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PubMed2006

[Isolation, culture and identification of rat retinal progenitor cells in vitro].

OBJECTIVE: To characterize embryonic rat retinal progenitor cells (RPCs) by flow cytometry (FACS), immunofluorescence and (3)H-Thymidine assay in vitro. METHODS: RPCs were prepared from the retina of embryonic day 19 Sprague Dawley rats and were cultured in DMEM: F12 medium with N2-supplement, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) for 0 to 4 days. Cell proliferation and cluster formation were quantified by (3)H-Thymidine assay and morphometric analysis, respectively. Antibodies used to characterize the RPCs included markers for neural progenitors (Nestin and Sox-2), astrocytes (glial fibrillary acidic protein [GFAP]), horizontal cells (Calretinin), ganglion cells (CD90), rods (Rhodopsin), biopolar cells (PKC) and amacrine cells (Syntaxin) by the use of FACS. RESULTS: In primary cells, 22.23%, 16.04% and 19.66% of cells were found to express Nestin, Sox-2 and Calretinin, respectively. GFAP, CD90, Rhodopsin, PKC, Syntaxin were not detected or trail. Meanwhile, more than 90% cells were Nestin positive by immunofluorescence. After 6 days culture in vitro, 43.36% cells express Nestin by FACS. Nestin and GFAP were expressed by use of immunofluorescence. There was significant proliferation of RPCs within the first 4 days as evaluated by (3)H-Thymidine assay. CONCLUSIONS: The results demonstrate that combine FACS, immunofluorescence and (3)H-Thymidine assay together could characterize RPCs. RPCs proliferate well at the early period of cell culture.

Animals↗

PubMed1983

Fine structure of the retinal rods and cones in the domestic pig.

The structure of the retinal photoreceptors of the domestic pig has been studied by both light and electron microscopy. In this species the photoreceptors can be traditionally and adequately differentiated into rods and cones, with rods being the more numerous. Rods are long, slender cells, while cones are shorter and stouter. Both rods and cones consist of an outer segment, a connecting cilium, an inner segment, a nuclear region and a synaptic process leading to a synaptic ending. Differences are noted between rods and cones for the various regions of these cells. What are presumed to be the displaced cell bodies of both rods and horizontal cells are often noted within the outer plexiform layer of this species.

Animals↗

PubMed1993

The visual system of the Florida garfish, Lepisosteus platyrhincus (Ginglymodi). I. Retina.

The retina and choriocapillaris of the Florida garfish, Lepisosteus platyrhincus (Ginglymodi), was examined at the light and electron microscopic levels. The inner limiting membrane is covered by an extensive system of vitreal blood vessels emanating from the hyaloid artery, which enters the eye ventrally at the proximal end of the elongated optic nerve head. Two size classes of ganglion cell soma are segregated by optic axon fascicles and Müller cell endfeet, all of which lie at the level of the ganglion cell layer. A third class of 'displaced' ganglion cells lies at the border of the inner plexiform and inner nuclear layers amidst tightly packed amacrine, bipolar and Müller cell soma. Two layers of horizontal cells lie vitread of a synaptic zone consisting of a complex arrangement of horizontal and bipolar dendrites invaginating rod spherules and cone pedicles to form single and multiple (three to six) synaptic ribbon connections, respectively. Immediately vitread of the photoreceptor nuclei lie a population of 'displaced' bipolars. Three types of photoreceptors are characterised: unequal double cones, single cones (large and small) and rods. These show retinomotor movements where the rods elongate in the light and are masked by the pigment epithelium and contract in the dark as the pigment migrates sclerad. Ultrastructurally, 4 types of dark-staining (osmophilic) granules are described: (1) Small glycogen granules (0.033 microns) aggregated at the bases of the photoreceptor nuclei and larger similar granules (0.078 microns and termed paraboloids) vitread to the ellipsoid; (2) tapetal granules (0.32 microns) distributed throughout the dorsal four-fifths of the retinal pigment epithelium (RPE); (3) pigment granules (0.5-2.0 microns) in the RPE, concentrated in ventral retina; (4) granules or melanosomes (0.813 microns) of the choriocapillaris. The second class of granules constitute a tapetum lucidum eliciting a yellow eyeshine when viewed in the dark. Two other tapeta also exist, a guanine tapetum (irregular guanine crystals) and a tapetum fibrosum (stacks of collagen fibrils). Functional correlations are made, and the putative ancestral (primitive) condition of particular visual characters is established for the ray-finned fishes by out-group comparisons.

Animals↗

PubMed1991

A detailed model of the primary visual pathway in the cat: comparison of afferent excitatory and intracortical inhibitory connection schemes for orientation selectivity.

In order to arrive at a quantitative understanding of the dynamics of cortical neuronal networks, we simulated a detailed model of the primary visual pathway of the adult cat. This computer model comprises a 5 degrees x 5 degrees patch of the visual field at a retinal eccentricity of 4.5 degrees and includes 2048 ON- and OFF-center retinal beta-ganglion cells, 8192 geniculate X-cells, and 4096 simple cells in layer IV in area 17. The neurons are implemented as improved integrate-and-fire units. Cortical receptive fields are determined by the pattern of afferent convergence and by inhibitory intracortical connections. Orientation columns are implemented continuously with a realistic receptive field scatter and jitter in the preferred orientations. We first show that realistic ON-OFF-responses, orientation selectivity, velocity low-pass behaviour, null response, and responses to spot stimuli can be obtained with an appropriate alignment of geniculate neurons converging onto the cortical simple cell (Hubel and Wiesel, 1962) and in the absence of intracortical connections. However, the average receptive field elongation (length to width) required to obtain realistic orientation tuning is 4.0, much higher than the average observed elongation. This strongly argues for additional intracortical mechanisms sharpening orientation selectivity. In the second stage, we simulated five different inhibitory intracortical connection patterns (random, local, sparse-local, circular, and cross-orientation) in order to investigate the connection specificity necessary to achieve orientation tuning. Inhibitory connection schemes were superimposed onto Hubel and Wiesel-type receptive fields with an elongation of 1.78. Cross-orientation inhibition gave rise to different horizontal and vertical orientation tuning curves, something not observed experimentally. A combination of two inhibitory schemes, local and circular inhibition (a weak form of cross-orientation inhibition), is in good agreement with observed receptive field properties. The specificity required to establish these connections during development is low. We propose that orientation selectivity is caused by at least three different mechanisms ("eclectic" model): a weak afferent geniculate bias, broadly tuned cross-orientation inhibition, and some iso-orientation inhibition. The most surprising finding is that an isotropic connection scheme, circular inhibition, in which a cell inhibits all of its postsynaptic target cells at a distance of approximately 500 microns, enhances orientation tuning and leads to a significant directional bias. This is caused by the embedding of cortical cells within a columnar structure and does not depend on our specific assumptions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2006

The challenge of axonal path-finding.

Congenital syndromes of altered nervous system connectivity are reviewed along with recent findings on axonal growth: achiasma, congenital nystagmus, congenital horizontal gaze palsy, mirror movements and the syndromes of Kallmann, Wildervanck, Duane and Marcus Gunn. Identical guidance molecules are most likely involved in making axonal connections after injury and during development. Thus, investigations into variants of connectivity may help develop strategies to treat disconnections of axons in the adult.

Axons↗

PubMed1993

The marker for nitric oxide synthase, NADPH-diaphorase, co-localizes with GABA in horizontal cells and cells of the inner retina in the carp retina.

NADPH-diaphorase histochemistry selectively stained discrete populations of neurons contributing to the inner plexiform layer of the carp retina. In addition H1 horizontal cells contributing to the outer plexiform layer were labeled. In these cells as in a subpopulation of the labeled cells in the inner retina, NADPH-activity co-localized with GABA immunoreactivity. NADPH-activity is a direct marker for NO synthesis and it is concluded that NO might be an important mediator of light-dependent adaptational processes in the outer retina.

Amino Acid Oxidoreductases↗

PubMed1994

Pharmacology of novel GABA receptors found on rod horizontal cells of the white perch retina.

A novel type of GABA receptor is present on rod-driven (H4) horizontal cells of the white perch retina (Qian and Dowling, 1993a). These receptors have been tentatively termed GA-BAC receptors. In this study, the pharmacological properties of these receptors were further investigated by applying several conformationally restricted GABAA receptor agonists, GABAA antagonists, and a GABAB agonist to the H4 horizontal cells. GABA analogs locked in a partially folded conformation had a variety of effects. Isonipecotic acid had no effect on these receptors, whereas isoguvacine activated them but with low potency (EC50 = 137 microM). THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) acted as a competitive antagonist on these receptors with an inhibition constant of 82.5 microM. P4S (piperidine-4-sulfonic acid) activated the receptors at high concentrations (> 1 mM), but at lower concentrations it was a competitive antagonist with an inhibition constant of 80.9 microM. 14AA (imidazole-4-acetic acid), a GABA analog with an extended conformation, potently inhibited the GABA responses on H4 horizontal cells with an inhibition constant of 1.67 microM. Muscimol, which can assume both partially folded and extended conformations, acted as a mixed agonist-antagonist. The GABA responses on H4 horizontal cells were resistant to several competitive GABAA receptor antagonists including bicuculline, hydrastine, and SR-95531, but they were very sensitive to picrotoxin (IC50 = 237 nM). The inhibition by picrotoxin was both competitive and noncompetitive in nature. On the other hand, TBPS (tert-butyl-bicyclophosphorothionate), another GABAA receptor channel blocker, had minimal effects on these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Unusual distribution of ganglion cells in the retina of the three-toed sloth (Bradypus variegatus).

1. The distribution and size of retinal ganglion cells labelled with horseradish peroxidase (HRP) were studied in flat-mounted retinas of three-toed sloths. 2. Massive injections of HRP solution were made throughout the thalamus and midbrain in anesthetized sloths in order to retrogradely label the retinal ganglion cell population. Twenty to thirty h later the eyes were excised and the retinas flat-mounted and reacted with phenylenediamine-HCl and H2O2 to label ganglion cells, thus distinguishing them from other cells in the same retinal layer. 3. Ganglion cell density gradually increased from about 500 cells/mm2 at the far periphery to a peak of about 1,500 cells/mm2 in an area, termed the area centralis, deep in the inferior temporal retina. The presence of a vertical visual streak was also noted. 4. The area centralis contained a higher frequency of small ganglion cells than the peripheral retina where large cells predominated. 5. The unusual position of the area centralis and visual streak in the retina can be explained by the sloth's unique ability to rotate its head 180 degrees while climbing upside-down along horizontal branches so that the head is right-side-up. If it is assumed that the branch directly above the sloth's head needs to be visualized for accurate claw placement then the branch would be imaged on the inferior temporal retina in an area corresponding to the maximum density region.

Animals↗

PubMed1990

Eye velocity is not the major factor that determines mossy fiber responses of rabbit floccular Purkinje cells to head and screen oscillation.

Single-unit activities were recorded from floccular Purkinje cells in 29 alert pigmented rabbits. The floccular areas specifically related to horizontal eye movement (H-zone) were identified by the effects of local stimulation and later confirmed histologically. Most of the 53 H-zone Purkinje cells responded to both vestibular stimulation with turntable oscillation (5 degrees peak-to-peak, 0.1 Hz) in darkness and optokinetic stimulation with dot screen oscillation (2.5 degrees, 0.33 Hz), often showing a preference either for turntable oscillation or screen oscillation. Since these responses were not correlated with the velocity of evoked eye movements or eye position shift, it is concluded that the major inputs to flocculus H-zone Purkinje cells are head velocity or retinal slip signals rather than eye velocity or position signals.

Action Potentials↗

PubMed2000

Role of architecture in determining passive electrical properties in gap junction-connected cells.

The electrical properties of gap junction-connected cells were analysed in terms of their architectural organization. Two major architectural categories were considered: trees and rings. Trees are described by means of Bethe lattices (lattices with no rings) with arbitrary co-ordination and rings by two-dimensional periodic lattices with fourfold (square) or sixfold (triangular) co-ordination. The Bethe lattice is solved analytically by the transfer constant method, which allows the introduction of several physiologically relevant effects in a very simple manner. The experimental data for the length constant and the input resistance were fitted by varying the coupling and membrane resistances for various morphologies. The large variations in the length constant observed experimentally in two systems (turtle retina horizontal cells with and without dopamine and pancreatic beta-cells in the active and silent phases) could not be explained by means of the Bethe lattice, indicating that the cell arrangements form rings. Subsequent analysis by means of a linear chain and the square and triangular lattices showed the crucial relevance of architecture in deriving the electrical characteristics of gap junction-connected cells from experimental data.

Animals↗

PubMed1995

Localisation of neuronal nitric oxide synthase-immunoreactivity in rat and rabbit retinas.

The distribution of neuronal nitric oxide synthase (NOS) immunoreactivity was examined in rat and rabbit retinas and was compared with the distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity and vasoactive intestinal peptide (VIP) immunoreactivity. An antibody raised against a C-terminal fragment of a cloned rat cerebellar NOS was used to localise NOS immunoreactivity. NOS immunoreactive cells were not detected in rat retinas at postnatal day 1 or 4, but were seen from postnatal day 7 onwards. NOS immunolabelling was seen in a small population of cells in the proximal inner nuclear layer. Most of the labelled cells had the position of amacrine cells and were seen to send processes into the inner plexiform layer. A few labelled cells were at times also seen in the ganglion cell layer, which are likely to correspond to displaced amacrine cells. The same NOS-labelling pattern was seen in rat and rabbit retinas. NADPH-diaphorase staining was observed in both species, in photoreceptor inner segments, in cells with the position of horizontal cells, in a subset of amacrine and displaced amacrine cells, in large cell bodies in the ganglion cell layer, in both plexiform layers, and in endothelium. Colocalisation of NOS immunoreactivity and NADPH-diaphorase staining was only observed among amacrine cells. However, not all NADPH-diaphorase-reactive amacrine cells were found to be NOS immunoreactive. VIP immunoreactivity was also localised in rat retinas in a subpopulation of amacrine cells, but no colocalisation of NOS and VIP immunoreactivity was observed. Our observations indicate that only amacrine cells contain the NOS form recognisable by the antibody used, and suggest that different isoforms of neuronal NOS may be present in retinal cells. Further, the onset of NOS expression in rat amacrine cells appears to occur independently of neuronal activity.

Amino Acid Oxidoreductases↗

PubMed1990

Patterns of glutamate immunoreactivity in the goldfish retina.

Postembedding silver-intensified immunogold procedures reveal high levels of glutamate immunoreactivity in "vertical" elements of the goldfish retina: (1) Red-sensitive and green-sensitive cones display strong glutamate immunoreactivity, especially in their synaptic terminals, but blue-sensitive cones are poorly immunoreactive. (2) All type Mb (on-center) and Ma (off-center) mixed rod-cone bipolar cells and all identifiable cone bipolar cells are highly glutamate immunoreactive. We find no evidence for bipolar cells that lack glutamate immunoreactivity. (3) The majority of the somas in the ganglion cell layer and certain large cells of the amacrine cell layer resembling displaced ganglion cells are strongly glutamate immunoreactive. (4) Despite their high affinity symport of acidic amino acids, the endogenous levels of glutamate in Müller's cells are among the lowest in the retina. (5) GABAergic neurons possess intermediate levels of glutamate immunoreactivity. Quantitative immunocytochemistry coupled with digital image analysis allows estimates of intracellular glutamate levels. Photoreceptors and bipolar and ganglion cells contain from 1 to 10 mM glutamate. The bipolar and ganglion cell populations maintain high intracellular glutamate concentrations, averaging about 5 mM, whereas red-sensitive and green-sensitive cones apparently maintain lower levels. Importantly, photoreceptor glutamate levels are extremely volatile, and in vitro maintenance is required to preserve cone glutamate immunoreactivity in the goldfish. GABAergic horizontal and amacrine cells contain about 0.3-0.7 mM glutamate, which matches the values predicted from the Km of glutamic acid decarboxylase. Müller's cells and non-GABAergic amacrine cells contain less than 0.1 mM glutamate. Though Müller's cells are known to possess potent glutamate symport, they clearly possess equally potent mechanisms for maintaining low intracellular glutamate concentrations.

Animals↗

PubMed2001

Visual pigments and optical habitats of surfperch (Embiotocidae) in the California kelp forest.

We studied the optical microhabitat use and visual pigment variation among a group of closely related teleosts (surfperch: Embiotocidae) living along the nearshore central California coast. We employed a diver-operated spectroradiometer to record the optical microhabitat use of eight surfperch species in Monterey Bay. and microspectrophotometry to measure visual pigment absorbance for nine surfperch species. Species were dichromatic with mixtures of A1- and A2-based visual pigments exhibiting extensive maximum absorbance (lambda(max)) variation across species: 455-482 nm for SWS cones and 527-546 nm for LWS cones. Interspecific variation in sidewelling irradiance measurements (mean lambdaFmaxs) significantly accounted for 63% of the variation in surfperch LWS visual pigments and 83% of the interspecific variation in SWS visual pigments using a phylogenetically-corrected regression technique. Optimality models for maximizing relative photon capture of background radiance demonstrate that the LWS cone lambda(max) values are tuned for maximizing photon capture of the species-specific horizontal visual field, while the SWS cone lambda(max), are well offset from the dominant background radiance. This study is one of the first to demonstrate species-specific differences in habitat usage at microhabitat scales accounting for differences in photoreceptor peak absorbance among closely related, sympatric species.

Animals↗

PubMed1985

The dendritic architecture of the visual pretectal nuclei of the rat: a study with the Golgi-Cox method.

The dendritic architecture of the neurons of the visual pretectal nuclei in the rat was studied with the Golgi-Cox method. The olivary pretectal nucleus (PO) is characterized by distinctive neurons with a gnarled, tufted, richly branched dendritic arbor forming a dense neuropil within the nucleus. The distinct dendritic morphology of the olivary pretectal neurons enables this nucleus to be identified at all levels of the pretectum in Golgi-impregnated preparations. Rostromedially, the PO is surrounded by peripheral neurons whose dendrites wrap around the surface of the PO. The nucleus of the optic tract (NTO) contains three types of cells: (1) superficial horizontal cells whose dendrites extend out transversely; (2) large multipolar neurons whose dendrites spread out predominantly in a transverse plane, and (3) small to medium multipolar neurons with varying dendritic architecture. The posterior pretectal nucleus (PP) is composed predominantly of (1) multipolar cells with horizontally and vertically oriented dendrites extending out transverse to the optic axons; (2) piriform cells with dendrites extending dorsally toward the brachium; and (3) small multipolar neurons. The presence of superficial horizontal and large multipolar neurons in the NTO distinguishes the NTO from the PP in Golgi preparations. The horizontally oriented dendrites of many of the multipolar neurons in the PP give this nucleus an appearance distinct from that of the NTO. The differences in dendritic morphology between the visual pretectal nuclei in the rat permit identification of these nuclei at all levels within the pretectum. The boundaries of these nuclei, as determined in the Golgi-Cox preparations, correlate quite well with the boundaries defined by studying retinal projections (Scalia and Arango, '79).

Animals↗

PubMed1981

The size and shape of rod and cone centres of cat retinal ganglion cells.

Receptive field centres of cat retinal ganglion cells, as mediated by rod and by cone inputs, were mapped as contours of iso-sensitivity at a mid-mesopic adapting luminance using, respectively, 452 nm-blue and 578 nm-yellow narrow-band lights at an intensity 1 log unit above threshold for the most sensitive locus. Based on the sizes and shapes of mapped or rod and cone centres for 74 ganglion cells, four receptive field centre categories were distinguished. Cone and rod centres were usually elliptical, and in almost 60% of cells the major axis through the receptive field centre was oriented within +/- 20 degrees of horizontal. In 69%, rod and cone centres were non-concentric, 66% rod and cone centres were non-concentric, 66% had larger rod than cone centres - area ratios ranging from 0.6 : 1 to 2.9 : 1, and in only two cases was the rod centre actually smaller than the cone centre.

Animals↗

PubMed2004

Regressive and reactive changes in the connectivity patterns of rod and cone pathways of P23H transgenic rat retina.

We have used the P23H line 1 homozygous albino rat to study how progressive photoreceptor degeneration affects rod and cone relay pathways. We examined P23H retinas at different stages of degeneration by confocal microscopy of immunostained sections and electroretinogram (ERG) recordings. By 21 days of age in the P23H rat retina, there is already substantial loss of rods and reduction in rod bipolar dendrites along with reduction of metabotropic glutamate receptor 6 (mGluR6) and rod-associated bassoon staining. The cone pathway is relatively unaffected. By 150 days, when rods are absent from much of the retina, some rod bipolars remain and dendrites of rod and cone bipolar cells form synaptic complexes associated with cones and horizontal cell processes. These complexes include foci of mGluR6 and bassoon staining; they develop further by 270 days of age. Over the course of degeneration, beginning at 21 days, bipolar axon terminals atrophy and the inner retina undergoes further changes including a reduced and disorganized AII amacrine cell population and thinning of the inner plexiform layer. Electroretinogram (ERG) results at 23 days show reductions in a-wave amplitude, in rod and cone-associated b-waves (using a double flash paradigm) and in the amplitude of oscillatory potentials (OPs). By 38 days, rod scotopic a-wave responses and OPs are lost. B-wave amplitudes decline until 150 days, at which point they are purely cone-driven and remain stable up to 250 days. The results show that during the course of photoreceptor loss in the P23H rat, there are progressive degenerative changes, particularly in the rod relay pathway, and these are reflected in the changing ERG response patterns. Later reactive changes involving condensation of cone terminals and neurotransmitter receptors associated with rod and cone bipolar dendrites and with horizontal cell processes suggest that at this stage, there are likely to be complex changes in the relay of sensory information through the retina.

Amacrine Cells↗

PubMed1992

Receptive field dynamics in adult primary visual cortex.

The adult brain has a remarkable ability to adjust to changes in sensory input. Removal of afferent input to the somatosensory, auditory, motor or visual cortex results in a marked change of cortical topography. Changes in sensory activity can, over a period of months, alter receptive field size and cortical topography. Here we remove visual input by focal binocular retinal lesions and record from the same cortical sites before and within minutes after making the lesion and find immediate striking increases in receptive field size for cortical cells with receptive fields near the edge of the retinal scotoma. After a few months even the cortical areas that were initially silenced by the lesion recover visual activity, representing retinotopic loci surrounding the lesion. At the level of the lateral geniculate nucleus, which provides the visual input to the striate cortex, a large silent region remains. Furthermore, anatomical studies show that the spread of geniculocortical afferents is insufficient to account for the cortical recovery. The results indicate that the topographic reorganization within the cortex was largely due to synaptic changes intrinsic to the cortex, perhaps through the plexus of long-range horizontal connections.

Afferent Pathways↗

PubMed1997

Variations in photoreceptor directionally across the central retina.

Cones show a differential sensitivity to light coming from different portions of the pupil, typically being most sensitive to light from the center of the pupil. We measured the directional properties of the cones across the central 6 deg of the retina, using an optical imaging technique. We find that the cones in the center of the fovea have the broadest tuning. The width of the angular tuning changes rapidly from 0 deg to 1 deg retinal eccentricity, with cones at 1 deg being much more narrowly tuned that the cones in the center of the fovea. Directional tuning of the cones remains relatively constant from 1 deg to 3 deg retinal eccentricity. Receptoral disarray contributes minimally to the measured directional properties of the foveal cones, and there is no evidence of asymmetry between horizontal and vertical retinal locations. There are only small differences among the five subjects in the change in angular tuning of the cones with retinal location. We find that at the foveal center the directional tuning of the cones is limited by the diameter of the cone apertures.

Adult↗