Francisco A. Prada

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).

Effects of ethanol on the inner layers of chick retina during development

Optic nerve hypoplasia is an important malformation of the fetal alcohol syndrome whose teratogenic mechanisms are unknown. In our experimental model we have quantified the concentration of ethanol and acetaldehyde in the retina and vitreous humor of the developing chick. The effect of ethanol alone during retinal development was analyzed by conventional histological techniques and by immunostaining. A single injection of ethanol in the vitelline sac at the beginning of retinal cell differentiation retarded synaptogenesis in the inner plexiform layer and produced abundant ganglion cell death and a sharp diminution of myelinic axons. Our observations could help to explain certain alterations described in children exposed to ethanol during the development of their nervous system.

Neuroprotection by melatonin from glutamate‐induced excitotoxicity during development of the cerebellum in the chick embryo

This work investigated the ability of melatonin to prevent cell damage in the cerebellar cortex of chick embryo caused by glutamate administration. Cell injury was evaluated estimating, at ultrastructural level, the phenomenon of cell death and the synaptogenesis of the Purkinje cells and the cerebellar glomerular synaptic complex. Administration of glutamate during cerebellar development of the chick provokes excitotoxic neuronal degeneration characterized by a phenomenon of neuronal cell death that exhibits essentially the features of a death pattern described as necrosis and the deletion of synaptogenic processes. Our results show that melatonin has a neuroprotective effect against glutamate‐induced excitotoxicity. This effect is morphologically revealed by the lack of neural cell death in the embryos treated with melatonin prior to glutamate injection and also by the degree of a synaptogenesis similar to that exhibited by the control group. Likewise, we corroborate the absence of teratological effects of melatonin on chick cerebellar development. Although the possible mechanisms involved in the neuroprotective effect of melatonin are discussed, i.e., direct antioxidant effects, up‐regulating endogenous antioxidant defenses, and inhibiting nitric oxide formation activated by glutamate, further studies are required to establish the actual mechanism involved in the neuroprotective effect of melatonin.

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.

Developmental study of axon formation in the horizontal neurons of the retina of the chick embryo

SummaryAmong the types of horizontal cells of the avian retina, one has been described that has an axon terminating, in a typical structure. The present study analyses the histogenesis of this axon whose initial outgrowth occurs on day 14 of incubation (HH-40). The axon terminal is first detectable, towards day 15 of incubation (HH-41), in the form of a varicose thickening possessing short filopodia. The formation of the axon and the growth of the axon terminal is coincident with a retraction of the perikaryal process. The axon usually originates from one of the pricipal dendrites and in these stages shows short and fine filopodia throughout its length. From day 16 onwards (synaptic) spines may be distinguished, both in the dendritic field and on the axon terminal. The growth of the axon, in the phase when the axon terminal still has not formed, may exhibit deflections and deviations in its course, the possible cause and mechanism of which are discussed.

Peripapillary glial cells in the chick retina: A special glial cell type expressing astrocyte, radial glia, neuron, and oligodendrocyte markers throughout development

Peripapillary glial cells of the chick are a special type of glia, not only because of their position, forming a boundary between the retina on one side and the optic nerve head (ONH) and the pecten on the other, but also because although they have the same orientation and similar shape as the retinal Müller cell (a type of radial glia) and express common markers for these cells and astrocytes, they do not express glutamine synthetase (GS) or carbonic anhydrase C (CA‐C), enzymes intensely expressed by Müller cells and astrocytes. In this study, we present further molecular characterization of these cells, using immunohistochemistry techniques. We show that peripapillary glial cells express a novel neuron antigen, 3BA8, that in the adult retina is located only in one neuron type (the amacrine cell) and in the inner plexiform layer (IPL). They also express an antigen specific to myelin and oligodendrocytes, MOSP, and a glial antigen, 3CB2, expressed by radial glia and astrocytes throughout the CNS. The study of the developmental expression of these three antigens in the peripapillary glial cell territory shows different spatiotemporal labeling patterns: 3CB2 and 3BA8 are expressed much earlier (embryonic days E3 and E5, respectively) than MOSP (E12), and during a developmental window (E6–E10) 3BA8 labels the peripapillary glial cells intensely and does not label the ONH or the optic nerve (ON), which are labeled later. The expression of 3CB2 is much more intense in the peripapillary glial cells than in Müller cells from early stages of development up to E16, and the expression of MOSP starts earlier in the peripapillary glial cells than in the Müller cells and is maintained with much higher intensity in the peripapillary glial cells throughout development. These findings show that Müller and peripapillary glial cells follow independent courses of differentiation, which together with the fact that the peripapillary glial cells express molecules typical of neurons, oligodendrocytes, radial glia, and astrocytes are evidence that peripapillary glial cells are a unique type of glia in the CNS.

Effect of ethanol on the morphohistogenesis and differentiation of cerebellar granule cells in the chick embryo

In this present study we analyse, with the help of the Golgi method, the effect of ethanol on the morphological differentiation of the cerebellar granule cells in the chick embryo. Ethanol seems to affect the process of cell migration from the early stages of differentiation. Some granule cells appear to differentiate in an inverted position. These observations also confirm, on the basis of their axon morphology, the existence of three types of granule cells in the chick cerebellum.

Centrifugal fibers in the chick retina. A morphological study.

This work is a morhological study of the centrifugal fibers in the chick retina. We have classified these fibers in two types: type I centrifugal fibers and type II centrifugal fibers. Type I centrifugal fibers constitute a new model of axonic terminal in the birds retina. These fibers terminate exclusively in the inner plexiform layer where they show long tangential trajectories. Type II centrifugal fibers are coincident with classical ones previously described in the avian retina. With the Golgi method we describe new levels of terminations of these type II centrifugal fibers.

Alcohol-Induced Lipid and Morphological Changes in Chick Retinal Development

BACKGROUND Alcohol exposure causes alterations in the lipid content of different organs and a reduction of long-chain fatty acids. During embryo development, the central nervous system is extremely vulnerable to the teratogenic effects of alcohol, and the visual system is particularly sensitive. METHODS White Leghorn chick embryos were injected with 10- and 20-microl alcohol doses into the yolk sac at day 6 of incubation. The lipid composition of the retina was analyzed in embryos at day 7 of incubation (E7), E11, E15, and E18. The percentages of phospholipids, free cholesterol, esterified cholesterol, diacylglycerides, and free fatty acids were estimated by using an Iatroscan thin layer chromatography flame ionization detector. Gas chromatography and mass spectrometry were used to determine fatty acid composition. The morphological study was performed at E7, E11, and E19 by means of semithin and immunohistochemical techniques. RESULTS In the retina, alcohol causes the total lipid content to change, with a remarkable increase in free cholesterol and a dramatic decrease in esterified cholesterol. Diacylglycerides and free fatty acids tend to increase. Phosphatidylcholine and phosphatidylethanolamine decrease, whereas phosphatidylserine, sphingomyelin, and phosphatidylinositol increase. The main fatty acids of the retina also undergo changes. At E7, myriotic acid increases, and oleic acid and polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid decrease. From E18 onward, there is some recovery, except for fatty acids, which recover earlier. From a morphological point of view, alcohol effects on retinal development are various: increase of intercellular spaces in all cell layers, pyknosis with loss of cellularity in the inner nuclear cell layer and ganglion cell layer, retarded or disorderly cell migration, early cell differentiation, and loss of immunoreactivity for myelin oligodendrocyte-specific protein. CONCLUSIONS Acute alcohol exposure during embryo development causes the lipid composition of the retina to change, with a trend to recovery in the last stages. These alterations are in line with the changes observed at a morphological level.