Changes in aquaporin-4 and Kir4.1 expression in rats with inherited retinal dystrophy.

Muller glial cells (MGC) are essential for normal functioning of retina. They are especially involved in potassium (K+) and water homeostasis, via inwardly rectifying K+ (Kir 4.1) and aquaporin-4 (AQP4) channels respectively. Because MGC appear morphologically and functionally altered in most retinal pathologies, we studied the expression of AQP 4 and Kir 4.1 during the time course of progressive retinal degeneration in Royal College of Surgeons (RCS) rats, an animal model for the hereditary human retinal degenerative disease Retinitis pigmentosa. Simultaneous detection of AQP4 and Kir 4.1 was performed by quantitative real-time polymerase chain reaction (QRT-PCR), Western blot and immunohistochemistry at birth and during progression of the pathology. Although small quantities of AQP4 and Kir 4.1 mRNA were detected at birth (postnatal day (PNd) 0) in both control and dystrophic rat retinas, proteins could not be detected at this age. Detectable proteins appeared in the second week of postnatal life. From PNd15 onwards, the time course in the expression of both AQP4 and Kir 4.1 mRNAs and protein was similar in dystrophic and control rats, with a progressive increase peaking at PNd60 and a subsequent decrease by one year. AQP4 protein and mRNA content were significantly lowered in dystrophic compared to control rats. Kir 4.1 protein levels were also lower in dystrophic retinas, while mRNA concentrations were unchanged and/or slightly higher in dystrophic rats. The discrepancies between Kir4.1 mRNA and protein suggest perturbation in protein translation due to the pathology. AQP4 and Kir 4.1/vimentin co-immunolabeling showed that: 1) apical radial processes of some MGC invaded the subretinal zone, and 2) MGC morphology was distorted in advanced pathology. MGC became hypertrophic both during the pathology and also with age in control rats. In conclusion, our results confirm that this inherited photoreceptor degeneration also leads to progressive alterations in physiological and morphological parameters of MGC which may aggravate retinal impairment.

Calbindin, calretinin and parvalbumin immunoreactivity in the retina of the chameleon (Chamaeleo chamaeleon).

Apart from the pioneering studies of Ramon y Cajal [1893] and Rochon-Duvigneaud [1943], few studies have been devoted to the detailed study of the cytological and biochemical structure of the chameleon retina. In the present study we analyzed the expression of calbindin (CB), calretinin (CR) and parvalbumin (PV) immunoreactivities in the chameleon retina, and compared their distribution with those found in the retinas of other vertebrate species. CB immunoreactivity is dense in photoreceptors, horizontal and some lower amacrine cells. The most intense immunoreactivity was observed for calretinin; CR-ir amacrine cells are distributed throughout the inner nuclear, inner plexiform, and ganglion cell layers of the retina. Horizontal cells also display immunoreactivity to CR. A few retinal interneurons are weakly PV-ir. Double-labeling shows that all PV-ir or CB-ir cells, except the photoreceptors, are also strongly CR-ir. The distributions of these calcium-binding proteins in the chameleon retina share similarities with those observed in mammalian and avian retinas. In addition, the widespread distribution and co-localization of CB and CR reinforces the idea that these proteins play a general role in buffering the intracellular calcium levels in retinal cells. Furthermore, CB- and CR-immunoreactivities have enabled us to identify for the first time axon-bearing horizontal cells in the peripheral retina of the chameleon, very similar to those described in mammals.

Gamma-aminobutyric acid-synthesizing cells in the retina of the chameleon Chamaeleo chameleon.

Antibodies directed against gamma-aminobutyric acid (GABA) and L-glutamic acid decarboxylases 65 and 67 kDa (GAD65 and -67) were used to study the GABAergic cell populations of the chameleon retina. GABA immunoreactivity was found in the two main types of retinal interneurons, amacrine and horizontal cells. Amacrine, displaced amacrine, and intra- and interplexiform cells displayed the strongest GABA immunoreactivity of all the retinal cell types. Horizontal cells formed a continuous GABA-immunoreactive cell layer lying against the outermost portion of the inner nuclear layer. In contrast to previous studies (Quesada et al. [1996] Cell Biol. Int. 20:395-400; [1999] Eur. J. Anat. 3:13-25), the present results demonstrate that the horizontal cells of the chameleon retina are GABA immunoreactive and that a subpopulation of these is immunolabelled by an antibody against GAD65. These results indicate that GABAergic synaptic transmission plays a key role in the outer plexiform layer of the vertebrate retina.

Differential expression of GAD(65) and GAD(67) during the development of the rat retina.

The development of synthetic enzymes in the GABAergic system (GAD(67) and GAD(65)) of the rat retina was analyzed from birth to the 4th postnatal week by the reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. As previously observed for GABA, immunoreactive GAD(67) profiles are seen clearly in the inner retinal layers at birth. At the end of the 1st week of postnatal life, immunolabeling is detected in amacrine and/or ganglion cells and in horizontal cells. GAD(67) immunoreactivity is transiently expressed in horizontal cells and disappears during the 3rd postnatal week. GAD(65) however does not develop until the 5th postnatal day. Immunolabeling is detected in the processes layering the inner plexiform layer (IPL) before being detected in the amacrine and/or ganglion cell bodies. The appearance of transcripts for GAD coincided with the appearance of the proteins. A transient form of mRNA transcripts of the GAD(67) gene containing an extra exon (ES-exon) is also observed which disappears progressively from birth to the 4th postnatal week. This form synthesizes a truncated, enzymatically inactive protein, which could participate in the regulation of GABA synthesis from glutamate present at high levels during retinogenesis.

Dopamine receptor localization in the mammalian retina.

After a short history of dopamine receptor discovery in the retina and a survey on dopamine receptor types and subtypes, the distribution of dopamine receptors in the retinal cells is described and correlated with their possible role in cell and retinal physiology. All the retinal cells probably bear dopamine receptors. For example, the recently discovered D1B receptor has a possible role in modulating phagocytosis by the pigment epithelium and a D4 receptor is likely to be involved in the inhibition of melatonin synthesis in photoreceptors. Dopamine uncouples horizontal and amacrine cell-gap junctions through D1-like receptors. Dopamine modulates the release of other transmitters by subpopulations of amacrine cells, including that of dopamine through a D2 autoreceptor. Ganglion cells express dopamine receptors, the role of which is still uncertain. Müller cells also are affected by dopamine. A puzzling action of dopamine is observed in the ciliary retina, in which D1- and D2-like receptors are likely to be involved in the cyclic regulation of intraocular pressure. Most of the dopaminergic actions appear to be extrasynaptic and the signaling pathways remain uncertain. Further studies are needed to better understand the multiple actions of dopamine in the retina, especially those that implicate rhythmic regulations.

Retinal TUNEL-positive cells and high glutamate levels in vitreous humor of mutant quail with a glaucoma-like disorder.

PURPOSE: To investigate whether retinal cell death observed in an avian glaucoma-like disorder occurs by apoptosis and whether an increase in excitotoxic amino acid concentration in the vitreous humor is associated temporally with cell death in the retina. METHODS: Presumptive retinal apoptotic nuclei were identified by histochemical detection of DNA fragmentation (by TdT-dUTP terminal nick-end labeling [TUNEL]), and vitreal concentrations of glutamate and several other amino acids were determined by high-pressure liquid chromatography with fluorometric detection in the al mutant quail (Coturnix coturnix japonica) in which a glaucoma-like disorder develops spontaneously. RESULTS: TUNEL-labeled nuclei were located mostly in the ganglion cell layer (GCL) in the retina of mutant quails 3 months after hatching. However, labeled nuclei were also observed in the inner and outer nuclear layers. At 7 months, most TUNEL-positive nuclei were detected in the inner nuclear layer, whereas labeled cells in the GCL were reduced in number. No TUNEL-labeled nuclei were detected in the retina of control quails at any age. Vitreal concentrations of glutamate and aspartate were significantly increased in 1-month-old mutant quails compared with control animals. Concentrations decreased at 3 months, and no significant differences were observed between strains at 7 months. CONCLUSIONS: Presumptive apoptotic cell death is detected from 3 months after hatching in mutant quails and is not restricted to retinal ganglion cells. Cell death appears just after a significant increase in excitotoxic amino acid concentrations in the vitreous humor, suggesting a correlation between both events.

Day and night dysfunction in intraretinal melatonin and related indoleamines metabolism, correlated with the development of glaucoma-like disorder in an avian model.

As previous studies have suggested that melatonin and serotonin may be involved in the regulation of intraocular pressure, retinal concentrations of melatonin, 5-HT, and related indoleamines measured at day and at night were studied during the development of a glaucoma-like disorder with increased intraocular pressure in the al mutant quail. Indoleamine levels were determined by HPLC with electrochemical detection in 1-month-, 3-month-, and 7-month-old al mutant and control quails. Morphology and numbers of melatonin-synthesizing and 5-HT-containing cells, labelled immunohistochemically with an anti-hydroxyindol-0-methyltransferase (HIOMT) antibody and an anti-5-HT antibody, respectively, were studied. Major findings were that: (1) no significant changes in morphology of melatonin-synthesizing cells or in the morphology and density of 5-HT-containing amacrine cells were observed during the development of glaucoma: (2) 5-HT metabolism was modified during the night at 1 month of age and during the day after 3 months; and (3) melatonin metabolism was modified during the night at 7 months and during the day after 3 months. These results demonstrate a relationship between the temporal evolution of this avian glaucoma and a dysfunction in indoleamine retinal metabolism.

Lamprey ganglion cells contact photoreceptor cells.

Lamprey retinal ganglion cells are localized in two separate layers: those close to the vitreous and those at the junction between the inner nuclear and inner plexiform layers, including some others in the inner nuclear layer, close to the photoreceptor cell layer. Whereas most ganglion cell dendrites arborize in the inner plexiform layer and contact amacrine, bipolar and retinopetal cell profiles, some of them, located in the inner nuclear layer, ascend radially through the outer plexiform layer and establish contacts with photoreceptor cells. This ganglion cell type might correspond to the biplexiform ganglion cells already described in gnathostome vertebrate species and could provide a fastforward signal from photoreceptors to ganglion cells, bypassing the usual bipolar cell interneuron.

Recoverin and hippocalcin distribution in the lamprey (Lampreta fluviatilis) retina.

Recoverin is a calcium-sensing protein which is involved in the transduction of light in vertebrate photoreceptors. It is also detected in other retina cell types in which its function is not yet elucidated, and is an autoantigen in a cancer-associated degenerative disease of the retina. Recently, hippocalcin, an homologous protein of recoverin, belonging to the same family of fatty acylated EF-hand calcium binding proteins was described in mammals. The immunohistochemical studies presented in this paper demonstrate, that, in the retina of the lamprey, an Agnathan considered the living ancestor of actual jawed vertebrates, recoverin was present in all photoreceptors and, to a lesser extent in subpopulations of amacrine and ganglion cells whereas hippocalcin was detected in numerous amacrine and ganglion cells and in the inner segments of long photoreceptors. The existence of these calcium-binding proteins shows that they have a high degree of conservation during evolution. Their presence in the same cells that in jawed vertebrates (photoreceptors and ganglion cells for recoverin; amacrine and ganglion cells for hippocalcin) suggests that some retinal functions are well conserved but because they were also found in different cell types than in other species (amacrine for recoverin; photoreceptors for hippocalcin), they may have functions more specific to the lamprey retina.

Determination of melatonin in rat pineal, plasma and retina by high-performance liquid chromatography with electrochemical detection.

A sensitive method for the routine measurement of endogenous melatonin (MEL) in pineal, retina and plasma rat tissues has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Quantification limit for MEL was 0.2 ng/mg protein in pineal, 15 pg/ml in plasma and 2.0 pg/mg protein in retina. To improve both MEL quantification and the reproducibility of the assay, an internal standard was used when an extraction in organic solvent was required, in contrast with other available chromatographic methods. MEL values and the circadian profile obtained in this study from both rat pineal and plasma agree with those reported previously. This method allows MEL detection in mammal retina, particularly in rat, where MEL levels are very low.

Molecular identification of a dopamine D1b receptor in bovine retinal pigment epithelium.

The rhythmic daytime inhibition of phagocytosis of shed photoreceptor outer segments (OS) by the retinal pigment epithelium (RPE) is related to increased cAMP in RPE cells. Dopamine (DA), the light-adaptive signal of the retinal oscillator can activate adenylyl cyclase through its D1-like receptors. It reduces OS phagocytosis by cultured bovine RPE, but a DA receptor was not demonstrated. Using primers selected from alignment of D1-like receptor genes already cloned, we have amplified by PCR two sequences in bovine genomic DNA. Phylogenetic analysis demonstrated that they correspond to D1a and D1b receptors. These receptors were then searched for using the reverse transcription-polymerase chain reaction (RT-PCR) in cultured bovine RPE. Only the D1b receptor subtype was demonstrated. It could mediate the DA-induced inhibition of phagocytosis.

Dopaminergic and GABAergic retinal cell populations in mammals.

A number of modern techniques now allow histologists to characterize subpopulations of retinal neurons by their neurotransmitters. The morphologies and connections of these chemically defined neurons can be analyzed precisely at both light and electron microscope levels and lead to a better understanding of retinal circuitry. The dopaminergic neurons form a loose population of special wide-field amacrine cells bearing intraretinal axons within the inner plexiform layer. One subtype, the interplexiform cell, sends an axon to the outer plexiform and outer nuclear layers. The number of interplexiform cells is variable throughout mammalian species. The GABAergic neurons form a dense and heterogeneous population of amacrine cells branching at all levels of the inner plexiform layer. The presence of GABA in horizontal cells seems to be species-dependent. Close relationships occur between dopaminergic and GABAergic cells. GABA antagonizes a number of dopaminergic actions by inhibiting both the release and synthesis of dopamine. This inhibition can be supported by GABA synapses onto dopaminergic cells, but GABA can also diffuse to its targets. Finally, GABA is also contained and synthesized in dopaminergic cells. This colocalization might be the basis of an intracellular modulation of dopamine by GABA.

Dopamine inhibits melatonin synthesis in photoreceptor cells through a D2-like receptor subtype in the rat retina: biochemical and histochemical evidence.

An intrinsic oscillator, using dopamine and melatonin as antagonist signals, controls rhythmic events in the retina of nonmammals. The purpose of the present work was to localize and characterize a dopamine receptor responsible for the nocturnal inhibition of melatonin synthesis in photoreceptor cells in a mammalian retina. An antibody against the D2 receptor stained photoreceptor cell inner segments of the rat retina. alpha-Methyl-p-tyrosine, a competitive inhibitor of tyrosine hydroxylase, enhanced the nocturnal content of melatonin, suggesting the dopamine control of melatonin synthesis as in non-mammals. Clozapine, a D2c/D4 antagonist, also enhanced the nocturnal level of melatonin, whereas raclopride, a D2A antagonist, did not. Taken together, these results support the control of melatonin levels by dopamine through a D2C/D4 receptor in photoreceptor cells of the rat retina. The presence of D4 receptors in the rat retina was confirmed by reverse transcription-PCR.

Changes in retinal dopaminergic cells and dopamine rhythmic metabolism during the development of a glaucoma-like disorder in quails.

PURPOSE: To examine the possible correlation between a dysfunction of the daily rhythm of retinal dopamine (DA) and the development of a glaucoma-like disorder in an animal model, the al mutant quail (Coturnix coturnix japonica). METHODS: The morphology and density of DA-containing cells labeled immunohistochemically with an anti-tyrosine hydroxylase (TH) antibody were correlated with the diurnal and nocturnal contents of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) determined by high-pressure liquid chromatography with electrochemical detection. RESULTS: The number of TH-immunoreactive cells was lower than normal in mutant quails suffering from the disorder. There were considerably fewer cells in the central retina, and the DA metabolism was reduced in parallel. The nocturnal DA content was lower than the diurnal level in normal quails, but there was no such circadian fluctuation in mutant quails. CONCLUSIONS: This glaucoma-like disorder in quails is correlated with the degeneration of DA-containing amacrine cells and a dysfunction of the circadian rhythmicity of DA synthesis.

Electron microscopic demonstration of tyrosine hydroxylase-immunoreactive interplexiform cells in the lamprey retina.

To clarify the controversies about the existence (or not) of dopaminergic interplexiform cells in the lamprey retina, we have performed an immunocytochemical electron microscopic study of the retina of the river lamprey Lampetra fluviatilis, using anti-tyrosine hydroxylase antibody. We demonstrate the presence of immunoreactive processes in both inner (IPL) and outer (OPL) plexiform layers. The external processes are in close contact with horizontal cell processes and photoreceptor terminals in the OPL, but do not make classical synapses. Some of them are ensheathed within Müller cell cytoplasm. Thus, dopaminergic interplexiform cells do exist in Lampetra fluviatilis, but the absence of synapses rather supports a paracrine action of dopamine in the outer retina.

Catecholamine-, indoleamine-, and GABA-containing cells in the chameleon retina.

Neurons containing catecholamine, indoleamine, and gamma-aminobutyric acid (GABA) were identified by immunohistochemistry in the chameleon retina. Tyrosine hydroxylase (TH) and serotonin (5HT) were observed mostly in two subtypes of orthotopic amacrine cells differing in their soma size and process distribution within the IPL. Some labelled cells were displaced either to the IPL (5HT) or the GCL (TH and 5HT). A multiplicity of retinal cell types contained GABA including cones, horizontal, amacrine, and ganglion cells. Our results confirmed those obtained in the retinas of other lizards except for the presence of interstitial and displaced amacrine cells containing TH or 5HT of which this is the first report.

Early development of GABA-like immunoreactive cells in the retina of turtle embryos.

Gamma aminobutyric acid (GABA) is one of the earliest neuroactive substances appearing in the developing central nervous system. The distribution and the time course of the appearance of GABA-like immunoreactivity in the retina of the turtle Emys orbicularis were investigated from embryonic stage 13 to hatching. The first GABA-like immunoreactive cells were observed at stage 14. These cells were located in both the scleral third of the neuroblastic layer and the inner layers of the retina. They were identified as presumptive immature horizontal cells and amacrine cells, respectively. The observation of numerous labelled fibers in the nerve fiber layer suggests that some of the GABA-like immunoreactive cells in the layers were ganglion cells. The development of GABA-like immunoreactive cells followed a gradient of maturation from central to peripheral retina. At hatching, the central retina appeared nearly morphologically mature. In conclusion, GABA is present before the morphofunctional maturation of the retina and this precocious existence supports the idea of its involvement in a neurotrophic role preceding the establishment of synaptic connections and neurotransmitter function.

Immunohistochemical localization of calbindin-D28K and calretinin in the lamprey retina.

Calbindin-D28K and calretinin are homologous cytosolic calcium binding proteins localized in many retinal neurons from different species. In this report, location of cells immunoreactive to both proteins was investigated in the retina of the lamprey, Lampetra fluviatilis. This organism constitutes one of the older representative vertebrates and possesses a peculiar organization, probably unique: two-thirds of the ganglion cells are in the classical amacrine cell layer and the nerve fiber layer is located in the scleral part of the inner plexiform layer. Calbindin-like immunoreactivity was demonstrated in large bipolar cells and in cell bodies located in the inner retina. Although the distinction between labelled ganglion cells and labelled amacrine cells was rendered difficult, we hypothesized that the majority of calbindin-immunoreactive cells observed in the inner retina are ganglion cells, because of the high number of labelled fibers in the nerve fiber layer. Calretinin-like immunoreactivity was detected in both large and small bipolar cells, and also in cells located in the inner retina. Since few calretinin-immunoreactive fibers were observed in the nerve fiber layer, we assume that the latter category of cells are amacrine cells. Horizontal cells were both negative for calbindin and calretin-like immunoreactivities. Calbindin and calretinin, which are present in cones from many species, could not be detected in the photoreceptor layer favouring the rod-dominated lamprey retina. Although their distribution differs from those observed in most vertebrates, the present results indicate the good conservation of both calcium binding proteins in the retina during the vertebrate evolution.

Immunoreactivity to glial fibrillary acid protein (GFAP) in the brain and spinal cord of the lamprey (Lampetra fluviatilis).

In contradiction to previous results, under certain conditions, it is possible to demonstrate GFAP immunoreactivity in the brain and spinal cord of a jawless vertebrate. The critical parameters appear to be (1) fixation with 4% paraformaldehyde at pH 7.4, (2) a very short period (2-6 hr) of postfixation, and (3) immunovisualization by the avidin/biotin/peroxidase technique rather than immunofluorescence or PAP techniques. Immunoreactivity appears throughout the brain and spinal cord, most frequently as fine prolongations normal to the pial surface, which can on occasion be traced to cell bodies near the ventricles or in the ependymal layer. The evolutionary implications of the presence of glial fibrillary acidic protein in a member of the Agnatha are discussed.

Dopaminergic interplexiform cells in the retina of pigmented and hypopigmented quails (Coturnix coturnix japonica).

Interplexiform cells (IPCs) have not been previously described as a component of the population of tyrosine hydroxylase (TH) immunoreactive (presumably dopaminergic) cells in the avian retina. In this study, carried out in both pigmented and imperfect albino mutant quails (Coturnix coturnix japonica), we initially describe TH immunoreactive cells in the inner nuclear layer whose internal dendritic arborization extends into strata 1, 3 and 4/5 of the inner plexiform layer. Then, we describe ascending processes arising from the somata or proximal dendrites of these cells. These sclerally directed processes (100-1,000 microns long) run across the inner nuclear layer to terminate within the outer plexiform layer, sometimes even reaching the outer nuclear layer. Hence, the cells bearing such processes correspond well with the definition of IPCs. The number of scleral processes is higher in mutant (48 +/- 19/retina) than in normal (12 +/- 10/retina) quails and are distributed throughout the retina except the area surrounding the pecten. Comparison of biochemical assays for dopamine in the two strains reveals a significantly higher dopamine content in the mutant quails which could be related to its increased number of dopaminergic IPC processes.