the immunolabeled terminals in the distal retina influencing rods directly, by which the quality of photopic vision is enhanced in the anuran retina.

Animals↗

Endogenous opioids regulate cell proliferation in the retina of developing rat.

The role of endogenous opioids and opioid receptors (endogenous opioid systems) in modulating cell proliferation in the developing mammalian retina was examined in 1-day-old rats. In contrast to a labeling index (LI) of 35.8% in control animals, administration of the opioid peptide [Met5]-enkephalin (100 micrograms/kg) significantly reduced (10.6%) the proportion of cells incorporating [3H]thymidine; concomitant injection of 1 mg/kg naloxone blocked the inhibitory effects of [Met5]-enkephalin on cell division. Naloxone (1 mg/kg) alone did not alter the LI. The interruption of endogenous opioid-opioid receptor interaction by naltrexone (50 mg/kg), a potent opioid antagonist, was accompanied by a significant increase (6.4%) in the LI relative to control levels. Immunocytochemical experiments revealed the presence of enkephalin-like immunoreactivity, with staining of the cortical cytoplasm of proliferating and differentiating retinal cells recorded; no immunoreactivity was noted in the adult retina. In vitro autoradiography using 125I-[Met5]-enkephalin indicated that [Met5]-enkephalin binding sites were localized to the developing retina; no binding of the radiolabeled ligand was recorded in the adult retina. These results demonstrate the presence of growth-related endogenous opioids and opioid receptors in the developing mammalian retina, but not in adult retina, and suggest that endogenous opioids serve as natural inhibitory trophic factors that tonically regulate cell proliferation.

Animals↗

Cholinergic and GABAergic neurons occur in both the distal and proximal turtle retina.

Turtle retinas were processed immunocytochemically and histochemically to detect the presence of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and glutamate decarboxylase (GAD). We observed cholinergic and gamma-aminobutyric acid (GABA)ergic neurons in the proximal retina, as expected, and in the distal retina as well. ChAT immunoreactivity in the distal retina was observed within the axons and pedicles of numerous cone photoreceptors, suggesting that a population of turtle cone photoreceptors uses ACh as a neurotransmitter. Type L2 horizontal cells were immunoreactive for GAD, and their dendrites invaginated into cone pedicles. AChE histochemistry revealed processes within the outer plexiform layer which formed a loosely organized lattice. In the proximal retina, labeling for ChAT and GAD was similar to that reported by previous investigators. Processes from ChAT-labeled amacrine cells in the inner nuclear layer formed a stratum within the distal inner plexiform layer (IPL) (at 16-21% relative IPL depth), and processes from ChAT-labeled amacrines in the ganglion cell layer formed a proximal ChAT stratum (at 55-58% relative IPL depth). In addition, six AChE-labeled bands and five GAD-labeled bands were observed within the IPL of stained retinas. Therefore, we determined that the two broadest AChE-labeled bands and the two broadest GAD-labeled bands overlapped the two labeled ChAT strata. The evidence for cholinergic and GABAergic processes in both the inner plexiform layer and the outer plexiform layer, combined with electrophysiological evidence from other investigators, raises the possibility that distal retinal neurons may be involved in the encoding of directional information.

Acetylcholine↗

The occurrence of three calcium-independent protein kinase C subspecies (delta, epsilon and zeta) in retina of different species.

The localisation and immunochemical identification of three different forms of calcium-independent protein kinase C (PKC-epsilon, PKC-delta and PKC-zeta) in retinas of different species were analysed by immunohistochemistry and SDS-PAGE/Western blotting, respectively. More than one component of different molecular weights reacted with the polyclonal antibodies in all retinal samples though in all instances a component of molecular weight corresponding to the individual PKCs was recognised and could be eliminated or reduced by preincubating the primary antibodies with the peptides used to generate the antibodies. PKC-zeta immunoreactivity was exclusively associated with the inner segments of the photoreceptors in both mammalian (guinea-pig, rabbit, rat) and non-mammalian (goldfish, chick) retinas. PKC-epsilon immunoreactivity is present in bipolar cells, particularly in their terminals of mammalian and goldfish retinas. In the chick retina immunoreactivity for this enzyme and for PKC-delta was with the inner segments of the photoreceptors. The Müller cells in mammalian retinas and a sub-population of ganglion cells in the goldfish retina exhibited positive immunoreactivity for PKC-delta. The immunoreactivities for all the PKC isoenzymes were eliminated or drastically reduced when the primary antibodies were first preincubated with the peptides used to generate the antibodies.

Amino Acid Sequence↗

Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina.

We have used monoclonal antibodies against each of the major mammalian brain microtubule-associated proteins (MAPs), MAP1, MAP2, MAP3, MAP5, and tau, to study the timing of appearance and the cytological distribution of these proteins during the development of the rat retina. Western blots of adult rat retina reveal MAPs that are characteristic of embryonic brain, i.e., MAP5 and the low-molecular-weight forms of MAP2 (MAP2c) and tau (juvenile tau). At the onset of neuronal differentiation within the embryonic retina, MAP5, MAP3, MAP2c, and tau are found in the perikarya or extending axons of ganglion cells. High-molecular-weight MAP2, a dendrite marker, does not appear in the retina until the second day of postnatal development, when ganglion cell dendrites ramify within the inner plexiform layer. MAP1, which is characteristic of adult brain, does not appear in the retina until 1 week after birth, and is limited to ganglion cells and their processes. In the adult retina, MAP5 and MAP2c are concentrated within the inner segments and cell bodies of photosensitive cells, whereas tau is found in horizontal cells and more internal cell layers. Since photosensitive cells are unique among retinal neurons in their constant regeneration of their primary processes, the photoreceptive outer segments, both MAP5 and MAP2c appear not only to be involved in events associated with the embryonic differentiation and growth of neurites, but also in process regeneration in adult neurons that maintain some embryonic characteristics.

Age Factors↗

Glutathione oxidation in retina: effects on biochemical and electrical activities.

This study investigates the possible role of glutathione (GSH) in defending the retina against oxidative damage. Freshly excised rat retina was found to contain 1.2 mumol/g wet wt GSH and an undetectable level of oxidized glutathione (GSSG). Whole retinas were either incubated or superfused with various concentrations of the GSH-oxidant diamide in order to study the effects of oxidation of GSH on the activity of the hexose monophosphate shunt (HMS) and on the receptor potential of the retina. It was found that exposure of the retina to diamide produced a stimulation of HMS activity up to 26-times that of the control. Significant changes in GSH content and receptor potential were observed at concentrations of diamide that produced more than a 5.4-fold stimulation of HMS activity. The diamide-induced electrical alterations included an increase in latency and peak time of the receptor potential, a delay in the onset of the off response and an increase in the time required for the potential to return to the baseline. It was found that nearly 80% of GHS could be regenerated and that most of the electrical effects of diamide could be reversed by superfusion with normal medium. The results indicate that the retina possesses an active system for maintaining GSH in the reduced state and that this may be essential for the normal function of this tissue.

Animals↗

Cyclic nucleotide distribution in identified layers of suprafused rabbit retinas.

To assess the effects of pharmacologic agents on metabolite levels in identified retinal layers we have devised an apparatus that allows suprafusion of everted eye cups (vitreal surface exposed) or isolated retinas (photoreceptor surface exposed), recording of electroretinograms (ERG), and rapid freezing in a configuration which permits obtaining frozen tangential sections of the retina. Samples from identified layers of the freeze-dried sections can be subsequently weighed and processed for biochemistry by Lowry methods (Lowry and Passonneau , 1972), or cyclic nucleotides can be extracted from weighed samples and assayed. To test the usefulness of the apparatus, we have compared levels of guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP), measured in defined layers of suprafused rabbit retinas, to concentrations reported for retinal layers from eyes removed from light- and dark-adapted rabbits and quickly frozen. For most layers the concentrations of cyclic nucleotides were similar in both the suprafused and whole eye preparations. In experimental applications we suprafused dark-adapted eye cups and retinas with a physiological saline whose free calcium level was sharply reduced by chelation, and observed that cGMP levels were elevated only in those retinal layers containing photoreceptor portions. Ethyleneglycol bis-(beta-amino-ethyl ether)N,N'-tetraacetic acid (EGTA) (3 mM) yielded a threefold increase in cGMP in 10 min when applied to the photoreceptor surface of isolated retinas. However, when the vitreal surfaces of everted eye cups were suprafused with calcium-chelated medium for 10 min, a threefold increase in cGMP required 20 mM EGTA, 3 mM EGTA being ineffective. In another study, vitreal surfaces of eye cups or photoreceptor surfaces of retinas were suprafused with physiological saline containing 3 mM 3-isobutyl-1-methylxanthine (IBMX) in order to alter cAMP retinal levels. In both cases, eightfold increases in cAMP were measured in the inner nuclear and inner plexiform layers, while three- to fourfold increases occurred in the outer nuclear and outer plexiform layers. This agent also caused a large increase in cAMP in the pigment epithelium.

1-Methyl-3-isobutylxanthine↗

Oxygen diffusion in the trout retina.

Oxygen tensions were measured in vivo within the different layers of the rainbow trout retina. Oxygen microelectrodes were advanced in 10 micron increments through the retinas and the PO2 measured at each location. Mean retinal PO2 ranged from 124 mmHg at the retinal-vitreal interface to 381 mmHg at the choriocapillaris. These data reaffirm that the cellular layers of the normal trout retina are continually exposed to supra-arterial oxygen tensions that are known to cause toxicity in other species. The intraretinal PO2 gradient was mathematically characterized and results indicate that the in vivo oxygen profile can be described by a two-component exponential function. In order to gain a better understanding of oxygen delivery to the retina, the data were also subjected to an analysis based upon the classical equation for planar diffusion. The trout retina is an excellent model for studying oxygen diffusion in vivo since this tissue is supplied with oxygen from a single source, thereby simplifying the mathematical analysis. Calculations yielded values of 1.86 X 10(-5) and 0.58 X 10(-5) ml O2 min-1 cm-1 atm-1 (at 9 degrees C) for the Krogh permeation coefficient (DS) for the photoreceptor region and the remaining neural retina, respectively.

Animals↗

Spatial distribution of visual pigment and dopamine in the bullfrog retina.

Visual pigments and a neurotransmitter, dopamine, were quantitatively investigated in the retina of adult bullfrog, Rana catesbeiana. The adult bullfrog (body length 15-16 cm, body weight 375 +/- 52 g, n = 10) had 21.4 +/- 4.2 nmol visual pigment and 209 +/- 28 pmol dopamine in retinal areas of 266 +/- 27 mm2. Greater pigment densities were recorded in a semicircular band around the optic disc, extending to the nasal and temporal peripheries of the ventral retina. The area with the highest concentration of visual pigment was found in the middle of the dorsal retina, 3-4 mm dorsal to the optic disc. A high concentration of vitamin A2-based pigment was found in the dorsal quarter of the retina (porphyropsin zone); the zone extended up to the most ventral part along peripheral regions of the retina. There was also a band with higher dopamine concentrations although it was not so prominent as that of the visual pigment; the highest concentration of dopamine was found in the area immediately dorsotemporal to the optic disc. Fluorescence micrography indicated that the distribution pattern of catecholamine-containing amacrine cells paralleled that of the dopamine content. The topographic map of dopamine was slightly different from that of visual pigment in the bullfrog retina.

Animals↗

Evidence of cell loss from the rat retina during senescence.

In order to determine whether neuronal cell death in the retina accompanies senescence, retinas from pigmented ACI rats of various ages were analysed for changes in dimensions, cell densities, DNA content, and rhodopsin levels. Between the ages of 4 and 30 months, there were significant declines in the densities of nuclei in all three nuclear layers of the retina. Over this age span, nuclear density in the outer nuclear layer (ONL) declined by 32-41%, depending on the region of the retina examined, while inner nuclear layer (INL) nuclear density decreased by 25-34%. Over the same age range, the nuclear density in the ganglion cell layer (GCL) declined by 25-28%. The eye increased in size over the 4-30 month age span, particularly between 4 and 12 months. The retinal stretching that resulted from this growth could only partially account for the age-related decline in retinal cell densities. Depending on the region of the retina examined, decreases in nuclear density of 17-26% for the ONL, 9-17% for the INL, and 16-20% for the GCL were apparently due to cell death. Measurements of retinal DNA content support the conclusion that significant cell loss from the retina occurs during senescence; between 4 and 30 months of age retinal DNA content decreased by about 13%. Age-related changes in retinal rhodopsin content did not parallel apparent changes in photoreceptor number. Between 4 and 11 months of age, rhodopsin levels rose by about 10%, and then remained constant up to 26 months. Over this same age span, there was an apparent decrease in photoreceptor number of about 15%.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

Characterization of neuropeptide Y-like immunoreactivity in vertebrate retina.

We have determined the endogenous levels of neuropeptide Y-like immunoreactivity (NPY-LI) in retinal extracts from pigs, cats, rabbits, chickens, and frogs by radioimmunoassay (RIA). The NPY-LI levels varied among the species. The highest concentration was found in frog retina, where seasonal variations were seen, 861 +/- 31 pmol g(-1) wet weight in the autumn and 334 +/- 26 pmol g(-1) wet weight in the spring. Lower levels were demonstrated in chicken and pig retina, 4.1 +/- 0.4 and 3.6 +/- 0.3 pmol g(-1) wet weight, respectively. The lowest concentration was demonstrated in rabbit retina, 2.0 +/- 0.3 pmol g(-1) wet weight. (All values are expressed as mean +/- S.E.M.). The NPY-LI in pig, rabbit, chicken and frog retina was characterized by high-performance liquid chromatography (HPLC). The main part of the extracted NPY-LI had an elution volume close to that to the porcine NPY. We have also analysed the evoked release of endogenous NPY from frog retina, induced either with light flashes (3 Hz, 300 lx), or with potassium depolarization of the neurons (40 mM). Light flashes and potassium induced an increased release of NPY-LI of 61 and 77%, respectively. NPY-LI in the efflux had the same HPLC retention time as that extracted directly from the retina.

Animals↗

Hydroxylamine-dependent inhibition of rhodopsin phosphorylation in the isolated retina.

Hydroxylamine (NH2OH), a substance known to accelerate the decay of the metarhodopsin II bleaching intermediate of rhodopsin, was examined for its effect on the light-dependent phosphorylation of rhodopsin in the intact, isolated retina. Groups of ovine and bovine retinas that had been pre-incubated in darkness with 32P-inorganic phosphate were supplemented with NH2OH at final concentrations of up to 20 mM, then irradiated and further incubated in darkness. Rod outer segments isolated from the incubated retinas were subjected to SDS-PAGE; the gel was analysed for 32P (autoradiography) and protein (Coomassie staining), to determine the specific radioactivity (ratio of 32P and protein levels; '32P/opsin') of the opsin monomer band. Among retinas of a given experimental group, 32P/opsin declined with increasing concentration of added NH2OH. The relative value of 32P/opsin exhibited by controls (0 mM NH2OH) was halved in the presence of about 1-2 mM NH2OH, and was reduced by greater than or equal to 80% in the presence of 20 mM NH2OH. Supplementation of the retina with 20 mM NH2OH 1 min after irradiation caused relatively little reduction in 32P/opsin. The results indicate that the light-dependent phosphorylation of rhodopsin in situ is substantially inhibited by NH2OH at millimolar levels. The data are discussed in relation to previous electrophysiological studies that have examined rod dark adaptation in NH2OH-treated retinas.

Animals↗

Immunocytochemical study of extracellular matrix components during lens and neural retina regeneration in the adult newt.

We have conducted an immunocytochemical study of fibronectin, laminin, heparan sulfate proteoglycans and nidogen-entactin during lens and neural retina regeneration in the adult newt from 0 to 60 days. In the normal eye, fibronectin was detected in the corneal stroma and Descemet's membrane, in dorsal and ventral irises and lens capsule but not in Bowman's membrane of the cornea. In normal neural retina, fibronectin was found in Bruch's and inner limiting membranes. Heparan sulfate proteoglycans gave a slight signal in both irises and the lens capsule. Nidogen-entactin distribution in the cornea was similar to that of fibronectin; it was absent from the stroma of both irises, and the signal was weak in the pigmented iris epithelium. Nidogen-entactin was not detected in the lens capsule and inner limiting membrane of the neural retina but was present in Bruch's membrane. During the first 15 days of lens regeneration, fibronectin and nidogen-entactin decreased but did not disappear from the pupillary margin of both irises, and no signal was obtained for laminin and heparan sulfate proteoglycans. From day 15 to day 60 fibronectin and nidogen-entactin increased in both irises and lens capsule. The signal for laminin was restricted to the lens capsule. Heparan sulfate proteoglycans gave a slight signal in both irises and in the lens capsule. During the first 25 days of neural retina regeneration, fibronectin was the first to appear in Bruch's membrane and the cell border of the new neuroepithelium and remained during the entire process. Laminin appeared after 41 days in the inner limiting and Bruch's membranes, but by day 50 it appeared as a weak signal only in the inner limiting membrane. Heparan sulfate proteoglycans were not detected at any of the regeneration stages studied. Nidogen-entactin was only detected in Bruch's membrane and around the cells and blood vessels of the new neural retina. Later it was detected in the inner limiting membrane but not in Bruch's membrane. Thus, the results obtained showed that extracellular matrix components do change during both lens and neural retina regeneration. These changes may play an important role during both regenerating processes.

Animals↗

Immunolocalization of basic fibroblast growth factor and its receptor in adult goldfish retina.

The neural retina of teleost fish can regenerate following surgical or neurotoxic lesions. As a first attempt to uncover the factors important for the regenerative response, we used immunocytochemistry to demonstrate the presence of basic fibroblast growth factor (bFGF) and its receptor in the goldfish retina. The bFGF-immunoreactivity was present throughout the retina, but was most intense in photoreceptor cells, especially cones, and Müller glia. Immunoreactivity for the bFGF receptor was strongest in the axon terminals of photoreceptors, both rods and cones. This pattern of immunolocalization is especially interesting since the proliferating cells that are thought to be responsible for generating the neural regenerate are located among the photoreceptor axon terminals. These proliferating cells have been identified as rod precursors because in the intact retina they give rise only to rod photoreceptors. When the neural retina is damaged, however, rod precursors are thought to be the source of proliferating neuroepithelial cells responsible for generating the retinal regenerate. The role played by bFGF in normal neurogenesis, cell differentiation, and/or neuronal regeneration in the fish retina has yet to be determined.

Animals↗

Fatty acid and lipid composition of the monkey retina in diet-induced hypercholesterolemia.

We determined the fatty acid composition of the lipids of cynomolgus monkey retina in animals fed commercial chow or a saturated fat, cholesterol-enriched atherogenic diet for 100 days. Doxosahexaenoic acid (22 : 6) accounted for 25.8% of the ethanolamine phosphoglyceride fatty acids, 17.6% of the serine plus inositol phosphoglyceride fatty acids, 8.4% of the choline phosphoglyceride fatty acids and 5.8% of the neutral lipid fatty acids in the retinas of the chow-fed animals. Therefore, monkey retinas, like those of other mammalian species, ordinarily contain large amounts of 22 : 6. Retinas from the monkeys fed the atherogenic diet contained less 22 : 6 as well as other polyunsaturates in each of the phospholipid classes. The decrease in polyunsaturates was compensated for by increases in palmitic, stearic, and oleic acids. There was no difference in the amount of phospholipid, the distribution of phospholipid classes, or the amount of cholesterol in the retinas of the monkeys fed the atherogenic diet. These results indicate that the single type of lipid alteration produced in the retina by a diet enriched in saturated fat and cholesterol is a decrease in the polyunsaturation of the retinal phospholipids. The reduction in retinal 22 : 6 content might have significance for photoreceptor function.

Animals↗