Germán Pinzón-Duarte

Brain-Derived Neurotrophic Factor Modulates the Development of the Dopaminergic Network in the Rodent Retina

Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF) (Cellerino and Kohler, 1997). To investigate whether BDNF can influence the development of the retinal dopaminergic pathway, we performed intraocular injections of BDNF during the second or third postnatal week and visualized the dopaminergic system with tyrosine hydroxylase (TH) immunohistochemistry. Both regimens of BDNF treatment caused an increase in TH immunoreactivity in stratum 1 and stratum 3 of the inner plexiform layer (IPL). D2 dopamine receptor immunoreactivity, a presynaptic marker of dopaminergic cells (Veruki, 1996), was also increased in stratum 1 and stratum 3 of the inner plexiform layer. These data suggest that BDNF causes sprouting of dopaminergic fibers in the inner plexiform layer. Other neurochemical systems, for example, the cholinergic amacrine cells, remained unaffected. Similar effects were observed after injections of neurotrophin-3 and neurotrophin-4, but not nerve growth factor. Analysis of whole-mounted TH-immunolabeled retinae revealed hypertrophy of dopaminergic cells (+41% in soma areas; p < 0.01) and an increase of labeled dopaminergic varicosities in stratum 1 of the IPL (+51%;p < 0.01) after BDNF treatment. The opposite was observed in mice homozygous for a null mutation of thebdnf gene: dopaminergic cells were atrophic (−22.5% in soma areas; p < 0.05), and the density of TH-positive varicosities in stratum 1 was reduced (57%;p < 0.01). We conclude that BDNF controls the development of the retinal dopaminergic network and may be particularly important in determining the density of dopaminergic innervation in the retina.

Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture

This study was focused on the analysis of cell type differentiation and synaptogenesis as well as outer segment formation in an organotypic culture of the neonatal rat retina during a 6-14 day period of in vitro development. Moreover, the effects of the retinal pigment epithelium (RPE) on these processes were investigated. The in vitro development resulted in a retinal architecture and lamination comparable to that of in vivo retinas. The RPE influences the proper alignment of photoreceptors as well as the formation of the outer limiting membrane (OLM), but not processes of cell differentiation, synaptogenesis and inner retinal lamination.

Defective Formation of the Inner Limiting Membrane in Laminin β2- and γ3-Null Mice Produces Retinal Dysplasia

PURPOSE Retinal basement membranes (BMs) serve as attachment sites for retinal pigment epithelial cells on Bruchs membrane and Müller cells (MCs) on the inner limiting membrane (ILM), providing polarity cues to adherent cells. The beta2 and gamma3 chains of laminin are key components of retinal BMs throughout development, suggesting that they play key roles in retinal histogenesis. This study was conducted to analyze how the absence of both beta2- and gamma3-containing laminins affects retinal development. Methods. The function of the beta2- and gamma3-containing laminins was tested by producing a compound deletion of both the beta2 and the gamma3 laminin genes in the mouse and assaying the effect on postnatal retinal development by using anatomic and electrophysiological techniques. Results. Despite the widespread expression of beta2 and gamma3 laminin chains in wild-type (WT) retinal BMs, the development of only one, the ILM, was disrupted. The postnatal consequence of the ILM disruption was an alteration of MC attachment and a resultant disruption in MC apical-basal polarity, which culminated in retinal dysplasia. Of importance, although their density was altered, retinal cell fates were unaffected. The laminin mutants have a markedly decreased visual function, resulting in part from photoreceptor dysgenesis. Conclusions. These data suggest that beta2 and gamma3 laminin isoforms are critical for the formation and stability of the ILM. These data also suggest that attachment of the MC to the ILM provides important polarity cues to the MC and for postnatal retinal histogenesis.

Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina

Pathologies of retinal blood vessels are among the major causes of blindness worldwide. A key cell type that regulates retinal vascular development is the astrocyte. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. Even though there is a strong correlation between astrocyte distribution and retinal vascular development, the factors that guide astrocytes into the retina remain unclear. In this study, we show that astrocytes migrate within a laminin-containing basement membrane - the inner limiting membrane. Genetic deletion of the laminin β2 and γ3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces β1 integrin expression in astrocytes. Culturing laminin-null retinal astrocytes on laminin substrates restores focal localization of β1 integrin. Finally, we show that laminins containing β2 and γ3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing β2 and γ3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo.

Effects of brain‐derived neurotrophic factor on cell survival, differentiation and patterning of neuronal connections and Müller glia cells in the developing retina

The aim of the present study was to determine the influence of brain‐derived neurotrophic factor (BDNF) on survival, phenotype differentiation and network formation of retinal neurons and glia cells. To achieve a defined concentration and constant level of BDNF over several days, experiments were performed in an organotypic culture of the developing rat retina. After 6 days in vitro, apoptosis in the different cell layers was determined by TUNEL staining and cell‐type‐specific antibodies were used to identify distinct neuronal cell types and Müller cells. Cultured retinas treated with BDNF (100 ng BDNF/mL medium) were compared with untreated as well as with age‐matched in vivo retinas. Quantitative morphometry was carried out using confocal microscopy. BDNF promoted the in vitro development and differentiation of the retina in general, i.e. the number of cells in the nuclear layers and the thickness of the plexiform layers were increased. For all neurons, the number of cells and the complexity of arborizations in the synaptic layers were clearly up‐regulated by BDNF. In control cultures, the synaptic stratification of cone bipolar cells within the On‐ and Off‐layer of the inner plexiform layer was disturbed and a strong reactivity of Müller cell glia was observed. These effects were not present in BDNF‐treated cultures. Our data show that BDNF promotes the survival of retinal interneurons and plays an important role in establishing the phenotypes and the synaptic connections of a large number of neuronal types in the developing retina. Moreover, we show an effect of BDNF on Müller glia cells.

Brain-derived neurotrophic factor regulates expression of vasoactive intestinal polypeptide in retinal amacrine cells

Brain‐derived neurotrophic‐factor (BDNF) is expressed in the retina and controls the development of subtypes of amacrine cells. In the present study we investigated the effects of BDNF on amacrine cells expressing vasoactive intestinal polypeptide (VIP). Rats received three intraocular injections of BDNF on postnatal days (P) 16, 18, and 20. The animals were sacrificed on P22, P40, P60, P80, and P120, and VIP expression in their retinas was detected by immunohistochemistry (P22, P40) and by radioimmunoassay (RIA; P22, P40, P60, P80, P120) to assess the time course of BDNF effects on VIP. A significant increase in the density of VIP‐positive amacrine cells was detected in BDNF‐treated retinas, and VIP concentration was up‐regulated by 150% both at P22 and at P40 with respect to untreated controls. VIP concentration then slowly declined in the treated retinas over a period of 3 months; however, a statistically significant increase of 50% was still detectable on P120. The impact of endogenous BDNF on the regulation of VIP expression in the retina was analyzed in mice homozygous for a targeted deletion of the BDNF gene locus (bdnf−/−). VIP immunohistochemistry revealed a marked reduction of VIP‐positive amacrine cells and of VIP‐immunopositive processes in the inner plexiform layer of the BDNF knockout mice. Mice lacking BDNF expressed only 5% of the VIP protein in their retinas compared with the retinas of wild‐type mice as measured by RIA. Our data show that BDNF is a major regulator of VIP expression in retinal amacrine cells and exerts long‐lasting effects on VIP content. J. Comp. Neurol. 467:97–104, 2003.

The γ3 Chain of Laminin is Widely But Differentially Expressed in Murine Basement Membranes: Expression and Functional Studies

Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an α, a β and a γ chain. Previous studies have shown that the γ3 chain is partnered with either the β1 chain (in placenta) or β2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the γ3 chain is expressed in both apical and basal compartments (Koch et al., 1999; Gersdorff et al., 2005; Yan and Cheng, 2006). This study investigates the expression pattern of the γ3 chain in mouse. We developed three new γ3-reactive antibodies, and we show that the γ3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the γ1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where γ3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the γ3 chain in mice; unlike other laminin null mice (α5, β2, γ1 nulls), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the γ3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system.

In Vivo and In Vitro Development of S- and M-Cones in Rat Retina

PURPOSE Organ cultures of the rodent retina could provide a powerful tool in the study of cone development and differentiation. Previous attempts, however, have failed to show M-cone development in organ cultures of the mouse and rat retina. This study mimicked the in vivo dynamics of S- and M-cone development in a culturing approach for the postnatal rat retina. METHODS Retinas of Brown Norway rats were collected at different developmental ages (postnatal day [P]0-P270) to study cone development in vivo. For culturing, the retinas were prepared from P0 to P2 animals and allowed to develop in organ culture for 2 to 15 days. Subsequently, opsin expression was analyzed immunohistochemically and morphometrically. RESULTS In control retinas, S-opsin was already expressed at birth, whereas M-opsin was detected after P4. The maximum density of S-opsin-positive cones was reached at P10 (∼17,000 cells/mm(2)) and of M-opsin-positive cones, at P12 (∼14,000 cells/mm(2)). The number of both cone types decreased gradually thereafter to ∼1,000 S-opsin cones/mm(2) and ∼4,000 M-opsin cones/mm(2) in the adult. In culture, both cone types developed with dynamics of appearance comparable to those in vivo, with a peak density of ∼12,300 cones/mm(2) for S-opsin and ∼7,500 cones/mm(2) for M-opsin labeling. CONCLUSIONS These results in rat retina showed for the first time that cone development and expression dynamics can be mimicked in organ culture. With this experimental approach, it will be possible to evaluate aspects of cone development under controlled experimental conditions and to elucidate factors crucial for proper cone differentiation.