NGF, TrkA-P and neuroprotection after a hypoxic event in the developing central nervous system

A decrease in the concentration of oxygen in the blood and tissues (hypoxia) produces important, sometimes irreversible, damages in the central nervous system (CNS) both during development and also postnatally. The present work aims at analyzing the expression of nerve growth factor (NGF) and p75 and the activation of TrkA in response to an acute normobaric hypoxic event and to evaluate the possible protective role of exogenous NGF. The developing chick optic tectum (OT), a recognized model of corticogenesis, was used as experimental system by means of in vivo and in vitro studies. Based on identification of the period of highest sensitivity of developmental programmed cell death (ED15) we show that hypoxia has a mild but reproducible effect that consist of a temporal increase of cell death 6 h after the end of a hypoxic treatment. Cell death was preceded by a significant early increase in the expression of Nerve Growth Factor (NGF) and its membrane receptor p75. In addition, we found a biphasic response of TrkA activation: a decrease during hypoxia followed by an increase −4 h later- that temporally coincide with the interval of NGF overexpression. To test the NGF - NGF receptors role in hypoxic cell death, we quantified, in primary neuronal cultures derived from ED15 OT, the levels of TrkA activation after an acute hypoxic treatment. A significant decline in the level of TrkA activation was observed during hypoxia followed, 24 h later, by significant cell death. Interestingly, this cell death can be reverted if TrkA inactivation during hypoxia is suppressed by the addition of NGF. Our results suggest that TrkA activation may play an important role in the survival of OT neurons subjected to acute hypoxia. The role of TrkA in neuronal survival after injury may be advantageously used for the generation of neuroprotective strategies to improve prenatal insult outcomes.Fil: Bogetti, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Pozo Devoto, Victorio Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Rapacioli, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Neurociencia Cognitiva. Fundación Favaloro. Instituto de Neurociencia Cognitiva; ArgentinaFil: Flores, Domingo Vladimir. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Neurociencia Cognitiva. Fundación Favaloro. Instituto de Neurociencia Cognitiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Fiszer de Plazas, Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Digital processing of in situ hybridization images: identification and spatial allocation of specific labels

In situ hybridization (ISH) method allows to reveal specific genes expression, identify specific cell types and detect areas or tissues, displaying differential gene expression. This work describes a standardized procedure of digital image processing that allows detailed analyses of ISH preparations. We have developed a software that allows through a graphical interface (a) to reliably identify and quantify ISH labels, (b) to locate each label within the image reference system (c) to assemble the total series of images obtained from a complete histological sections of a biological structure, and d) to locate all the labels within a unique reference system that corresponds to the complete biological structure. As each ISH label is positioned within a spatial coordinates system coinciding with an intrinsic biological reference axis, this software can be used to analyze the spatial pattern of distribution of specific genes expression during the embryonic development. The software allows the construction of numerical space series that can be used to analyze the variability and the dynamics of genes expression as a function of space and/or time during the embryonic development.Facultad de Informátic

The developing optic tectum: An asymmetrically organized system and the need for a redefinition of the notion of sensitive period

The present article summarizes the main events involved in the isthmic organizer and optic tectum determination and analyses how optic tectum patterning is translated, by the organized operation of several specific cell behaviors, into the terminally differentiated optic tectum. The paper proposes that this assembling of temporally/spatially organized cell behaviors could be incorporated into a wider notion of patterning and that, given the asymmetric organization of the developing optic tectum, the notion of “sensitive period” does not capture the whole complexity of midbrain development and the pathogenesis of congenital disorders. The cell behaviors involved in the optic tectum development are organized in time and space by the isthmic organizer. A comprehensive description of the normal optic tectum development, and also its alterations, should consider both domains. Significantly, the identity of each neuronal cohort depends critically on its “time and place of birth”. Both parameters must be considered at once to explain how the structural and functional organization of the optic tectum is elaborated. The notion of “patterning” applies only to the early events of the optic tectum development. Besides, the notion of “sensitive period” considers only a temporal domain and disregards the asymmetric organization of the developing optic tectum. The present paper proposes that these notions might be re-defined: (a) a wider meaning of the term patterning and (b) a replacement of the term “sensitive period” by a more precise concept of “sensitive temporal/spatial window”.Fil: Rapacioli, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Neurociencia Cognitiva. Fundación Favaloro. Instituto de Neurociencia Cognitiva; ArgentinaFil: Fiszer de Plazas, Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Flores, Domingo Vladimir. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Neurociencia Cognitiva. Fundación Favaloro. Instituto de Neurociencia Cognitiva; Argentin

An improved method to obtain a soluble nuclear fraction from embryonic brain tissue.

This paper describes modifications of the standard methods for obtaining a soluble nuclear fraction from embryonic brain tissue. The main improvements are: (1) the inclusion of a low speed centrifugation step to prevent the appearance of high density contaminants, (2) a sucrose density gradient to remove perinuclear mitochondria and ER membranes and (3) a protein extraction approach which significantly enhances protein yield. To demonstrate the effectiveness of the method, pellets were analyzed by light and electron microscopy and purity of the soluble extracts was immunologically tested. Finally, to illustrate the applicability of this approach, the induction of the transcription factor HIF-1 (hypoxia-inducible factor-1) was assessed by Western blot using soluble nuclear fractions and by immuno-electron microscopy using purified nuclear fractions, both obtained from the optic lobes of chick embryos. In conclusion, the procedure presently described appears to be reliable and convenient for obtaining a pure soluble nuclear fraction from a discrete amount of embryonic brain tissue

Temporal-spatial correlation between angiogenesis and corticogenesis in the developing chick optic tectum.

The developing chick optic tectum is a widely used model of corticogenesis and angiogenesis. Cell behaviors involved in corticogenesis and angiogenesis share several regulatory mechanisms. In this way the 3D organizations of both systems adapt to each other. The consensus about the temporally and spatially organized progression of the optic tectum corticogenesis contrasts with the discrepancies about the spatial organization of its vascular bed as a function of the time. In order to find out spatial and temporal correlations between corticogenesis and angiogenesis, several methodological approaches were applied to analyze the dynamic of angiogenesis in the developing chick optic tectum. The present paper shows that a typical sequence of developmental events characterizes the optic tectum angiogenesis. The first phase, formation of the primitive vascular bed, takes place during the early stages of the tectal corticogenesis along which the large efferent neurons appear and begin their early differentiation. The second phase, remodeling and elaboration of the definitive vascular bed, occurs during the increase in complexity associated to the elaboration of the local circuit networks. The present results show that, apart from the well-known influence of the dorsal-ventral and radial axes as reference systems for the spatial organization of optic tectum angiogenesis, the cephalic-caudal axis also exerts a significant asymmetric influence. The term cortico-angiogenesis to describe the entire process is justified by the fact that tight correlations are found between specific corticogenic and angiogenic events and they take place simultaneously at the same position along the cephalic-caudal and radial axes

Radial organization of the OT cortex during DS1 (ED3; HH20).

<p>(A and C: Hoechst; B: β-III-Tubulin; D: NeuroD). The first post-mitotic neurons accumulate below the outer limiting membrane (OLM) and form an incipient premigratory zone (PMZ). They display the β-III-Tubulin+ cytoplasm labeling typical of the early differentiation neurons (B); they also display the intense NeuroD+ nuclear labeling that characterize the neuronal lineage (D). Dashed line: boundary between the generation zone (GZ) and the PMZ. (Bars: 20 µm).</p

Ingression of primitive vessels into the OT (Early DS2; ED3–6; HH20–29).

<p>(A-D and F-H: H-E; E and I: Notch and Hoechst). (A) Endothelial cells attach to the outer limiting membrane (OLM) and degrade the basal membrane (dotted line). (B) PAS-Stained section reveals the interruption (between arrows) of the basement membrane at a site where a vessel sprouts traverses the pial surface and penetrates the neuroepithelium. (C) Solid sprouts (arrowhead) from the perineural vascular plexus (PNP) penetrate the neuroepithelium and pass through the marginal zone (MZ) toward the premigratory zone (PMZ). (D) Early primitive solid radial vessel (arrowhead) entering the PMZ. (E-F) After entering the PMZ the tip of the growing vessel develops a lumen (arrows). (G-J) Transverse sections of primitive radial vessels in different stages of development (Bars: A: 5 µm; B-J: 10 µm).</p

Space-dependent differences in the OT corticogenesis and vasculature development along the cph-cd axis (ED6; DS2-DS4; HH29) (A-C: Notch).

<p>(A) OT caudal pole: formation of primitive radial vessels (arrowheads); (B) Middle zone: generation of oblique or tangential terminal branches (arrows); and (C) OT cephalic pole: increase in complexity of the vascular network (Bars: 20 µm).</p

Recording of the angles that define the vessels growth direction.

<p>(A) Spatial reference axes of the OT cortex. (B) Ascending (pial-caudal) collateral branches. (C) Descending (ventricular-cephalic) collateral branches (Bars: 20 µm).</p