Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid

Purpose. In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. Methods. Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. Results. Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [3H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. Conclusions. These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.Instituto Multidisciplinario de Biología Celula

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid

Purpose. In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. Methods. Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. Results. Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [3H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. Conclusions. These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.Instituto Multidisciplinario de Biología Celula

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid

Purpose. In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. Methods. Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. Results. Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [3H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. Conclusions. These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.Instituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

Laser-Degeneration Study of Nerve Fibers in the Optic Nerve

Knowledge about wiring of neurons is one of the most important goals of neurobiology. Neuronal processes -axons and dendrites- degenerate when they are severed from their cell body. Since different staining procedures distinguish between the degenerating axons and their healthy neighbors, most neuroanatomical pathways have been mapped through the follow-up of degenerating axons after spontaneous or experimental lesions at some point of the pathway. Mapping of neuroanatomical connections has been enormously enriched during the past few years, thanks to new labelling techniques with great resolution power [1]. However, the resolution of the older degeneration procedures is only limited by the extent of the lesion and the resolution of the differential staining of degenerating axons. As we will show in this report, the use of a laser to produce small lesions in the retina of birds, coupled to the detection of degenerating axons in semi-thin plastic sections [2] is allowing us to understand the relationship between axons along the optic pathway with a resolution comparable to that of “in vivo” labelling techniques

Two distributions of axons in the optic nerve of quails: a study of nerve degeneraron after láser lesions of the retina

Order in the optic pathway has been under cióse scrutiny since the last century. In fishes, the position of the axons along the optic nerve is a function of the position or of the age of the corresponding neu- ronal body in the retina5’12’13. However, in cats, optic axons only show a crude retinotopic order8. Our observations in the quail showed the existence of at least two kinds of axons according to their position in the optic nerve. Although some axons were found in cióse spatial relationship with others originating in the same región of the retina, other axons occupied positions independent of their site of origin in the retina

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

Distribution of nerve fibers in the optic nerve of the quail: a laser-degeneration study

Normal visual function requires specific connections between the retina and the visual centers. Connections arise after migration of the optic axons to the brain; however, the biological basis of their specificity are mostly unknown. Som9 of the postulated mechanisms require a recognition between axons and brain neurons, whereas others depend on the maintenance of a retinotopic order along the optic pathways. (P&aacute;rrafo extra&iacute;do a modo de resumen)</em

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