Juan M. Lara

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.

Characterization of Pax2 expression in the goldfish optic nerve head during retina regeneration.

The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2+ astrocytes located in the ONH and S100+ astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2+ and S100+ astrocytes have different responses. At 7 days after injury the number of Pax2+ cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100+ astrocytes in the retina surrounding the ONH and S100+ processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100+ astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2+ cells increase. At the same time, S100+/GFAP+/GS+ astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2+/PCNA+ astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2+ astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process.

The Neuroglia in the CNS of Teleosts

Although the concept of neuroglia, or “nervenkitt” (Virchow, 1846) is prior to the identification in the nervous system of cells distinct from neurons (Deiters, 1865), only once the concept of tissue and cellular theory are accepted can the glia be considered as an integral part of the nervous tissue, differentiated from the neurons, but closely associated with them, both spatially and functionally.