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Protein kinase C association with the retinal cytoskeleton and phosphorylation of vimentin.

Protein kinase C plays an important role in regulation of the cytoskeleton. We found that protein kinase C activity is associated with the retinal cytoskeleton, and that it catalyses the stoichiometric phosphorylation of two cytoskeletal proteins, with apparent molecular masses of 56 kDa and 100 kDa. The 56-kDa substrate was identified as vimentin on the basis of its apparent molecular mass, pI, solubility, immunoreactivity, pattern of proteolysis by Lys-C and a partial amino acid sequence. Immunomicroscopy was consistent with previous reports that in the retina vimentin has the unusual property of being present in neuronal cells--horizontal cells--as well as non-neuronal cells. The characteristics of protein kinase C phosphorylation of vimentin that was enriched in neuronal vimentin were determined. Hyperactivation of protein kinase C by treatment of retinas with phorbol myristate acetate resulted in the phosphorylation of vimentin in situ, indicating that the phosphorylation is physiologically relevant. In vitro, purified retinal protein kinase C catalysed the incorporation of nearly 2 mol phosphate per mole of monomeric vimentin. The phosphorylation was highly dependent on the presence of phosphatidylserine. Thus, protein kinase C functions in the retinal cytoskeleton, where a major role is in the modification of vimentin. The characterization of the phosphorylation of outer retinal vimentin by protein kinase C provides a basis for further studies on the regulation and function of this cytoskeletal element.

Animals↗

Inversion of direction-selectivity to anterior fields in neurons of nucleus of the optic tract in rabbits with ocular albinism.

Extracellular recordings were made of 46 well-isolated single units in the nucleus of the optic tract (NOT) from 27 rabbits which were paralyzed but unanesthetized. Rabbits were selected from 3 varieties with ocular albinism. These units showed short latencies (mean = 2.2 msec, S.D. = 0.4) to chiasmic stimulation suggestive of monosynaptic innervation from retinal ganglion cells. Receptive fields were large and receptive field centers were confined to the projection of the visual streak. All units showed direction selectivity in which preferred stimulus motions were always nearly horizontal in the anterior or posterior direction. The direction selectivity of the units was broad in that excitatory acceptance angles were 180 degrees. Single units recorded from NOT in albino rabbits could be classified into 4 types based on regions of direction selectivity. The most common (64%) type of unit had receptive fields with two regions of opposed direction selectivity. The preferred direction of motion was anterior in lateral and posterior visual fields and was posterior in anterior visual fields. Two smaller groups of units (13% and 11%) had receptive fields with only one preferred direction. When the receptive field was anterior, the preferred direction was posterior. When the receptive field was lateral or posterior, the preferred direction was anterior. The least commonly encountered type of unit (7%) had receptive fields with two regions of direction selectivity. Anterior visual fields gave rise to anterior direction selectivity and lateral and posterior visual fields gave rise to posterior direction selectivity. The characteristics of NOT units are similar to those described for NOT units in pigmented rabbits except for an inversion of direction selectivity to stimulation from anterior visual fields. NOT units in albino rabbits show posterior direction selectivity when stimulation arises from anterior visual fields. The difference in directional preference in the visual receptive fields of the units in the NOT of albino rabbit may be responsible for the inversion of optokinetic pursuit eye movements characteristic of albino rabbits.

Albinism↗

Rapid postmortem changes in the cellular localisation of amino acid transmitters in the retina as assessed by immunocytochemistry.

We have assessed by means of immunocytochemistry, the cellular distributions of the amino acid transmitters GABA, glycine and glutamate, and the free-radical scavenger taurine, in the retinae of adult rabbits at various times after death. Within 10 min of death, horizontal cells began to display immunoreactivity for GABA, whilst displaced amacrine cells began to display immunoreactivity for glycine. By 40 min postmortem, GABA was present in glial cells. Glutamate, which is not normally detectable in retinal glia, was detected in such glia by 20 min postmortem. By contrast immunocytochemically detectable glycine did not accumulate in glia. There was a gradual diminution of immunoreactivity for taurine in glial cells and photoreceptors. By 2 h postmortem, most immunoreactivity had disappeared from the retina. We conclude that amino acid transmitters show rapid changes in their distributions immediately after death, which may be related to changes in the patterns of transmitter release and uptake, and changes in degradation mechanisms. The rapid changes in cellular localisation of amino acid immunoreactivity illustrated in this study, indicate that the fixation of nervous tissues must be performed rapidly. Moreover, the massive loss of immunoreactivity by 2 h postmortem suggests that any assays for content of these transmitters at this, and subsequent time-points, will bear little resemblance to the values obtained at the time of death.

Amino Acids↗

Dopamine hyperpolarizes and reduces the light responses of rod ON-centre bipolar cells in the retina of the dogfish, Scyliorhinus canicula.

Intracellular recordings were obtained from horizontal cells and bipolar cells of the dark-adapted, virtually all-rod retina of the dogfish. Eyecups were superfused with Ringer's solution containing micromolar concentrations of dopamine (DA). It was found that 10 microM DA hyperpolarized rod ON-centre bipolar cells and reduced their responses to light flashes. Detectable effects could be obtained with DA concentrations as low as 1 microM. A decrease in the light responses of bipolar cells due to the effects of DA was concomitant with a decrease in the amplitude of the b-wave of the electroretinogram. DA had no effect on the membrane potential or the light response of rod horizontal cells. These results demonstrate a selective action on rod ON-centre bipolar cells and suggest the existence of a dopaminergic pathway which affects the sensitivity of the rod visual system at the retinal level.

Animals↗

Localisation of annexins in the retina of the rainbow trout-light and electron microscopical investigations.

We present a first description of annexin immunoreactivity within the teleost retina. Antibodies against annexins V and VI were used in light and electron microscopic sections of light- and dark-adapted retinae. Strong immunoreactivity could be found in retinal layers with high synaptic input, such as the outer and inner plexiform layers and dendritic regions within the inner plexiform layer, in cells that are involved in negative feedback control such as horizontal and amacrine cells, in the membrane metabolism of photoreceptor outer segments, and in close relation to cytoskeletal components. Our findings suggest that both annexins V and VI are involved in the regulation of transmitter release, particularly of transmitters that are not directly involved in phototransduction. The annexins appear also to be involved with structures that support morphological changes in light and dark adaptation.

Animals↗

Immunocytochemical localization of calretinin containing neurons in retina from rabbit, cat, and dog.

Calcium homeostasis is critical for many neuronal functions, yet the distribution of calcium-binding protein is not always conserved among species, even between closely related species. We decided therefore to study the distribution of one of these calcium-binding proteins calretinin, in retina from rabbit, cat, and dog. Calretinin was localized using antibody immunocytochemistry. Calretinin immunoreactivity was found in numerous cell bodies in the ganglion cell layer in all three animals. These cells had small to medium-sized somas. Large ganglion cells, however, were not labeled using antiserum against calretinin. In the inner nuclear layer, calretinin immunoreactivity was found in many neurons in all three species. The regular distribution of neurons, the inner marginal location of their cell bodies in the inner nuclear layer, and the distinctive bilaminar morphologies of their dendritic arbors in the inner plexiform layer suggested that these calretinin-positive cells were AII amacrine cells. Calretinin immunoreactivity was observed in both A- and B-type horizontal cells in cat and dog retina. However, horizontal cells in the rabbit retina were not labeled by this antibody. Neurons in the photoreceptor cell layer were not labeled by this antibody. The present study suggests that calretinin immunoreactivity is present in several populations in the retina. In particular, calretinin labels AII amacrine cells and a subpopulation of ganglion cells in all three animals. Horizontal cells, however, were not labeled in rabbit.

Animals↗

The dynamic characteristics of the feedback signal from horizontal cells to cones in the goldfish retina.

1. The dynamic properties of the microcircuitry formed by cones and horizontal cells in the isolated goldfish retina were studied. Cones project to horizontal cells and horizontal cells feed back to cones via a relatively slow negative feedback pathway. 2. The time constant of the feedback signal in cones and of the effect this feedback signal had on the responses of second-order neurons was determined using whole-cell patch clamp and intracellular recording techniques. 3. It was found that the feedback signal in cones had a time constant of around 80 ms, whereas the time constant of the effect this feedback signal had on the second-order neurons ranged from 36 to 116 ms. This range of time constants can be accounted for by the non-linearity of the Ca(2+) current in the cones. In depolarized cones, the feedback-mediated response in second-order neurons had a similar time constant to that of the direct light response of the cone, whereas in hyperpolarized cones, the time constant of the feedback-mediated response in second-order neurons was considerably larger. 4. Further, it was shown that there was no delay in the feedback pathway. This is in contrast to what has been deduced from the response properties of second-order neurons. In one type of horizontal cell, the responses to red light were delayed relative to the responses to green light. This delay in the second-order neurons can be accounted for by the interaction of the direct light response of the medium-wavelength-sensitive cones (M-cones) with the feedback response of the M-cones received from the horizontal cells.

Animals↗

Oscillations in rod and horizontal cell membrane potential: evidence for feed-back to rods in the vertebrate retina.

1. Rods and horizontal cells were studied with intracellular recordings in the retina of the toad, Bufo marinus; 161 cells were from the eyecup preparation and thirty were from the isolated perfused retina. 2. Of these cells, 39% exhibited either transient or sustained oscillations of membrane potential. Light flashes either evoked transient oscillations or temporarily abolished sustained oscillations. The amplitudes of the oscillations could be as large as 27 mV. The frequency of the oscillations at 25 degrees C was between 1-5 and 3-5 Hz and was strongly dependent on temperature and background illumination. 3. The rod oscillation amplitude and the peak of the horizontal cell light response increased similarly with increasing test flash diameters. They continued to grow for diameters much larger than those which increased the peak of the rod light response. 4. Perfusion of the isolated retina with 2 mM aspartate had only a small effect on the rod light response but it completely eliminated the horizontal cell light response as well as the oscillations recorded in both rods and horizontal cells. 5. It is believed that the oscillations result from a reverberating interaction between rods and neurones post-synaptic to rods. Thus, rods can be both post- as well as presynaptic retinal elements.

Adaptation, Ocular↗

Effect of temporal frequency and contrast on peripheral grating resolution.

We measured resolution at 10 degrees in the inferior temporal retina using horizontal and vertical gratings of different contrasts which modulated in time at temporal frequencies from 0 to 40 Hz. Since peripheral grating resolution is directly related to the sampling density of retinal ganglion cells this allowed us to observe changes in responding ganglion cell density as the stimulus properties changed to selectively stimulate magnocellular pathway cells (M cells) instead of parvocellular pathway cells (P cells). At all contrasts there was no decline in resolution performance up to 10 Hz, after which resolution deteriorated significantly, this effect being greater at lower contrasts. The predicted psychophysical resolution limit for M cells calculated from the anatomical data of Dacey indicates that resolution for high temporal frequency (30 Hz) or low contrast (5%) stimuli is too good to be mediated by M cells alone and there is still significant P cell stimulation by these stimuli.

Contrast Sensitivity↗

The occurrence of dopaminergic interplexiform cells correlates with the presence of cones in the retinae of fish.

Using light-microscopic immunocytochemistry against tyrosine hydroxylase, we have investigated the morphology of dopaminergic cells in 23 species of fishes representing various systematic classes and subclasses and which live in very different habitats. We have, for the first time, observed teleosts with dopaminergic amacrine cells. Thus, in both bony and cartilaginous fishes, dopaminergic cells are differentiated as interplexiform and amacrine cells. The differentiation of dopaminergic cells into amacrine or interplexiform cells in fishes correlates with the absence or presence of cones. In pure-rod retinae, they occur as amacrine cells, and in mixed rod/cone retinae, they occur as interplexiform cells. We conclude therefore that the differentiation of retinal dopaminergic cells in fish does not depend on the evolutionary or systematic classification of a given species. Rather, it is correlated with the occurrence of rods and/or cones, and thus linked more closely to the habitat. We argue that, in fish, the presence of cones and cone-specific horizontal cells may be responsible for inducing dopaminergic cells to differentiate as interplexiform cells. Possible functions of dopamine in all-rod retinae, which may not require adaptation, may include neuromodulation in the inner plexiform layer for the sensitization of the rod pathway, the shaping of biological rhythms, and the control of eye growth.

Animals↗

Neuronal and glial cell types revealed by NADPH-diaphorase histochemistry in the retina of a teleost fish, the grass goby (Zosterisessor ophiocephalus, Perciformes, Gobiidae).

The grass goby is a mud-burrowing fish with a rich retinal vasculature appropriate to its hypoxic habitat. NADPH-diaphorase histochemistry was performed on retinal sections and wholemounts to reveal cells that contain nitric oxide synthase and so may be presumed to synthesise nitric oxide, a gaseous intercellular messenger with many roles including vasodilation. Structures that were consistently stained by this method included cone ellipsoids, horizontal cells, Müller cells and their processes, large displaced ganglion cells in the inner nuclear layer (identified by their axons), large interstitial ganglion cells in the inner plexiform layer, and capillary endothelial cells. In wholemounts, horizontal cells were seen to form a regular pattern, contacting each other at their dendritic terminals. Some cells in the ganglion cell layer were weakly stained, but stained bipolar and amacrine cells were not seen. The diaphorase-positive large ganglion cells all formed large, sparsely branched dendritic trees, arborizing near the scleral border of the inner plexiform layer. The displaced and interstitial cells seemed to belong to distinct morphological types, the interstitial cells having smaller somata and trees. Analysis of their spatial distributions in one representative retina confirmed this: the displaced cells formed a highly regular mosaic with a mean spacing (nearest-neighbour distance) of 303 microm, whereas the interstitial cells formed a separate mosaic, almost as regular but with a smaller mean spacing of 193 microm, rising to 217 microm in a sample that excluded the area retinae temporalis. Spatial correlogram analysis showed that these two mosaics were spatially independent. Nitric oxide probably has many roles in the retina. The presence of its synthetic enzyme in Müller cells, which communicate with retinal blood vessels, is consistent with a role in the control of retinal blood flow. Its function in large, mosaic-forming retinal ganglion cells is unknown.

Animals↗

Development of the human retina in the absence of ganglion cells.

Retinal development was studied in eyes from fetal and neonatal human anencephalic (AnC) and normal age-matched infants to determine the time of retinal ganglion cell (GC) loss and its effect on the development of other retinal neurons. At fetal week (Fwk) 14, GC loss was evident in central retina and by Fwk 19-20 almost all GC were absent, although immunocytochemical labeling for GC markers brain 3, neurofilament M and parvalbumin detected a few GC in the AnC far periphery at older ages. The inner nuclear and inner plexiform (IPL) layers showed variable amounts of thinning but all normal bipolar (BP) and horizontal cell markers were still present. The amacrine (AM) labels calbindin and calretinin were markedly reduced. Lamination for these markers in the IPL was less organized than in normal retinas, with BP and AM markers extending into the degenerated GC layer. Cone and rod photoreceptors had normal morphology and topography in AnC retina and each expressed normal phototransduction and synaptic markers. The prospective fovea was identified in AnC neonatal retina by cone packing and the absence of immunolabeled rod photoreceptors. In one AnC neonatal retina, blood vessels and astrocytes extended across the inner retina in the putative fovea and there was no evidence of a pit. In another AnC neonatal retina, blood vessels and astrocytes formed a foveal avascular zone in the inner retina and a shallow pit was present within this zone. However, both foveas showed evidence for the onset of cone elongation and packing. These findings support the model of Springer and Hendrickson [2005; Vis. Neurosci. 22, 171] in which the foveal avascular zone is critical for pit formation, but suggest that mechanisms inherent to the outer retina may be involved in early stages of foveal cone packing.

Anencephaly↗

Regional difference in density of monoamine-accumulating cells of carp and catfish retinas.

By means of a histofluorescence technique, a comparative study was conducted on the regional density of dopaminergic (DA) and indoleamine-accumulating (IA) cells in carp (Cyprinus carpio) and catfish (Ictalurus punctatus) retinas. In order to enhance detection of fluorescent cells, noradrenaline (NA; 5.0 micrograms) or a mixture of NA (2.5 micrograms) and 5,6-dihydroxytryptamine (5,6-DHT; 2.5 micrograms) was intravitreally injected into the eyes 2-3 hr before enucleation. DA and IA cells were counted systematically in space on flat-mounted preparations. Both classes of cells were found to be distributed similarly in the two species of fish; the cell density is highest in the circumferential margin of the retina, and is slightly higher in a region dorsal to the optic disc than in the surrounding area. Differences in the distribution pattern of the cells between carp and catfish retinas were as follows: (a) the DA cell density is higher over the whole retinal field in carp (the mean density +/- SD = 34 +/- 16 cells/mm2) than in catfish (13 +/- 7 cells/mm2); (b) the region where the density is slightly higher than in the surrounding area is restricted to a small area immediately dorsolateral to the optic disc in carp, while it is relatively broadly placed dorsal to the optic discs, forming a horizontal band in catfish; (c) the density ratio of DA cells to IA cells is 1:1 in carp but 1:2 in catfish; and (d) catfish DA cells seem to be more irregular than carp DA cells in shape, size, dendritic arborization, uptake preference for monoamines intravitreally injected, and also in depth location seen in radial cryosections.

Animals↗

Experimental retinal detachment in the cone-dominant ground squirrel retina: morphology and basic immunocytochemistry.

The cellular responses of the cone-dominant ground squirrel retina to retinal detachment were examined and compared to those in rod-dominant species. Retinal detachments were made in California ground squirrels. The retinas were prepared for light, electron, and confocal microscopy. Tissue sections were labeled with antibodies to cone opsins, rod opsin, glial fibrillary acidic protein (GFAP), vimentin, synaptophysin, cytochrome oxidase, and calbindin D 28K. Wax sections were probed with the MIB-1 antibody to detect proliferating cells. By 10 h postdetachment many photoreceptor cells in the ground squirrel already show structural signs of apoptosis. At 1 day many photoreceptors have collapsed inner segments (IS), yet others still have short stacks of outer segment discs. At 3 days there is a marked increase in the number of dying photoreceptors. Rod and medium-/long-wavelength opsins are redistributed in the cell membrane to their synaptic terminals. At 7 days photoreceptor cell death has slowed. Some regions of the outer nuclear layer (ONL) have few photoreceptor somata. IS remnants are rare on surviving photoreceptors. At 28 days these trends are even more dramatic. Retinal pigmented epithelium (RPE) cells do not expand into the subretinal space. The outer limiting membrane (OLM) appears flat and uninterrupted. Müller cells remain remarkably unreactive; they show essentially no proliferation, only negligible hypertrophy, and there is no increase in their expression of GFAP or vimentin. Horizontal cells show no dendritic sprouting in response to detachment. The speed and extent of photoreceptor degeneration in response to detachment is greater in ground squirrel than in cat retina-only a small number of rods and cones survive at 28 days of detachment. Moreover, the almost total lack of Müller cell and RPE reactivity in the ground squirrel retina is a significant difference from results in other species.

Animals↗

A unifying model for activity-dependent and activity-independent mechanisms predicts complete structure of topographic maps in ephrin-A deficient mice.

Axons of retinal ganglion cells establish orderly projections to the superior colliculus of the midbrain. Axons of neighboring cells terminate proximally in the superior colliculus thus forming a topographically precise representation of the visual world. Coordinate axes are encoded in retina and in the target through graded expression of chemical labels. Additional sharpening of projections is provided by electric activity, which is correlated between neighboring axons. Here we propose a quantitative model, which allows combining the effects of chemical labels and correlated activity in a single approach. Using this model we study a complete structure of two-dimensional topographic maps in mutant mice, in which the label encoding the horizontal retinal coordinate ephrin-A is reduced/eliminated. We show that topographic maps in ephrin-A deficient mice display a granular structure, with the regions of smooth mapping separated by linear discontinuities reminiscent of fractures observed in the maps of preferred orientation.

Animals↗

Responses of neurons of the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic tract in the awake monkey.

The nucleus of the optic tract (NOT) and the dorsal terminal nucleus of the accessory optic tract (DTN) are essential nuclei for the generation of slow-phase eye movements during horizontal optokinetic nystagmus. We recorded from 101 neurons (all directionally selective) in four NOT/DTN of three trained and behaving rhesus monkeys. Neuronal activity increased when stimuli moved ipsiversively with respect to the recording site and decreased below spontaneous activity when stimuli moved contraversively. While the monkey fixated a small spot, some NOT/DTN neurons did not respond at all to the retinal image slip of a whole-field random dot pattern; others showed a monotonic increase of activity to increasing velocities of that stimulus. The velocity range tested was up to 100 degrees/s. During the execution of optokinetic nystagmus, 39 of 73 cells tested showed a velocity-tuned response with an average optimum at 21 degrees/s retinal image slip. Following saccades during optokinetic nystagmus (quick phases), the NOT/DTN neuronal activity briefly attained the level of spontaneous activity, as predicted from the velocity selectivity during optokinetic nystagmus. Immediately upon cessation of optokinetic stimulation in the preferred direction, NOT/DTN activity returned to the spontaneous level and did not reflect the ongoing optokinetic afternystagmus in darkness. Most NOT/DTN neurons displayed direction selectivity also during smooth pursuit. Twenty-one of 50 cells tested (42%) always responded to the retinal slip of the target (target velocity cells), 16 cells (32%) responded to the retinal slip of the background (background velocity cells), and 13 cells (26%) did not respond at all during smooth pursuit. We conclude from our results that the NOT/DTN is an essential structure for the processing of the direction and speed of retinal image slip. This information is then used for the generation and maintenance of slow eye movements, preferentially during horizontal optokinetic nystagmus but also during pursuit eye movements.

Animals↗

Immunocytochemical localization of parvalbumin- and neurofilament triplet protein immunoreactivity in the cat retina: colocalization in a subpopulation of AII amacrine cells.

Using antibodies against parvalbumin and neurofilament triplet protein, colocalization of these two neuronal markers was revealed in all of type A horizontal cells and alpha ganglion cells and in a small number of AII amacrine cells of the cat retina. Besides the double-labeled neurons, parvalbumin alone was present in type B horizontal cells, in small numbers of starburst- and A13-like amacrine cells and in the somata of unidentified ganglion cells. The processes of the double- or single-labeled amacrine cells did not have a continuous retinal cover. Although the parvalbumin- and neurofilament-immunolabeled amacrine cells belonged to groups of neurons with well-defined cell morphologies, their neurochemical features differed from other AII, starburst and A13 amacrine cells. The presence of these cells may be due to an accidental expression of an unusual combination of neurochemical features during retinal development. It is also possible that these cells support the functioning of ganglion cells with rarely occurring complex receptive fields.

Animals↗

Distribution of parvalbumin immunoreactivity in the vertebrate retina.

Parvalbumin, a calcium-binding protein thought to buffer intracellular calcium, is expressed in selected neuronal and non-neuronal cell populations. We used a well-characterized antibody directed against parvalbumin to investigate the distribution of parvalbumin in the retina of twelve vertebrate species to evaluate patterns of cellular expression for recurrent functional features. Parvalbumin immunoreactivity was displayed by subpopulations of ganglion, amacrine, bipolar and horizontal cells in different species-specific combinations. In the pigeon retina, subpopulations of amacrine, ganglion and bipolar cells were immunoreactive for parvalbumin. Parvalbumin immunoreactive bipolar cells in this species were mostly confined to the temporal dorsal region of the retina. In the owl, no immunoreactive amacrine cells were found, but many bipolar cells displayed parvalbumin immunoreactivity. In the teleost retina, amacrine and ganglion cells were found to be immunoreactive for parvalbumin. A high degree of species-specific variation was encountered in the mammalian retina. The most consistent finding within this class was that subpopulations of parvalbumin-immunoreactive amacrine cells were consistently observed in every species. In the rabbit, horizontal and ganglion cells displaying parvalbumin immunoreactivity were also seen. In rodents (hamster, ground squirrel), parvalbumin immunoreactivity was displayed by subpopulations of amacrine cells and, in the squirrel, by some ganglion cells as well. In the cat and in the baboon retina, parvalbumin immunoreactivity was found in horizontal cells, ganglion cells and a subpopulation of amacrine cells. The distribution of parvalbumin immunoreactive neurons in the vertebrate retinae studied showed no systematic correlation with phylogenetic proximity. The expression of parvalbumin within the systems of retinal neurons may therefore reflect the functional needs of different visual behaviors.

Animals↗