Carmen Prada

Spatial and Temporal Patterns of Neurogenesis in the Chick Retina.

Chick embryo retinas were labelled in ovo by single injections of [3H]thymidine at selected times between days 2 and 12 of incubation. Embryos were later removed, at different stages of development, and the retinas processed for autoradiography of either serial sections or dissociated cell preparations. Analysis of unlabelled cells shows that neurogenesis starts, on day 2 of incubation, in a dorsotemporal area of the central retina, close to the posterior pole and to the optic nerve head. A gradient of neurogenesis spreads from this central area to the periphery, where neurogenesis ends, shortly after day 12, when the last few bipolar cells withdraw from the cell cycle. Additional dorsal‐to‐ventral and temporal‐to‐nasal gradients can be discerned in our autoradiographs. In all retinal sectors, ganglion cells start first to withdraw from the cell cycle, followed, with substantial overlapping, by amacrine, horizontal, photoreceptor plus Müller, and bipolar neuroblasts. Ganglion cells are also the first to reach the 50% level of unlabelled cells, followed this time by horizontal, photoreceptor, amacrine, Müller and bipolar cells. Finally, 100% levels of unlabelled cell populations are attained simultaneously by ganglion, horizontal and photoreceptor cells, followed by amacrine, then by Müller, and last by bipolar cells. Although all classes of neurons, in varying proportions, are being produced most of the time, our results also demonstrate that, in any given retinal area, the first cells leaving the cycle are determined to become ganglion cells, and the last ones bipolar cells, and not other types.

Early maternal deprivation in rats induces gender-dependent effects on developing hippocampal and cerebellar cells

Adult animals submitted to a single prolonged episode of maternal deprivation [24 h, postnatal day 9–10] show behavioral alterations that resemble specific symptoms of schizophrenia. According to the neurodevelopmental theory, these behavioral deficits might be mediated by detrimental neurodevelopmental processes that might be associated, at least partially, with stress‐induced corticosterone responses. In order to address this hypothesis, we have focused on the hippocampus and cerebellar cortex, two brain regions that show high density of glucocorticoid receptors, and analyzed possible neuronal and glial alterations by immunohistochemical techniques. To evaluate the presence of degenerated neurons we used Fluoro‐Jade‐C (FJ‐C) staining and for the study of astrocytes we employed glial fibrillary acidic protein (GFAP). Within control animals, females showed significantly more GFAP positive cells than males and a trend towards more FJ‐C positive cells. Maternal deprivation induced neuronal degeneration and astroglial changes in the hippocampus and cerebellar cortex of neonatal rats that, in general, were more marked in males. This differential effect may be attributable to a greater vulnerability of males to this kind of early environmental insult and/or to sex‐dependent differences in the onset and/or progression of the effects. The present experimental procedure may be instrumental in elucidating sex‐dependent mechanisms of neurodevelopmental psychiatric disorders with a basis in early environmental insults.

Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: A possible role for the endocannabinoid system

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal days (PND) 9–10] show behavioral alterations that resemble specific symptoms of schizophrenia. These behavioral impairments may be related to neuronal loss in the hippocampus triggered by elevated glucocorticoids. Furthermore, our previous data suggested functional relationships between MD stress and the endocannabinoid system. In this study, we addressed the effects of MD on hippocampal glial cells and the possible relationship with changes in plasma corticosterone (CORT) levels. In addition, we investigated the putative involvement of the endocannabinoid system by evaluating (a) the effects of MD on hippocampal levels of endocannabinoids (b) The modulation of MD effects by two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N‐arachidonoyl‐serotonin (AA‐5‐HT), and the endocannabinoid reuptake inhibitor, OMDM‐2. Drug treatments were administered once daily from PND 7 to PND 12 at a dose of 5 mg/kg, and the animals were sacrificed at PND 13. MD induced increased CORT levels in both genders. MD males also showed an increased number of astrocytes in CA1 and CA3 areas and a significant increase in hippocampal 2‐arachidonoylglycerol. The cannabinoid compounds reversed the endocrine and cellular effects of maternal deprivation. We provide direct evidence for gender‐dependent cellular and biochemical effects of MD on developmental hippocampus, including changes in the endocannabinoid system.

Glutamine synthetase (GS) activity and spatial and temporal patterns of GS expression in the developing chick retina: relationship with synaptogenesis in the outer plexiform layer.

The profile of glutamine synthetase (GS) activity in the neural retina of chicken embryos and adults was studied alongside the in vivo spatio‐temporal patterns of generation and morphogenesis of Müller cell and of retinal synaptogenesis. The rise of GS activity during development is not related to Müller cell differentiation but to synaptogenesis in the outer plexiform layer (opl).

The maternal deprivation animal model revisited.

Early life stress, in the form of MD (24h at pnd 9), interferes with brain developmental trajectories modifying both behavioral and neurobiochemical parameters. MD has been reported to enhance neuroendocrine responses to stress, to affect emotional behavior and to impair cognitive function. More recently, changes in body weight gain, metabolic parameters and immunological responding have also been described. Present data give support to the fact that neuronal degeneration and/or astrocyte proliferation are present in specific brain regions, mainly hippocampus, prefrontal cortex and hypothalamus, which are particularly vulnerable to the effects of neonatal stress. The MD animal model arises as a valuable tool for the investigation of the brain processes occurring at the narrow time window comprised between pnd 9 and 10 that are critical for the establishment of brain circuitries critical for the regulation of behavior, metabolism and energy homeostasis. In the present review we will discuss three possible mechanisms that might be crucial for the effects of MD, namely, the rapid increase in glucocorticoids, the lack of the neonatal leptin surge, and the enhanced endocannabinoid signaling during the specific critical period of MD. A better understanding of the mechanisms underlying the detrimental consequences of MD is a concern for public health and may provide new insights into mental health prevention strategies and into novel therapeutic approaches in neuropsychiatry.

Neuronal and glial alterations in the cerebellar cortex of maternally deprived rats: Gender differences and modulatory effects of two inhibitors of endocannabinoid inactivation

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal day 9–10] show behavioral alterations that resemble specific symptoms of schizophrenia. Accordingly, this experimental procedure has been proposed as an animal model of schizophrenia based on the neurodevelopmental hypothesis. We have recently reported that MD‐induced sex‐dependent alterations in the hippocampus of neonatal rats. In view of recent evidence for important implications of the cerebellum in neurodevelopmental psychiatric diseases, we have now addressed possible degenerative changes in the cerebellar cortex of neonatal Wistar rats of both genders. To evaluate the presence of degenerated nerve cells, we used Fluoro‐Jade C staining and for the study of astrocytes, we employed glial fibrillary acidic protein. Further, we analyzed the modulatory actions of two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N‐arachidonoyl‐serotonin, AA‐5‐HT, and the endocannabinoid reuptake inhibitor, OMDM‐2 (daily subcutaneous injections during the postnatal period 7–12). The animals were sacrificed at postnatal Day 13. MD induced significant increases in the number of Fluoro‐Jade C positive cells (indicative of degenerating neurons) and in the number of glial fibrillary acidic protein positive cells, only in males. The two cannabinoid compounds reversed or attenuated these effects. The present results provide new insights regarding the psycopathological implications of the cerebellum, the role of the endocannabinoid system in neural development, and the possible neurodevelopmental basis of gender differences in schizophrenia.

Shape diversity among chick retina Müller cells and their postnatal differentiation.

It is currently believed that in each vertebrate species Müller cells in the central retina constitutes a fairly homogeneous population from the morphologic point of view and that particularly the chick Müller cell attains full shape differentiation at prenatal stages. However, in this study of the chick retina, from day 1 to day 55 of life, we show that there is a large variety of Müller cell shapes and that many of them complete shape differentiation postnatally. We used a cell dissociation method that preserves the whole shape of the Müller cells. Unstained living and unstained fixed cells were studied by phase‐contrast microscopy, and fixed cells immunostained for intermediate filaments of the cytoskeleton were studied by fluorescence microscopy. Our results show that (1) Müller cell shapes vary in the origination of the hair of vitread processes, in the shape of the ventricular (outer or apical) process, in the presence or absence of an accessory process, as well as in the number and shape of processes leaving from the ventricular process at the level of the outer nuclear and outer plexiform layers (ONL/OPL); (2) during the first month of life, many Müller cells differentiate the portion of the ventricular process that traverses the ONL, most Müller cells differentiate the ONL/OPL processes, and all Müller cells differentiate the thin short lateral processes leaving from the vitread hair processes at the level of the inner plexiform layer (IPL). The number of cells differing in the shape of the ventricular process and that of cells with and without accessory process were estimated. The spatial relationship between the outer portion of the ventricular process of the Müller cell and the photoreceptor cells was also studied. Our results show that the branching of the ventricular process and the refinement of Müller cell shape is achieved without apparent participation of growth cones. We give a schematic view of how the branching of the ventricular process might take place and propose the size increase of photoreceptor soma as a factor responsible for this branching. J. Comp. Neurol. 438:32–49, 2001

Spatiotemporal gradients of differentiation of chick retina types I and II cholinergic cells: identification of a common postmitotic cell population.

The chick retina has three types of cholinergic amacrine cells. We have found that Types I and II differentiate from a common population of postmitotic cells temporarily located in the inner plexiform layer (IPL cells). Golgi staining and immunocytochemistry for choline acetyltransferase (ChAT) and gamma‐aminobutyric acid (GABA) were used to trace the development and fate of IPL cells. Transformation of the shape of IPL cells into those typical of both conventional amacrine cells and those displaced to the ganglion cell layer are seen. All IPL cells are doubly immunoreactive, for ChAT and GABA, from the time they appear as a cell population within the inner plexiform layer (IPL) until their separation into the two amacrine cell populations. Polarization and early stages of shape differentiation of both types occur while they are in the IPL, starting in the dorsocentral area in the temporal retina and spreading to the rest of the retina. Three spatial gradients of differentiation are observed: from central‐to‐peripheral, dorsal‐to‐ventral, and temporal‐to‐nasal retina. Our findings suggest that the fate of both types of cells in the chick is determined locally, whereas their postmitotic precursors are within the IPL. The presence of GABA and acetylcholine in both types of amacrine cells at early stages of their morphogenesis, well before they have synaptic interactions, suggests a morphogenetic role for these molecules in inner retinal differentiation. J. Comp. Neurol. 410:457–466, 1999.

Displaced ganglion cells in the chick retina

New morphological and cytological data on the displaced ganglion cells (DGCs) in the chick retina are presented. Analysis of the topographic distribution, cellular number, dendritic field, perikaryon size and ultrastructural characteristics are included. The DGCs were found predominantly in the peripheral retina. The sizes of the DGCs, 18-42 microns, observed either by Normarskys interferential contrast or by silver impregnation techniques, spanned the size range of the other retinal neurons. The results support the hypothesis that DGCs, in the chick retina, may constitute a specific morphofunctional system, and therefore they might not be considered as neurons that fail to attain the normal location of ganglion cells during the developmental process of migration.

Pax2 is expressed in a subpopulation of Müller cells in the central chick retina

Müller cells in the chick retina are generally thought to be a homogeneous population. We show that the transcription factor Pax2 is expressed by Müller cells in the central chick retina and its expression was first observed at stage 32 (embryonic day [E] 7.5). Birth‐dating indicated that the majority of Pax2‐positive Müller cells are generated between stage 29 and 33 (E5.5–E8). At stage 42 (E16), several Müller cell markers, such as Sox2 and 2M6, had reached the peripheral retina, while the Pax2 labeling extended approximately half‐way. A similar pattern was maintained in the 6‐month‐old chicken. Neither the Pax2‐positive nor the Pax2‐negative Müller cells could be specifically associated to proliferative responses in the retina induced by growth factors or N‐methyl‐D‐aspartate. Pax2 was not detected in Müller cells in mouse, rat, guinea‐pig, rabbit, or pig retinas; but the zebrafish retina displayed a similar pattern of central Pax2‐expressing Müller cells. Developmental Dynamics 239:1858–1866, 2010.