Regulation of axonal EphA4 forward signaling is involved in the effect of EphA3 on chicken retinal ganglion cell axon growth during retinotectal mapping.

The Eph and ephrins are involved in the genesis of topographic ordered connections at the visual system. Previously we demonstrated that tectal EphA3 stimulates axon growth of nasal retinal ganglion cells (RGCs) toward the caudal tectum preventing them from branching in the rostral tectum. Now we investigated whether tectal EphA3 plays this role by modulating the axonal EphA4 forward signaling or throughout axonal ephrin-As reverse signaling. For this purpose we used cultures of nasal retinal explants and dissociated retinal neurons from chicken embryos. We treated them with clustered EphA3-Fc, Fc (control), PI-PLC (sheds ephrin-As) or KYL (inhibits ephrin-As-mediated EphA4 activation). We achieved in vitro and in vivo electroporations of chicken embryo retinas with wild type EphA4, Ki-EphA4 (kinase inactive dominant negative EphA4) or EGFP in pMES expression vector. We performed immunocytochemistry, immunoprecipitation and Western blot against Eph/ephrin-As system. Our results showed that: 1) shedding of ephrin-As and the inhibition of ephrin-A-mediated EphA4 activity increase axon length and decrease axonal interstitial filopodia density of nasal RGCs; and 2) a dominant negative form of EphA4 increases axon growth in vitro and induces nasal RGC axons to grow passing throughout their target area in the caudal tectum meanwhile overexpression of EphA4 produces the opposite effects. All together, these results demonstrate that ephrin-A-mediated EphA4 forward signaling decreases the level of axon growth and increases the density of axonal interstitial filopodia of nasal RGCs. Besides, our results showed that: 3) EphA3 ectodomain increases axon growth and decreases the density of axonal interstitial filopodia and branching in vitro and in vivo and 4) EphA3 ectodomain diminishes the ephrin-A2/EphA4 colocalization, and the EphA4 and ephexin1 phosphorylation. All together, these results show that the EphA3 ectodomain produces the opposite effects than the EphA4 forward signaling, by decreasing this signaling pathway throughout competing with EphA4 for ephrin-As binding. Furthermore, it is proposed that tectal EphA3 participates in the establishment of retinotectal mapping throughout this mechanism and that EphAs can regulate axon growth and branching by modulating other EphA receptors forward signaling.

Embryonic neural stem cells in a 3D bioassay for trophic stimulation studies.

Progenitors were discovered in the corpus striatum several years ago, but little is known about their proliferation and differentiation. The aim of this study was to analyze embryonic progenitor cells from the corpus striatum using a bioassay with trophic stimulation. Primary cells obtained from brains of rat embryos at E13-14 were dissected from striatum niches and cultured in stem cell media. These floating dispersed cells clumped together to forming floating bodies like irregular spheres (spheroids), which were placed in type I collagen gel and cultured under basal conditions or with the addition of NGF, NT-3, or NTN. Optimum growth of neurites was obtained, and after 24 and 48 h, they were measured for number and length. The expression of proliferation markers such as PCNA and Ki67, and of neural progenitor markers such as GFAP, nestin, vimentin, O4, A2B5, Pax6, S100, TubIII, and NeuN, was then analyzed. The initial behavior in cell cultures showed distinguishable spheroids that, when placed in 3D gels and with trophic support, generated neurites. A similar effect was observed in glial cell outgrowth from the spheroids. Our assay showed high reproducibility, short culture time, and high resolution for tracing neuron-neurite outgrowth or visualizing glial outgrowth in a few hours.

Anaglyph of retinal stem cells and developing axons: selective volume enhancement in microscopy images.

Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high-quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three-dimensional (3-D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high-quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3-D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Key roles of Ephs and ephrins in retinotectal topographic map formation.

Cellular and molecular mechanisms involved in the development of topographic ordered connections in the central nervous system (CNS) constitute a key issue in neurobiology because neural connectivities are the base of the CNS normal function. We discuss the roles of the Eph/ephrin system in the establishment of retinotopic projections onto the tectum/colliculus, the most detailed studied model of topographic mapping. The expression patterns of Ephs and ephrins in opposing gradients both in the retina and the tectum/colliculus, label the local addresses on the target and give specific sensitivities to growth cones according to their topographic origin in the retina. We postulate that the highest levels of these gradients could signal both the entry as well as the limiting boundaries of the target. Since Ephs and ephrins are membrane-bound molecules, they may function as both receptors and ligands producing repulsive or attractant responses according to their microenvironment and play central roles in a variety of developmental events such as axon guidance, synapse formation and remodeling. Due to different experimental approaches and the inherent species-specific differences, some results appear contradictory and should be reanalyzed. Nevertheless, these studies about the roles of the Eph/ephrin system in retinotectal/collicular mapping support general principles in order to understand CNS development and could be useful to design regeneration therapies.

Glial-derived neurotrophic factor (GDNF) prevents ethanol (EtOH) induced B92 glial cell death by both PI3K/AKT and MEK/ERK signaling pathways.

We investigated the neuroprotective effect of glial-derived neurotrophic factor (GDNF) upon alcohol-exposed B92 cultures, as well as the role of the cytoskeleton and mitogen-activated protein kinase (MAPK) pathways in this effect. Ethanol (EtOH) was added to cultures, either alone or in combination with 30 ng/ml GDNF. Exposure to EtOH (86 and 172 mM; 60 and 120 min) increased the frequency of apoptotic cells identified by nuclear DNA staining with 4,6-diamidino-2-phenylindole (DAPI). Cultures treated with GDNF showed a decrease in ethanol-induced apoptosis. A jun N-terminal kinase (JNK) pathway is activated by EtOH and their pharmacological inhibition (by SP600125) neutralized ethanol-induced apoptosis, suggesting a role for JNK in EtOH neurotoxicity. Immunocytochemically detected phospho-JNK (p-JNK) showed an unusual filamental expression, and localized together with actin stress fibers. Examination of the cytoskeleton showed that EtOH depolymerized actin filaments, inducing p-JNK dissociation and translocation to the nucleus, which suggests that released p-JNK may contribute to glial cell death after EtOH exposure. Treatment with GDNF, in turn, may neutralize the ethanol-induced cell death pathway. Either a phosphatidylinositol 3-kinase (PI3K)/AKT pathway inhibitor (LY294002) or an inhibitor of the extracellular signal-regulated kinase (ERK) 1, 2 pathways (UO126) failed to neutralize GDNF protective effects. However, the simultaneous use of both inhibitors blocked the protective effect of GDNF, suggesting a role for both signaling cascades in the GDNF protection. These findings provide further insight into the mechanism involved in ethanol-induced apoptosis and the neurotrophic protection of glial cells.

GLIA: A reassessment based on novel data on the developing and mature central nervous system.

Several studies on neurobiology have contributed to our understanding of the genesis, survival and death of neurons, unquestionably the stars of the Central Nervous System (CNS). However, they would not be so famous without their close associates: the glial cells. Since novel studies have demonstrated new and important functions for glial cells, they are beginning to gain significant importance in brain research to allow us to reinterpret long known functions on the basis of new concepts. Here, we review recent progress in our understanding of the role of glial cells in the biology of tissue development and maturation.