Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study

We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.     

Effect of prenatal exposure to alcohol on membrane-bound enzymes during astrocyte development in vivo and in primary culture

In the present work we have analyzed the effect of prenatal ethanol exposure on the activity of several glial marker and functional enzymes during the development of astrocytes isolated from rat brain as well as in primary culture. The activity of marker enzymes glutamine synthetase and butylcholinesterase showed no differences between isolated astrocytes from 15 and 70 day old control rats. However, the activity of the membrane-bound enzymes (Na+K)ATPase and 5'-nucleotidase was higher in astrocytes from 70 day old control rats than in those from 15 day old animals. Although the pattern found in astrocytes from alcohol-exposed rats was similar to that of controls, the levels of activity of the enzymes were lower in alcoholic than in control animals. When control astrocytes in primary culture were used, the activity of (Na+K)ATPase and 5'-nucleotidase increased throughout the entire culture period. In contrast, the maximal activity of glutamine synthetase was found at 7 days of culture. Ethanol also induced a decrease in the activity of all enzymes, which was more evident at the end of the culture period. These results indicate that the activity of the enzyme markers analyzed increased mainly during the first weeks of life and remained constant after this period. By contrast, the membrane-bound enzymes studied showed a progressive increase with age. In conclusion, since these astrocyte enzymes are important in the regulation of several neuronal functions through the control of the composition of extracellular fluid, the effect of ethanol on their activities could explain some of the neuronal alterations reported in children and animals exposed to ethanol during development.     

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina

By gently scraping off the surface of the lateral ventricles of adult bovine brains, we obtained sheets containing the ependymal layer and some attached sub-ependymal cells. Explants were cultured in serum-free medium or in two media enriched with 20% fetal calf serum or 20% adult bovine cerebrospinal fluid, and processed for different time intervals from 4 h to 60 days. For characterization of the ependymal cells we used antisera against S-100 protein, vimentin and glial fibrillary acidic protein (GFAP). For comparison, the ependyma of adult bovines and of fetuses from days 60 to 120 post coitum was studied in situ. The adult ependyma consisted of a ciliated, cuboid cell monolayer with short basal processes; it displayed S-100 immunoreactivity but only scarce deposits of vimentin and no GFAP. The fetal ependyma had the appearance of a pseudostratified epithelium with elongated nuclei and basal processes containing S-100 and vimentin from day 80 post coitum and GFAP from day 100 post coitum. In explants, no differences were seen between the three culture media; the ependyma became pseudostratified, developed basal processes and showed increasing amounts of S-100 and vimentin first, and subsequently also GFAP. These changes were concomitant with the onset of mitotic activity in the subependymal layer leading to the production of numerous cells. The morphological and immunocytochemical features of ependymal cells in cultured explants resembled those of fetal ependyma. Our results indicate that the culture of ependymal explants from adult bovine lateral ventricles is an useful model system for morphological and functional studies of the ependyma and for the analysis of cell proliferation in the subependymal layer.     

Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia     

Cortical radial glial cells in human fetuses: depth-correlated transformation into astrocytes

In the human brain, the transformation of radial glial cells (RGC) into astrocytes has been studied only rarely. In this work, we were interested in studying the morphologic aspects underlying this transformation during the fetal/perinatal period, particularly emphasizing the region-specific glial fiber anatomy in the medial cortex. We have used carbocyanine dyes (DiI/DiA) to identify the RGC transitional forms and glial fiber morphology. Immunocytochemical markers such as vimentin and glial fibrillary acidic protein (GFAP) were also employed to label the radial cells of glial lineage and to reveal the early pattern of astrocyte distribution. Neuronal markers such as neuronal-specific nuclear protein (NeuN) and microtubule-associated protein (MAP-2) were employed to discern whether or not these radial cells could, in fact, be neurons or neuronal precursors. The main findings concern the beginning of RGC transformation showing loss of the ventricular fixation in most cases, followed by transitional figures and the appearance of mature astrocytes. In addition, diverse fiber morphology related to depth within the cortical mantle was clearly demonstrated. We concluded that during the fetal/perinatal period the cerebral cortex is undergoing the final stages of radial neuronal migration, followed by involution of RGC ventricular processes and transformation into astrocytes. None of the transitional or other radial glia were positive for neuronal markers. Furthermore, the differential morphology of RGC fibers according to depth suggests that factors may act locally in the subplate and could have a role in the process of cortical RGC transformation and astrocyte localization. The early pattern of astrocyte distribution is bilaminar, sparing the cortical plate. Few astrocytes (GFAP+) in the upper band could be found with radial processes at anytime. This suggests that astrocytes in the marginal zone could be derived from different precursors than those that differentiate from RGCs during this period.     

Expression of vimentin and glial fibrillary acidic protein in human developing spinal cord

Summary Expression of intermediate filament proteins was studied in human developing spinal cord using immunoperoxidase and double-label immunofluorescence methods with monoclonal antibodies to vimentin and glial fibrillary acidic protein (GFAP). Vimentin was found in the processes of radial glial cells in 6-week embryos, while GFAP appeared in vimentin-positive astroglial cells at 8–10 weeks. GFAP and vimentin were present in approximately equal amounts in differentiating astrocytes in 23-week spinal cord. In 30-week fetuses, astrocytes reacted strongly for GFAP, while both the reaction intensity and the number of vimentin-positive cells fluctuated predominantly in the grey matter. No clear-cut transition from vimentin to GFAP was noticed during the development of astrocytes. The majority of ependymal cells in 23-week fetuses contained vimentin but only a few of them reacted for GFAP. The expression of vimentin continued during the whole development of the ependymal layer, in contrast to the reactivity for GFAP which disappeared between the 30th week and term.     

Glial fibrillary acidic protein in the brain of prenatally stressed rats

The content of glial fibrillary acidic protein (GFAP) was studied in the brain structures of rats borne by intact females and females that underwent stress. In the offspring of stressed rats, the GFAP content in the brain gray and white matter on the 15th postnatal day noticeably dropped. On the 30th postnatal day, the GFAP content in the cortex and pons increased, while it somewhat decreased in the striatum and cerebellum. The results suggest that formation of the intermediate astrocyte filaments in the animals subjected to prenatal stress is markedly disturbed.     

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo.

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)-positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.     

Cortical radial glial cells in human fetuses: Depth‐correlated transformation into astrocytes

In the human brain, the transformation of radial glial cells (RGC) into astrocytes has been studied only rarely. In this work, we were interested in studying the morphologic aspects underlying this transformation during the fetal/perinatal period, particularly emphasizing the region‐specific glial fiber anatomy in the medial cortex. We have used carbocyanine dyes (DiI/DiA) to identify the RGC transitional forms and glial fiber morphology. Immunocytochemical markers such as vimentin and glial fibrillary acidic protein (GFAP) were also employed to label the radial cells of glial lineage and to reveal the early pattern of astrocyte distribution. Neuronal markers such as neuronal‐specific nuclear protein (NeuN) and microtubule‐associated protein (MAP‐2) were employed to discern whether or not these radial cells could, in fact, be neurons or neuronal precursors. The main findings concern the beginning of RGC transformation showing loss of the ventricular fixation in most cases, followed by transitional figures and the appearance of mature astrocytes. In addition, diverse fiber morphology related to depth within the cortical mantle was clearly demonstrated. We concluded that during the fetal/perinatal period the cerebral cortex is undergoing the final stages of radial neuronal migration, followed by involution of RGC ventricular processes and transformation into astrocytes. None of the transitional or other radial glia were positive for neuronal markers. Furthermore, the differential morphology of RGC fibers according to depth suggests that factors may act locally in the subplate and could have a role in the process of cortical RGC transformation and astrocyte localization. The early pattern of astrocyte distribution is bilaminar, sparing the cortical plate. Few astrocytes (GFAP+) in the upper band could be found with radial processes at anytime. This suggests that astrocytes in the marginal zone could be derived from different precursors than those that differentiate from RGCs during this period. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 288–298, 2003     

Expression of glial fibrillary acidic protein in the developing human brain]

It was shown that the glial fibrillary acidic protein (GFAP) content in developing (fetal) human brain is sharply increased. The expression of GFAP was observed already on the 7th-8th week after gestation, the GFAP concentration being less than 0.05% in comparison with adult brain. GFAP can be immunohistochemically detected in radial glial cells. At early stages of development the presence of antigenic determinants of 68 kDa and 100 kDa polypeptides interacting with monoclonal antibodies alongside with native GFAP (51 kDa) and its low molecular weight forms was demonstrated. These antigenic determinants cannot be detected at later stages of development and are absent in adult brain. The data obtained testify to changes in the gene expression of intermediate filament proteins at early stages of human brain ontogenesis.     

Radial glia-like cells at the base of the lateral ventricles in adult mice

During development radial glia (RG) are neurogenic, provide a substrate for migration, and transform into astrocytes. Cells in the RG lineage are functionally and biochemically heterogeneous in subregions of the brain. In the subventricular zone (SVZ) of the adult, astrocyte-like cells exhibit stem cell properties. During examination of the response of SVZ astrocytes to brain injury in adult mice, we serendipitously found a population of cells in the walls of the ventral lateral ventricle (LV) that were morphologically similar to RG. The cells expressed vimentin, glial fibrillary acidic protein (GFAP), intermediate filament proteins expressed by neural progenitor cells, RG and astrocytes. These RG-like cells had long processes extending ventrally into the nucleus accumbens, ventromedial striatum, ventrolateral septum, and the bed nucleus of the stria terminalis. The RG-like cell processes were associated with a high density of doublecortin-positive cells. Lesioning the cerebral cortex did not change the expression of vimentin and GFAP in RG-like cells, nor did it alter their morphology. To study the ontogeny of these cells, we examined the expression of molecules associated with RG during development: vimentin, astrocyte-specific glutamate transporter (GLAST), and brain lipid-binding protein (BLBP). As expected, vimentin was expressed in RG in the ventral LV embryonically (E16, E19) and during the first postnatal week (P0, P7). At P14, P21, P28 as well as in the adult (8–12 weeks), the ventral portion of the LV retained vimentin immunopositive RG-like cells, whereas RG largely disappeared in the dorsal two-thirds of the LV. GLAST and BLBP were expressed in RG of the ventral LV embryonically and through P7. In contrast to vimentin, at later stages BLBP and GLAST were found in RG-like cell somata but not in their processes. Our results show that cells expressing vimentin and GFAP (in the radial glia-astrocyte lineage) are heterogeneous dorsoventrally in the walls of the LV. The results suggest that not all RG in the ventral LV complete the transformation into astrocytes and that the ventral SVZ may be functionally dissimilar from the rest of the SVZ.     

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

Fibronectin is expressed by astrocytes cultured from embryonic and early postnatal rat brain

In early primary cultures from newborn rat brain, few glial fibrillary acidic protein (GFAP)-positive glial cells expressed intracytoplasmic immunoreactivity for fibronectin. After the second week in culture, however, fibronectin was expressed by a distinct population of GFAP-positive flat astrocytes, irrespective of which brain region was studied. In cerebellar cultures, these cells were more abundant than in cortical or neostriatal cultures and often formed a major population of the GFAP-positive cells. The difference in fibronectin expression between cerebellum and the other areas studied was statistically significant. When cultures were started from 9-day-old postnatal rat brain, fibronectin-positive astrocytes appeared earlier than in those from newborn animals, in all areas studied. Further, especially in the case of cerebellum, the number of fibronectin-positive astrocytes increased as a function of time in culture. In cultures started from whole brains of 12-day-old rat embryos, fibronectin was expressed within 24 h in culture by all the cells with morphology of flat astrocytes, positive for vimentin but negative for GFAP. These results indicate that astrocytes cultured from newborn and early postnatal rat brain are a heterogeneous population of cells: depending on the brain region studied and also depending on the age of brain tissue or the time in culture, less than 1-60% of the GFAP-positive flat astrocytes expressed fibronectin. This, together with the fact that fibronectin was present in early embryonic brain cells in culture, suggests that fibronectin may be a prerequisite for the development or interactions of brain cells.     

Astroglial cells in the central nervous system of the adult brown anole lizard, Anolis sagrei, revealed by intermediate filament immunohistochemistry

We analyzed the distribution of intermediate filament molecular markers, glial fibrillary acidic protein (GFAP), and vimentin in the brain and spinal cord of the adult brown anole lizard, Anolis sagrei. The GFAP immunoreactivity is strong and the positive structures are basically represented by fibers of different lengths and thicknesses which are arranged in a regular radial pattern throughout the central nervous system. In the brain regions that have a thicker neural wall, the radial orientation is not so evident as in the thinner areas. These fibers emerge from radial ependymoglia (tanycytes) whose cell bodies are generally GFAP-immunopositive. The glial fibers give rise to endfeet that are apposed to the subpial surface and to blood vessel walls. In the spinal cord, the optic tectum and the lateroventral regions of the mesencephalon and medulla oblongata, star-shaped astrocytes coexist with radial structures. Vimentin-immunoreactive structures are absent in the brain and spinal cord. In A. sagrei the immunohistochemical response of the astroglial intermediate filaments appears typical of a mature astroglial cell lineage, since they fundamentally express GFAP immunoreactivity. A Western-blot analysis reveals a GFAP-positive single band, common to the different nervous areas. This immunohistochemical study shows that the star-shaped astrocytes have a different distribution in saurians and while the glial pattern of A. sagrei is more evolved than in urodeles it remains immature as compared with crocodilians, avians, and mammals. This condition suggests that reptiles represent a fundamental step in the phylogenetic evolution of the vertebrate glial cells.     

Ganglioside Modulation of Cyclic AMP-Dependent Protein Kinase and Cyclic Nucleotide Phosphodiesterase In Vitro

The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4-56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7-32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7-18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7-32 days), alpha-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.