Almudena Velasco

Response of microglial cells after a cryolesion in the peripheral proliferative retina of tench

We studied the glial response after inducing a lesion in the zone of the peripheral retina of tench, where there is proliferative neuroepithelium. In the retina and optic nerve, the microglial response was analysed with tomato lectin and the macroglial response with antibodies against GFAP and S-100. In lesioned retinas, there was a temporal-spatial distribution pattern of microglia. One day after lesion, primitive ramified cells appeared in the nerve fibre layer. These cells appeared progressively from the vitreal to the scleral layers until day 7 when cells appeared in all layers, with the exception of the outer plexiform layer. From this point, labelling decreased. In the optic nerve, 3 days after lesion, an increase in the number of microglial cells was observed, first in the nerve folds and from day 15 in specific areas of the optic nerve. In the central retina, in the optic nerve head and within the optic nerve itself, the appearance of microglial cells, after the lesion, near the blood vessels, could indicate a vascular origin of microglia, as has been proposed by many authors. However, we cannot discount the idea that some of the reactive microglial cells arise by proliferation of the microglia existing in the normal state. Using GFAP and S-100 antibodies, no important changes in the retina were observed, however in the optic nerve there was response to the lesion. Thus, the macroglial cells appeared to be involved in reorganisation of the optic nerve axons after lesion.

A Comparative Study of Protein Kinase C-Like Immunoreactive Cells in the Retina

The present study is a morphological and quantitative analysis of protein kinase C-like immunoreactive (PKC-L ir) bipolar cells in the retinas of five different vertebrate species (chicken, tench, zebrafish, goldfish and rat). The morphology of PKC-L-ir bipolar cell axon terminals in fish differs significantly from those of chicken and rat retinas. Fish have bulky terminals whereas chicken and rat have their terminals in the form of small knob-shaped branches. In tench and goldfish, PKC-L-ir bipolar cells gradually decrease in size from the medial (i.e., in tench: mean ± SD soma area of 30.09 ± 5.98 µm2) to the peripheral (i.e., in tench: 19.93 ± 1.73 µm2) retinal regions. This is not observed in chicken, rat or zebrafish where there is more homogeneity in s oma and axon terminal sizes between different retinal regions. Except in chicken, cell density increases from the central (i.e., in tench: mean ± SD 1795.88 ± 242.35 cells/mm2) to the peripheral (i.e., in tench: 4295.41 ± 279.23 cells/mm2) retina. This study provides data that show relevant differences in the PKC-L-ir bipolar morphology and density among birds, fish and mammals. Moreover, these structural variations could mean not only differences in the cellular physiology, but also in the patterns of development and maintenance of the retina in each species.

Protein kinase C-like immunoreactive cells in embryo and adult chicken retinas.

Morphological evidence of a temporal parallelism between the appearance of the alpha isoform of protein kinase C (PKC) and some processes such as synaptogenesis in the plexiform layers of the chicken retina is offered. Immunostaining experiments were performed throughout embryonic, young and adult chicken life. The results help to understand the development of rod bipolar cells.

Enzyme histochemical identification of microglial cells in the retina of a fish (Tinca tinca).

Histochemistry for nucleoside diphosphatase was used to study the microglial cells in the adult tench retina. An abundant population of microglial cells was located in the vascular membrane, nerve fibre layer, inner and outer plexiform layers and scattered cells were observed in the inner nuclear layer. Rounded and amoeboid cells could be seen close to the vessel in the vascular membrane, bipolar cells in the nerve fibre layer and ramified cells in the rest of the layers. Several microglial forms could correspond to developing cells. The pattern of distribution was similar to that described in other vertebrates, but with several differences, such as the presence of microglial cells in the vascular membrane and inner nuclear layer and the overlap of processes in the plexiform layers.

Modifications of the retina neuronal populations of the heterozygous mutant small eye mouse, the SeyDey

We analyzed the modifications of the retinal neurons in a heterozygous mutant small eye mouse, the Sey(Dey). This mouse presents a mutation in chromosome 2 which affects the gene Pax6 and other nearby genes, such as the Wt1 gene and the gene of the Reticulocalbin. The eyes of these animals do not have lenses and their retinas present important morphological alterations: in the anterior portion they are joined to the cornea, they are found detached from the pigment epithelium, they present folds that form rosettes in some zones and alteration of the lamination can be observed. The partial loss of the genes affected does not prevent the formation of the different layers of the retina, but does affect its thickness, principally of the plexiform layers; moreover, the internal limiting membrane is found disorganized. All the neuronal populations are present in the retina of these animals and express the same neurochemical markers as the control animals, but the number of Pax6(+) cells is notably reduced. In these retinas a marked disorganization of the distribution of the dendrites and axons is observed and a notable reduction in the axons of ganglion cells. These results suggest that, although it does not appear determinant in the differentiation of the distinct neuronal types of the retina, the partial lack of genes of the heterozygotes +/Sey(Dey) provokes important morphological and neurochemical modifications in the cytoarchitecture of the retina.

Cytoarchitectonic and neurochemical differentiation of the visual system in ethanol-induced cyclopic zebrafish larvae.

Embryonic exposure to ethanol leads to malformations such as cyclopia. Cyclopic embryos present fused eyes and lack of the ventral specification of the brain, with physiological and morphological defects in the visual system, which provides a useful model for teratology and neurotoxicity assessments. We analysed the differentiation of the visual areas in the ethanol-induced cyclopic animals. For this purpose we exposed zebrafish embryos to 1.5% ethanol from 4 hours post-fertilisation (hpf) to 24 hpf in order to get cyclopic embryos. We monitored cytoarchitecture and quantified both the proliferation rate and cell differentiation from 2 days post-fertilisation (dpf) onwards, focusing on the main components of the visual system (retina, optic nerve and optic tectum) of normal and cyclopic zebrafish embryos. The visual system of the zebrafish embryos is affected by exposure to ethanol; two optic nerves that fuse before leaving the eyes are present in cyclopic specimens but an optic chiasm is not evident. Cell differentiation is severely delayed throughout the visual system at 2 dpf. At 5 dpf, lamination in the cyclopic retina and optic tectum is completed, but they are filled with pyknotic nuclei demonstrating cell death. At this stage the proliferation rate and expression patterns are unaltered and glial and neuronal neurochemical differentiations are similar to untreated animals. We found that the alterations produced by exposure to ethanol are not only cell-selective, but also tissue-selective. Cyclopia is the most severe phenotype induced by ethanol, although cell differentiation and proliferation can reach normal patterns after a certain period of time, which points to a neural plasticity process. Zebrafish embryos may possess a compensation mechanism against the ethanol effect, which would account for their use for pharmacogenetic and chemical screenings in the analysis of new molecules that could improve visual problems.

Growing and regenerating axons in the visual system of teleosts are recognized with the antibody RT97

We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual system tissues (105, 115, 160, 200, 325 and 335 kDa approximately). By using immunoprecipitation and Western blot we have found that after crushing the optic nerve the immunoreactivity to anti RT97 increased markedly in the optic nerve. In immunohistochemical analysis we also found a different pattern of labeling in normal and regenerating visual pathways. In normal tench RT97 is a good marker for the horizontal cells in the retina, for growing ganglion cell axons which run along the optic nerve from the retina to the optic tectum and of the axon terminals in the stratum opticum and stratum fibrosum and griseum superficiale in the optic tectum. After optic nerve crush, no immunohistochemistry modifications were observed in the retina. However, in accordance with Western blot experiments, in the optic nerve intensely stained groups of regenerating axons appeared progressively throughout the optic nerve as far as the optic tectum. We conclude that the antibody RT97 is an excellent marker of growing and regenerating axons of the optic nerve of fish.

Characterisation of neuronal and glial populations of the visual system during zebrafish lifespan

During visual system morphogenesis, several cell populations arise at different time points correlating with the expression of specific molecular markers We have analysed the distribution pattern of three molecular markers (zn‐1, calretinin and glial fibrillary acidic protein) which are involved in the development of zebrafish retina and optic tectum. zn‐1 is a neural antigen expressed in the developing zebrafish central nervous system. Calretinin is the first calcium‐binding protein expressed in the central nervous system of vertebrates and it is widely distributed in different neuronal populations of vertebrate retina, being a valuable marker for its early and late development. Glial fibrillary acidic protein (GFAP), which is an astroglial marker, is a useful tool for characterising the glial environment in which the optic axons develop.

Sox10 Expression in Goldfish Retina and Optic Nerve Head in Controls and after the Application of Two Different Lesion Paradigms

The mammalian central nervous system (CNS) is unable to regenerate. In contrast, the CNS of fish, including the visual system, is able to regenerate after damage. Moreover, the fish visual system grows continuously throughout the life of the animal, and it is therefore an excellent model to analyze processes of myelination and re-myelination after an injury. Here we analyze Sox10+ oligodendrocytes in the goldfish retina and optic nerve in controls and after two kinds of injuries: cryolesion of the peripheral growing zone and crushing of the optic nerve. We also analyze changes in a major component of myelin, myelin basic protein (MBP), as a marker for myelinated axons. Our results show that Sox10+ oligodendrocytes are located in the retinal nerve fiber layer and along the whole length of the optic nerve. MBP was found to occupy a similar location, although its loose appearance in the retina differed from the highly organized MBP+ axon bundles in the optic nerve. After optic nerve crushing, the number of Sox10+ cells decreased in the crushed area and in the optic nerve head. Consistent with this, myelination was highly reduced in both areas. In contrast, after cryolesion we did not find changes in the Sox10+ population, although we did detect some MBP- degenerating areas. We show that these modifications in Sox10+ oligodendrocytes are consistent with their role in oligodendrocyte identity, maintenance and survival, and we propose the optic nerve head as an excellent area for research aimed at better understanding of de- and remyelination processes.

Effects of retinoic acid exposure during zebrafish retinogenesis

Retinoic acid (RA) is an important morphogen involved in retinal development. Perturbations in its levels cause retinal malformations such as microphthalmia. However, the cellular changes in the retina that lead to this phenotype are little known. We have used the zebrafish to analyse the effects of systemic high RA levels on retinogenesis. For this purpose we exposed zebrafish embryos to 0.1μM or 1μM RA from 24 to 48h post-fertilisation (hpf), the period which corresponds to the time of retinal neurogenesis and initial retinal cell differentiation. We did not find severe alterations in 0.1μM RA treated animals, but the exposure to 1μM RA significantly reduced retinal size upon treatment, and this microphthalmia persisted through larval development. We monitored histology and cell death and quantified both the proliferation rate and cell differentiation from 48hpf onwards, focusing on the retina and optic nerve of normal and 1μM treated animals. Retinal lamination and initial neurogenesis are not affected by RA exposure, but we found widespread apoptosis after RA treatment that could be the main cause of microphthalmia. Proliferating cells increased their number at 3days post-fertilisation (dpf) but decreased significantly at 5dpf maintaining the microphthalmic phenotype. Retinal cell differentiation was affected; some cell markers do not reach normal levels at larval stages and some cell types present an increased number compared to those of control animals. We also found the presence of young axons growing ectopically within the retina. Moreover although the optic axons leave the retina and form the optic chiasm they do not reach the optic tectum. The alterations observed in treated animals become more severe as larvae develop.