Differential expression of connexins during histogenesis of the chick retina

Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species. (C) 2008 Wiley Periodicals, Inc.

Identification of small Sca-1(+), Lin(-), CD45(-) multipotential cells in the neonatal murine retina

OBJECTIVE: Bone marrow contains a subset of stem cells that give rise to nonhematopoietic lineages. These nonhematopoietic stem cells appear heterogeneous and contain cells committed to mesenchymal and endothelial lineages, as well as more primitive multipotential cells resembling progenitors of germ cells and very small embryonic/epiblast-like stem cells (VSELs). Nonhematopoietic stem cells can be mobilized from the bone marrow in response to tissue injury, and cells with similar properties have been found in cord blood and normal adult organs. However, the relationship between bone marrow cells and these adult organ stem cells is still unclear. The differentiation potential of some adult stem cells is organ-restricted, but other populations appear to retain multipotential capacity. MATERIALS AND METHODS: A population of small Sca-1(+), lineage-negative (Lin(-)), CD45(-) cells resembling VSELs were isolated from neonatal mouse retina by cell sorting. Differentiation of the cells in culture was achieved by exposure to embryonic stem cell differentiation protocols. RESULTS: VSEL-like cells comprise 1.5% of the neonatal mouse retina. They remain quiescent during retinal differentiation, and thus they do not contribute to normal retinal development. However, they display eye cell differentiation potential in culture and they are also multipotential and can give rise to cells representative of all three embryonic layers. CONCLUSIONS: The neonatal retina is an abundant postnatal source of multipotential VSEL-like cells that can differentiate in culture into a variety of lineages.

Cell fate determination in the vertebrate retina.

In the vertebrate central nervous system, the retina has been a useful model for studies of cell fate determination. Recent results from studies conducted in vitro and in vivo suggest a model of retinal development in which both the progenitor cells and the environment change over time. The model is based upon the notion that the mitotic cells within the retina change in their response properties, or "competence", during development. These changes presage the ordered appearance of distinct cell types during development and appear to be necessary for the production of the distinct cell types. As the response properties of the cells change, so too do the environmental signals that the cells encounter. Together, intrinsic properties and extrinsic cues direct the choice of cell fate.

Does Müller cell differentiation occur prior to the emergence of synapses in embryonic turtle retina?

Müller cells are the main glial cells in the retina, and are related to plexiform layer activity. Recent studies have demonstrated that Müller cells are involved in the synaptic conservation, plasticity, development and metabolism of glutamate. During turtle retinal development, layers, cells and synapses appear at different times. The aim of this research is to study the emergence of Müller cells during embryonic development and their relationship with the synaptogenesis. The authors used retinas from Trachemys scripta elegans embryos at stages S14, 18, 20, 23, and 26. Some retinas were processed with immunocytochemistry in order to detect the presence of glutamine synthetase in Müller cells, which was used as a marker of these cells. Other retinas from the same stages were processed for ultrastructural studies. Samples were observed in confocal and transmission electron microscopes, respectively. The present results show that glutamine synthetase expression in Müller cells occurs at S18, before the emergence of the retinal layers and the early synapses.

Recessive Mutations in RTN4IP1 Cause Isolated and Syndromic Optic Neuropathies

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The role of Bmi1 in CNS development and in retinal degeneration

SUMMARY : The present work addresses several aspects of cell cycle regulation, cell fate specification and cell death in the central nervous system (CNS), specifically the cortex and the retina. More precisely, we investigated the role of Bmi1, a polycomb family gene required for stem cell proliferation and self-renewal, in the development of the cerebral cortex, as well as in the genesis of the retina. These data, together with studies published during the last two decades concerning cell cycle re-activation in apoptotic neurons in the CNS, raised the question of a possible link between regulation of the cell cycle during development and during retinal degeneration. 1. The effects of Bmi1 loss in the cerebral cortex : Consistently with our and others' observations on failure of Bmi9-/- stem cells to proliferate and self-renew in vitro, the Bmi9-/- cerebral cortex presented slight defects in proliferation in stem/progenitor cells compartments in vivo. This was in accordance with the pattern of Bmi1 expression in the developing forebrain. The modest proliferation defects, compared to the drastic consequences of Bmi9 loss in vitro, suggest that cell-extrinsic mechanisms may partially compensate for Bmi1 deletion in vivo during cortical histogenesis. Nevertheless, we observed a decreased proliferating activity in neurogenic regions of the adult telencephalon, more precisely in the subventricular zone, showing that Bmi1 controls neural stem/progenitor proliferation during adulthood in vivo. Our data also highlight an increased production of astrocytes at birth, and a generalized gliosis in the adult Bmi9-/- brain. Importantly, glial progenitors and astrocytes retained the ability to proliferate in the absence of Bmi1. 2. The effects of Bmi1 loss in the retina : The pattern of expression of Bmi1 during development and in the adult retina suggests a role for Bmi1 in cell fate specification and differentiation rather than in proliferation. While the layering and the global structure of the retina appear normal in Bmi1 /adult mice, immunohistochemìcal analysis revealed defects in the three major classes of retinal interneurons, namely: horizontal, bipolar and amacrine cells. Electroretinogram recordings in Bmi9-/- mice are coherent with the defects observed at the histological level, with a reduced b-wave and low-profile oscillatory potentials. These results show that Bmi1 controls not only proliferation, but also cell type generation, as previously observed in the cerebellum. 3. Cell cycle events and related neuroprotective strategies in retinal degeneration : In several neurodegenerative disorders, neurons re-express cell cycle proteins such as cyclin dependent kinases (Cdks) prior to apoptosis. Here, we show for the first time that this is also the case during retinal degeneration. Rd1 mice carry a recessive defect (Pdeóbrd/rd) that causes retinal degeneration and serves as a model of retinitis pigmentosa. We found that photoreceptors express Cdk4 and Cdk2, and undergo DNA synthesis prior to cell death. To interfere with the reactivation of Cdk-related pathways, we deleted E2fs or Brni1, which normally allow cell cycle progression. While deleting E2f1 (downstream of Cdk4/6) in Rd1 mice provides only temporary protection, knocking out Bmi1 (upstream of Cdks) leads to an extensive neuroprotective effect, independent of p16ink4a or p19arf, two tumor suppressors regulated by Bmi1. Analysis of Cdks and the DNA repair-related protein Ligase IV showed that Bmi1 acts downstream of DNA repair events and upstream of Cdks in this neurodegenerative mechanism. Expression of Cdks during an acute model of retinal degeneration, light damage-induced photoreceptor death, points to a role for Bmi1 and cell cycle proteins in retinal degeneration. Considering the similarity with the cell cycle-related apoptotic pathway observed in other neurodegenerative diseases, Bmi1 is a possible general target to prevent or delay neuronal death. RESUME : Ce travail aborde plusieurs aspects de la régulation du cycle cellulaire, de la spécification du devenir des cellules et de la mort cellulaire dans le système nerveux centrale (SNC), plus particulièrement dans le cortex cérébral et dans la rétine. Nous nous sommes intéressés au gène Bmi1, appartenant à la famille polycomb et nécessaire à la prolifération et au renouvellement des cellules souches. Nous avons visé à disséquer son rôle dans le développement du cortex et de la rétine. Ces données, ainsi qu'une série de travaux publiés au cours des deux dernières décennies concernant la réactivation du cycle cellulaire dans les neurones en voie d'apoptose dans le SNC, nous ont ensuite poussé à chercher un lien entre la régulation du cycle cellulaire pendant le développement et au cours de la dégénérescence rétinienne. 1. Les effets de l'inactivation de Bmi1 dans le cortex cérébral : En accord avec l'incapacité des cellules souches neurales in vitro à proliférer et à se renouveler en absence de Bmi1, le cortex cérébral des souris Bmi1-/- présente de légers défauts de prolifération dans les compartiments contenant les cellules souches neurales. Ceci est en accord avec le profil d'expression de Bmi1 dans le télencéphale. Les conséquences de la délétion de Bmi1 sont toutefois nettement moins prononcées in vivo qu'in vitro ; cette différence suggère l'existence de mécanismes pouvant partiellement compenser l'absence de Bmi1 pendant la corticogenèse. Néanmoins, l'observation d'une réduction de la prolifération dans la zone sous-ventriculaire, la zone majeure de neurogenèse dans le télencéphale adulte, montre que Bmi1 contrôle la prolifération des cellules souche/progénitrices neurales chez la souris adulte. Nos résultats démontrent par ailleurs une augmentation de la production d'astrocytes à la naissance ainsi qu'une gliose généralisée à l'état adulte chez les souris Bmi1-/-. Les progéniteurs gliaux et les astrocytes conservent donc leur capacité à proliférer en absence de Bmi1. 2. Les effets de l'inactivation de Bmi1 dans la rétine : Le profil d'expression de Bmi1 pendant fe développement ainsi que dans la rétine adulte suggère un rôle de Bmi1 dans la spécification de certains types cellulaires et dans la différentiation plutôt que dans la prolifération. Alors que la structure et la lamination de la rétine semblent normales chez les souris Bmi1-/-, l'analyse par immunohistochimie amis en évidence des défauts au niveau des trois classes d'interneurones rétiniens (les cellules horizontales, bipolaires et amacrines). Les électrorétinogrammes des souris Bmi1-/- sont cohérents avec les défauts observés au niveau histologique et montrent une réduction de l'onde « b » et des potentiels oscillatoires. Ces résultats montrent que Bmi1 contrôle la génération de certaines sous-populations de neurones, comme démontré auparavant au niveau de cervelet. 3. Réactivation du cycle cellulaire et stratégies théraoeutiaues dans les dégénérescences rétiniennes : Dans plusieurs maladies neurodégénératives, les neurones ré-expriment des protéines du cycle cellulaire telles que les kinases cycline-dépendantes (Cdk) avant d'entrer en apoptose. Nous avons démontré que c'est aussi le cas dans les dégénérescences rétiniennes. Les souris Rd1 portent une mutation récessive (Pde6brd/rd) qui induit une dégénérescence de la rétine et sont utilisées comme modèle animal de rétinite pigmentaire. Nous avons observé que les photorécepteurs expriment Cdk4 et Cdk2, et entament une synthèse d'ADN avant de mourir par apoptose. Pour interférer avec la réactivation les mécanismes Cdk-dépendants, nous avons inactivé les gènes E2f et Bmi1, qui permettent normalement la progression du cycle cellulaire. Nous avons mis en évidence que la délétion de E2f1 (en aval de Cdk4/6) dans les souris Rd1 permet une protection transitoire des photorécepteurs. Toutefois, l'inactivation de Bmi1 (en amont des Cdk) est corrélée à une neuroprotection bien plus durable et ceci indépendamment de p16ink4a et p19arf, deux suppresseurs de tumeurs normalement régulés par Bmi1. L'analyse des Cdk et de la ligase IV (une protéine impliquée dans les mécanismes de réparation de l'ADN) a montré que Bmi1 agit en aval des événements de réparation de l'ADN et en amont des Cdk dans la cascade apoptotique dans les photorécepteurs des souris Rd1. Nous avons également observé la présence de Cdk dans un modèle aigu de dégénérescence rétinienne induit par une exposition des animaux à des niveaux toxiques de lumière. Nos résultats suggèrent donc un rôle général de Bmi1 et des protéines du cycle cellulaire dans les dégénérescences de la rétine. Si l'on considère la similarité avec les événements de réactivation du cycle cellulaire observés dans d'autres maladies neurodégénératives, Bmi1 pourrait être une cible thérapeutique générale pour prévenir la mort neuronale.

Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for 125I-a-bungarotoxin was reduced. Despite the presence of a8-like immunoreactivity at DIV4, functional responses mediated by a-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the a-bungarotoxin-sensitive response at DIV5. Therefore, a-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an a-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by a-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express a-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse.

The mechanisms regulating retinal ganglion cell (RGC) development are crucial for retinogenesis and for the establishment of normal vision. However, these mechanisms are only vaguely understood. RGCs are the first neuronal lineage to segregate from pluripotent progenitors in the developing retina. As output neurons, RGCs display developmental features very distinct from those of the other retinal cell types. To better understand RGC development, we have previously constructed a gene regulatory network featuring a hierarchical cascade of transcription factors that ultimately controls the expression of downstream effector genes. This has revealed the existence of a Pou domain transcription factor, Pou4f2, that occupies a key node in the RGC gene regulatory network and that is essential for RGC differentiation. However, little is known about the genes that connect upstream regulatory genes, such as Pou4f2 with downstream effector genes responsible for RGC differentiation. The purpose of this study was to characterize the retinal function of eomesodermin (Eomes), a T-box transcription factor with previously unsuspected roles in retinogenesis. We show that Eomes is expressed in developing RGCs and is a mediator of Pou4f2 function. Pou4f2 directly regulates Eomes expression through a cis-regulatory element within a conserved retinal enhancer. Deleting Eomes in the developing retina causes defects reminiscent of those in Pou4f2(-/-) retinas. Moreover, myelin ensheathment in the optic nerves of Eomes(-/-) embryos is severely impaired, suggesting that Eomes regulates this process. We conclude that Eomes is a crucial regulator positioned immediately downstream of Pou4f2 and is required for RGC differentiation and optic nerve development.

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Vascular endothelial growth factor-B and retinal vascular development in the mouse

Purpose: Vascular endothelial growth factor-A (VEGF-A) is crucial to retinal vascular growth, both normal and pathological. VEGF-B, recently characterized, is reported to be expressed in retinal tissues, but the importance of VEGF-B to retinal vascular development remained unknown. The aim of this study was to analyse retinal vascular growth in the Vegfb (-/-) knockout mouse. Methods: Retinal vascular growth was measured in Vegfb (-/-) knockout mice raised under normal conditions, and Vegfb (-/-) knockout mice with an oxygen-induced proliferative retinopathy. Wild type Vegfb (+/+) mice served as controls. Vessels were perfused with ink and retinal flatmounts secondarily labelled with FITC-lectin (BS-1, Griffonia simplicifolia ). Area and diameter of retinal growth and retinal vascular growth were recorded over days 0-20, and capillary density and mean diameter recorded from day 17 pups. Results: A variety of techniques confirmed that Vegfb (+/+) mice expressed VEGF-B and that VEGF-B expression was absent in Vegfb (-/-) mice. Vegfb (-/-) mice raised in room air showed no significant differences from Vegfb (+/+) controls. No differences were found in oxygen-induced retinopathy between Vegfb (-/-) and Vegfb (+/+) pups in either the extent of the initial oxygen-induced ablation, or in the regrowth of retinal vessels or vitreal (neovascular) sprouts; vitreal sprouts are important markers of the abnormal proliferative response, and are maximally expressed on day 17 in this model of oxygen-induced retinopathy. Conclusions: These results indicate that a lack of VEGF-B does not significantly affect development of the retinal vasculature under normal conditions, nor does it appear to affect the proliferative retinal responses seen in oxygen-induced retinopathy.

Retinal serotonin, eye growth and myopia development in chick

Myopia (short-sightedness) is a visual problem associated with excessive eye growth and vitreous chamber expansion. Within the eye serotonin (5-hydroxytryptamine, 5-HT) appears to have a variety of effects, it alters retinal amacrine cell processing, increases intraocular pressure, constricts ocular blood vessels, and is also mitogenic. This study sought to determine the role of the retinal serotonin system in eye growth regulation. Myopia was produced in 7-day-old chicks using -15 D spectacle lenses (LIM) and form deprivation (FDM). The effect on LIM and FDM of daily intravitreal injections of a combination of 5-HT receptor antagonists (1, 10, 50 mu M), 5-HT2 selective antagonist (Mianserin 0.5, 20 mu M) were assessed. Counts were performed of serotonin and tyrosine hydroxylase positive neurons and the relative density used to account for areal changes due to eye growth. The effect of LIM and lens-induced hyperopia (LIH) on the numbers of 5-HT-containing amacrine cells in the retina were then determined. The combination of the 5-HT receptor antagonists inhibited LIM by approximately half (1 mu M RE: -7.12 +/- 1.0 D, AL: 0.38 +/- 0.06 mm vs. saline RE: -13.19 +/- 0.65 D, AL: 0.64 +/- 0.03 mm. RE: p < 0.01, AL: p < 0.01), whereas FDM was not affected (1 mu M RE: -8.88 +/- 1.10 D). These data suggest that serotonin has a stimulatory role in LIM, although high doses of serotonin were inhibitory (1 mu M RE: -9.30 +/- 1.34 D). 5-HT immunoreactivity was localised to a subset of amacrine cell bodies in the inner nuclear layer of the retina, and to two synaptic strata in the inner plexiform layer. LIM eyes had increased numbers of 5-HT-containing amacrine cells in the central retina (12.5%). Collectively, these results suggest that manipulations to the serotonin system can alter the eye growth process but the role of the transmitter system within this process remains unclear. (c) 2005 Elsevier Ltd. All rights reserved.