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.

Confirmation of the retinopetal/centrifugal nature of the tyrosine hydroxylase-immunoreactive fibers of the retina and optic nerve in the weaver mouse.

The number of tyrosine hydroxylase-immunoreactive fibers in the nerve fiber layer is increased in the retina of the weaver compared to control mice (Dev. Brain Res. 121 (2000) 113). To confirm the retinopetal/centrifugal nature of these fibers, a newly devised whole-mounted optic nerve technique allowed us to determine, during development, their first appearance within the optic nerve (post-natal day 12) compared to retina (post-natal day 13). One such fiber was also observed looping in the retina of a monkey fetus.

Iron, ferritin, transferrin, and transferrin receptor in the adult rat retina.

PURPOSE: The retina and other tissues need iron to survive. However, the normal iron metabolism in rodent retinas had not been characterized. This study was intended to investigate iron and iron homeostasis protein (ferritin, transferrin [Tf] and transferrin receptor [Tf-R]) distribution in 20- to 55-day-old rat retinas. METHODS: Iron was revealed on retinal sections directly by proton-induced x-ray emission (PIXE) and indirectly by electron microscopy (EM). Ferritin, Tf, and Tf-R proteins were localized by immunohistochemistry. Transferrin expression was localized by in situ hybridization (ISH). Transferrin and ferritin proteins and mRNA were analyzed by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS: Iron is widely and unevenly distributed throughout the adult rat retina. The highest concentration was observed by PIXE in the choroid and the retinal pigmented epithelial cell (RPE) layer, and in inner segments of photoreceptors (IS). Outer segments of photoreceptors (OS) also contain iron. EM studies suggested the presence of iron inclusions inside the photoreceptor discs. Choroid, RPE, and IS showed a strong immunoreactivity for ferritin. Transferrin accumulated mainly in the IS and OS areas and in RPE cells but can also be detected slightly in retinal capillaries. Western blot analysis for Tf and ferritin confirmed their presence in the adult neural retina. By RT-PCR, H- and L-chains of ferritin and Tf mRNAs were expressed in neural retina, but the main sites of Tf synthesis observed by ISH were the RPE and choroid cell layers. Tf-R immunoreactivity was detected in the ganglion cell layer, inner nuclear layer, outer plexiform layer, IS, RPE, and choroid. These results were similar for all stages studied. CONCLUSIONS: For the first time, the present study characterized both iron and iron homeostasis proteins in rodent retinas. In the outer retina, iron and ferritin shared the same distribution patterns. In contrast, Tf, mainly synthesized by RPE cells and detected in OS and IS areas, probably helps to transport iron to photoreceptors through their Tf-R. This is a likely pathway for filling iron needs in the outer retina.

Paradoxical increase of tyrosine hydroxylase-immunoreactive retinopetal fibers in the weaver mouse.

Weaver mice undergo apoptosis of the granule cell precursors of the cerebellum and nonapoptotic death of mesencephalic dopaminergic cells during post-natal development. In contrast, the number of retinal dopaminergic cells was transiently increased in weaver compared to control mice [C. Savy, E. Martin-Martinelli, A. Simon, C. Duyckaerts, C. Verney, C. Adelbrecht, R. Raisman-Vozari, J. Nguyen-Legros, Altered development of dopaminergic cells in the retina of weaver mice, J. Comp. Neurol. 1999;412:656-668]. While re-examining the retinas, we observed, in the nerve fiber layer, retinopetal tyrosine hydroxylase-immunoreactive fibers, which were dramatically increased in number throughout development and adulthood in the weaver compared to control mice.

Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium.

The discovery of disc protein renewal in rod outer segments, in 1960s, was followed by the observation that old discs were ingested by the retinal pigment epithelium. This process occurs in both rods and cones and is crucial for their survival. Photoreceptors completely degenerate in the Royal College of Surgeons mutant rat, whose pigment epithelium cannot ingest old discs. The complete renewal process includes the following sequential steps involving both photoreceptor and pigment epithelium activity: new disc assembly and old disc shedding by photoreceptor cells; recognition and binding to pigment epithelium membranes; then ingestion, digestion, and segregation of residual bodies in pigment epithelium cytoplasm. Regulating factors are involved at each step. While disc assembly is mostly genetically controlled, disc shedding and the subsequent pigment epithelium phagocytosis appear regulated by environmental factors (light and temperature). Disc shedding is rhythmically controlled by an eye intrinsic circadian oscillator using endogenous dopamine and melatonin as light and dark signal, respectively. Of special interest is the regulation of phagocytosis by multiple receptors, including specific phagocytosis receptors and receptors for neuroactive substances released from the neuroretina. The candidates for phagocytosis receptors are presented, but it is acknowledged that they are not completely known. The main neuromodulators are adenosine, dopamine, glutamate, serotonin, and melatonin. Although the transduction mechanisms are not fully understood, attention was brought to cyclic AMP, phosphoinositides, and calcium. The chapter points to the multiplicity of regulating factors and the complexity of their intermingling modes of action. Promising areas for future research still exist in this field.

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.

Altered development of dopaminergic cells in the retina of weaver mice.

Postnatal degeneration of dopaminergic (DA) cells is known to occur in mesencephalic nuclei of mutant weaver mice, whereas retinal DA content is reported to be unchanged in the adult animal. To determine whether morphological changes occur in the weaver retinal DA system, we compared weaver and control developing and adult retinas after tyrosine hydroxylase (TH) immunohistochemistry. The density and distribution of DA cells were analyzed using Dirichlet tessellation. Not only was no DA cell loss found in adult weaver retinas, but we even observed an increase in DA cells in weaver compared to control retinas between postnatal days 14 and 30. Furthermore, some unusual features were found during the latter period: atypical cells (representing a maximum of 12% of the whole DA cell population) were observed, and these differed from typical DA cells in terms of both location (slightly more external within the inner nuclear layer) and appearance (flat somata, round and clear nuclei, thick dendritic trunks emerging laterally and giving rise to horizontal processes). Some of the atypical cells were intermingled in a delicate network lying in a more outer focal plane than the main DA plexus. The expression of GIRK2, a G protein-related inward rectifying K(+) channel responsible for the weaver syndrome, was investigated. Although no GIRK2 labeling was demonstrated in DA cells, its possible involvement in the transient disturbances observed in the weaver DA retinal system is discussed.

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.

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.

Immunocytochemical localization of dopamine D1 receptors in the retina of mammals.

Dopamine is one of the major neurotransmitters in the retina. It is released from amacrine and interplexiform cells into both inner (IPL) and outer (OPL) plexiform layers. Several dopaminergic actions are known to occur through D1 receptors (D1R) but the precise location of these receptors has not been established. An antibody that recognizes the intracytoplasmic C-terminal of the rat D1R was used to detect D1R, immunohistochemically, in rats (Wistar and RCS), mouse, hamster, and macaque monkey retinas. The OPL was heavily stained in each species, consistent with the known actions of dopamine on horizontal cells. Three to five bands were observed in the IPL, depending on species. Three were in the a sublayer, the outermost of which was close to the amacrine cell layer, and may represent the massive dopamine input to the AII rod-amacrine cells. As observed in mice, where bipolar cells are D1-immunoreactive, the band located in sublayer 3 of the IPL may contain cone-bipolar cell terminals. A band of D1R-immunoreactivity in the b sublayer of the IPL contains ON-bipolar cell terminals and a second site of interaction between dopaminergic cells and the AII amacrine cells. This sublayer was absent from the RCS rat retina, suggesting a severe impairment of the rod-driven pathway following rod degeneration in these mutant rats. Cells in the ganglion cell layer exhibited relatively heavy staining, and may be ganglion cells or displaced amacrine cells. Some extrasynaptic localizations of D1R in the retina are suggested.

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.

[Dopamine slows phagocytosis of rods from bovine pigment epithelium in vitro trough D1 receptor]. / La dopamine ralentit la phagocytose des bâtonnets par l'épithélium pigmentaire de boeuf in vitro grâce à un récepteur D1.

Photoreceptor disc shedding and their phagocytosis by the retinal pigment epithelium undergo a daily rhythm entrained by an intrinsic oscillator involving melatonin and dopamine in non-mammals. Such a mechanism is not demonstrated in mammals, but the rhythm of photoreceptor renewal can be modulated by exogenous melatonin and dopamine. The present experiments were designed to show whether a direct action of DA occurs on pigment epithelial cells, and to identify the receptor mediating this action. Primary cultures of bovine retinal pigment epithelium were incubated with bovine rod outer segments in the presence of dopamine, D1 and D2 agonists, D1 antagonist and forskolin. Dopamine, D1 agonist and forskolin decreased phagocytosis, while D2 agonist was inactive. Thus dopamine slows pigment epithelium phagocytosis in vitro through a D1 receptor. Increased phagocytosis following blockade of the receptor by an antagonist suggests a more complex modulation of phagocytosis by dopamine.

The effects of prenatal exposure to cocaine on the dopaminergic cells in the rat retina. An immunocytochemical and neurochemical study.

There is a growing consensus that the development of the eye is affected by prenatal exposure to cocaine. Considering that the retina is affected by prenatal cocaine exposure, that this drug affects the dopaminergic systems, that the dopaminergic cells in the retina show a well-defined pattern of development and that they can be specifically stained in wholemounts by the antibody anti-tyrosine hydroxylase (TH), this study was undertaken to evaluate the effects of in utero cocaine exposure on the dopaminergic cells of the rat retina. Pregnant Wistar rats were given 60 mg (kg body weight)-1 day-1 of cocaine hydrochloride, subcutaneously, from gestational days 8 to 22. Control groups of pregnant rats were pair-fed. At PND14, 30 and 90, male offspring from different litters were perfused with fixative and the retinas processed as wholemounts and immunostained with the antibody anti-TH. Rats from other groups were decapitated at the same post-natal ages, the retinas dissected and processed by neurochemical techniques to measure the concentrations of dopamine, its metabolites and the turnover of dopamine. There was a significant increase of the retina surface area between PND14-30 in the control group, which was not found in the cocaine group. The density of the immunostained small TH cells was lower in the cocaine groups. No drug-effects were detected in the density of the large TH cells. The densities of the total large and small cells in the superior, inferior and nasal hemiretinas were similar to those found in the whole retinas; however, in the temporal hemiretinas of the cocaine groups, the density of the large TH cells was higher and of the small TH cells was lower than in controls, resulting in an absence of effects on the total density of TH-cells in this hemiretina. A transient increase in the level of dopamine metabolite (DOPAC) and of the turnover of dopamine at PND14 was detected in the cocaine groups. All quantitative parameters reached normal values, in all groups, at PND90. These results show that, during the critical periods in which catecholamines can influence the development of neurons, cocaine transiently affects the pattern of dopaminergic neurons in the retina. This may have functional importance due to the role of this neurotransmitter as a regulatory and/or trophic factor in developing neuronal circuitries.

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.

Are there dopaminergic ganglion cells in the mammalian retina?

New improvements of the tyrosine hydroxylase immunocytochemical labelling in wholemounts allowed us to visualize tyrosine hydroxylase-immunoreactive fibres in the nerve fibre layer of the mouse, rat and macaque monkey retinas. These fibres could not be related to labelled somata in the retina. Their possible origin from ganglion cells or brain nuclei is discussed.

Distribution and spatial geometry of dopamine interplexiform cells in the retina. II. External arborizations in the adult rat and monkey.

The morphology and distribution of dopaminergic interplexiform cells in adult rat and monkey retinas were analyzed to determine any correlation with the function of dopamine in the outer retinal layers. The retinas were processed as whole mounts for tyrosine hydroxylase immunohistochemistry. There was a network formed by the sclerally directed processes of interplexiform cells in the inner nuclear, outer plexiform, and outer nuclear layers running throughout the retina. Their density was higher in the superior retina than in the inferior retina of the rat and was especially high in the superior temporal quadrant. The external network in this quadrant was significantly less dense in the monkey than in the rat, as are the interplexiform cells. The somata of interplexiform and other dopaminergic cells were about the same size in both rats and monkeys. Computer-assisted reconstruction of external arborizations of individual cells showed that external processes lay very close to horizontal and photoreceptor cells and also to blood capillaries. Because they were long, thin, and highly varicose; branched at right angles; and often arose from an axon hillock, the external processes were identified as axons. Therefore, we define the dopaminergic interplexiform cells as multiaxonal neurons, with at least one outwardly directed axon that reaches the outer plexiform layer. The function of the network of external processes from the interplexiform dopaminergic cells is discussed in terms of modulating the release of dopamine to external layers.