Distribution of glial cells in the central nervous system of the pulmonate snail Megalobulimus oblongus identified by means of a glial fibrillary acidic protein marker

The distribution of the glial cells in the pulmonate gastropod Megalobulimus oblongus was studied by means of an immunohistochemical procedure. These cells expressed glial fibrillary acidic protein in their cell bodies as well as in their processes. In all ganglia of the central nervous system, four types of glial cells were identified. The glial lacunar network and the perineuronal glial cells were found in the cortical region of the ganglia, and the perisynaptic and the fibrous glial cells in the neuropilar region. However, in the procerebrum of the cerebral ganglion the glial cells only had a reticular distribution throughout the cellular area. These observations provide morphological evidence of glial cell functions. These cells are probably involved in the support of neurones, the uptake and/or degradation of neurotransmitters, the transfer of metabolic substrates to neurones, as well as the regulation of ionic constituents of extracellular space. As occurs in vertebrates, there is a strong relationship between the different cellular components of the central nervous system of this invertebrate.     

Distribution and ontogeny of glial fibrillary acidic protein in the snail Megalobulimus abbreviatus

Glial fibrillary acidic protein (GFAP) is the major component of intermediate glial filaments in the central nervous system of many vertebrates and invertebrates. In vertebrates, this protein is mainly expressed in mature astrocytes and provides structural cell stability. The highly conserved structure and glial specificity of this protein have allowed studies of ontogeny and phylogeny using antibodies. The present study investigated the ontogenetic profile and molecular weight of GFAP in the snail, Megalobulimus abbreviatus, particularly in cerebral ganglia and subesophageal mass, by immunohistochemistry and immunoblotting. Our results confirm and extend previous studies about glial intermediate filaments in snails, showing: (i) a higher GFAP content in cerebral ganglia than in subesophageal mass; (ii) a developmental increase of GFAP immunocontent in cerebral ganglia, as described in Vertebrates; and (iii) an electrophoretic band for GFAP of approximately 55 kDa.     

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of the African lungfish, Protopterus annectens (Dipnoi: Lepidosirenidae)

The distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP), and vimentin, in the brain and spinal cord of the African lungfish, Protopterus annectens, was examined by light microscopy immunoperoxidase cytochemistry. Glial fibrillary acidic protein immunoreactivity is clear and is evident in a radial glial system. It consists of fibers of different lengths and thicknesses that are arranged in a regular radial pattern throughout the central nervous system (CNS). They emerge from generally immunopositive radial ependymoglia (tanycytes), lining the ventricular surface, and are directed from the ventricular wall to the meningeal surface. These fibers give rise to endfeet that are apposed to the subpial surface and to blood vessel walls forming the glia limitans externa and the perivascular glial layer, respectively. GFAP-immunopositive star-shaped astrocytes were not found in P. annectens CNS. In the gray matter of the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependymal layer. Vimentin-immunopositive structures are represented by thin fibers mostly localized in the peripheral zones of the brain and the spinal cord. While a few stained fibers appear in the gray matter, the ependymal layer shows no antivimentin immunostaining. In P. annectens the immunocytochemical response of the astroglial intermediate filaments is typical of a mature astroglia cell lineage, since they primarily express GFAP immunoreactivity. This immunocytochemical study shows that the glial pattern of the African lungfish resembles that found in tetrapods such as urodeles and reptiles. The glial pattern of lungfishes is comparable to that of urodeles and reptiles but is not as complex as that of teleosts, birds, and mammals.     

Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin

In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.     

Developmental expression of the Glial fibrillary acidic protein (GFAP) gene in the mouse retina

1. In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes. 2. In the mammalian retina, Muller cells, the major class of retinal glia, do not express GFAP or contain only low amounts of this protein. In retinas with photoreceptor degeneration, however, high levels of GFAP are found. It is possible that GFAP synthesis in these retinas could result from "dedifferentiation" of Muller cells as a consequence of disruption of normal neuron-glia interactions. 3. We have carried out immunocytochemical and in situ hybridization studies to examine whether GFAP or its mRNA is expressed by retinal cells early in embryonic development. 4. Our results show that GFAP-containing cells, which are probably astrocytes, are found only in the ganglion cell and nerve fiber layers and that these cells appear after postnatal day-1 (P-1) and continue to form until P-10. 5. Astrocyte formation starts from the optic disc and moves toward the periphery of the retina at a rate of approximately 160-200 microns per day. 6. An unexpected result from these studies is that GFAP mRNA levels are high in the first week of birth and decline rapidly as the animal develops. 7. Finally, we did not find either GFAP or GFAP mRNA in retinal cells other than astrocytes during normal development.     

High ammonia diet: Its effect on the glial fibrillary acidic protein (GFAP)

The effect of a recent hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, on glial fibrillary acidic protein (GFAP) in the rat spinal cord and on blood ammonia levels has been studied. The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. GFAP contents were determined by means of immunoblotting analysis. The results demonstrated that this high ammonia diet model neither induces significant changes in GFAP immunoreactivity, nor modifies total protein concentration, and only induces significant blood hyperammonemic levels in the first days of treatment. An adaptative response to the diet is suggested and discussed to explain these results. A relation between ammonia and GFAP expression is suggested because transient hyperammonemia induces transient, although no significant, changes on GFAP expression.     

Immunohistochemical localization of glial fibrillary acidic protein (GFAP) and vimentin in the subcommissural organ of the Mongolian gerbil (Meriones unguiculatus)

Summary The chemical composition of intermediate filaments (IF's) in the ependyma of the subcommissural organ (SCO) of the Mongolian gerbil (Meriones unguiculatus) was investigated immunohistochemically in paraffin-embedded tissue. Antibodies against glial fibrillary acidic protein (GFAP), vimentin, neurofilament proteins and cytokeratins were used. Only GFAP and vimentin were detected in the non-specialized diencephalic ependyma and in the ependymocytes of the SCO. Staining could be observed in apical and basal processes of the SCO-cells. The latter processes extended into the posterior commissure up to the subpial surface, thus establishing a well-developed leptomeningeal route of ependymal projections. In contrast to the homogeneous vimentin-labeling, the SCO was particularly immunoreactive for GFAP in its lateral aspects and in the supraand precommissural parts. The coexpression of GFAP and vimentin in a subclass of SCO-ependymocytes was demonstrated on differentially immunostained semithin sections. The present study confirms the glial nature of the SCO-ependyma, which has been a matter of debate recently. It appears from this investigation that the high degree of secretory activity in the SCO does not necessarily lead to the disappearance of glial IF proteins. Moreover, the SCO-cells belong to the expanding group of mature astroglia, which is characterized by coexpression of GFAP and vimentin. The morphological similarity between SCO-ependymocytes and tanycytes is underscored by their common immunoreactivity against these two IF proteins. In view of the absence of GFAP from the rat SCO, interspecific differences must be considered in the evaluation of the IF protein composition.     

Targeting malignant gliomas with a glial fibrillary acidic protein (GFAP)-selective oncolytic adenovirus

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein abundantly expressed in malignant gliomas. We have constructed a novel oncolytic adenovirus, Ad5-gfa2(B)3-E1, for treatment of these tumors. In this construct, the E1 region is under control of the tissue-specific GFAP promoter (gfa2) with three additional copies of the glial specific 'B' enhancer. Infection of a GFAP-positive cell line with Ad5-gfa2(B)3-E1 resulted in E1A and E1B expression at 75% and 30% of the levels obtained after wtAd5 infection. Q-PCR showed that Ad5-gfa2(B)3-E1 replicated 4.5 times more efficiently in the GFAP-positive than in the GFAP-negative cell lines. Cell viability assays showed efficient elimination of GFAP-positive cells by Ad5-gfa2(B)3-E1, in some cell lines as efficiently as wtAd5, while the elimination was attenuated in GFAP-negative cell lines. When tested in human tumor xenografts in nude mice, Ad5-gfa2(B)3-E1 effectively suppressed the growth of GFAP-positive SNB-19 glial tumors but not of GFAP-negative A549 lung tumors. In Ad5-gfa2(B)3-E1, the E3 region was deleted to create space for future insertion of heterologous therapeutic genes. Experiments with dl7001, an E3-deleted variant of wtAd5, confirmed that the specificity of Ad5-gfa2(B)3-E1 replication was based on the promoter driving E1 and not on the E3 deletion. Strategies to further improve the efficacy of Ad5-gfa2(B)3-E1 for the treatment of malignant gliomas include the insertion of therapeutic genes in E3 or retargeting to receptors that are more abundantly expressed on primary glioma cells than CAR.     

Ependyma: phylogenetic evolution of glial fibrillary acidic protein (GFAP) and vimentin expression in vertebrate spinal cord

The phylogenetic evolution was studied of both glial fibrillary acidic protein (GFAP) and vimentin expression in the ependyma of the adult vertebrate spinal cord. Eleven species from different vertebrate groups were examined using different fixatives and fixation procedures to demonstrate any differences in immunoreactivity. GFAP expression in the ependymal cells showed a clear inverse relation with phylogenetic evolution because it was more elevated in lower than in higher vertebrates. GFAP positive cells can be ependymocytes and tanycytes, although depending on their structural characteristics and distribution, the scarce GFAP positive ependymal cells in higher vertebrates may be tanycytes. Ependymal vimentin expression showed a species-dependent pattern instead of a phylogenetic pattern of expression. Vimentin positive ependymal cells were only found in fish and rats; in fish, they were tanycytes and were quite scarce, with only one or two cells per section being immunostained. However, in the rat spinal cord, all the ependymocytes showed positive immunostaining for vimentin. The importance of the immunohistochemical procedure, the cellular nature of GFAP positive ependymal cells and the relationship between tanycytes and ependymocytes are discussed, as well as GFAP and vimentin expression.     

Glial fibrillary acidic protein (GFAP) shows circadian oscillations in crayfish Procambarus clarkii putative pacemakers

Although several studies of glia have examined glial fibrillary acid protein (GFAP) and its relationship to the circadian rhythms of different organisms, they have not explored the daily GFAP oscillations in the putative pacemakers of the crayfish Procambarus clarkii or in other crustaceans. In this study we investigated the daily variations in GFAP concentrations in the eyestalk and brain, which are considered to be putative pacemakers in adult P. clarkii. In both structures, the glial GFAP was quantified using the indirect enzyme-linked immunosorbent assay (ELISA), and double labeling immunofluorescence was used to detect it and its co-localization with protein Period (PER), an important component of the circadian clock, in various regions of both structures. The ELISA results were analyzed using Cosinor and one-way ANOVA with Bonferroni and Scheffé’s post hoc tests. The results of this analysis showed that the GFAP levels present circadian oscillations in both structures. Moreover, GFAP was localized in different structures of the eyestalk and brain; however, co-localization with PER occurred only in the lamina ganglionaris, specifically in the cartridges of the eyestalk and in some of the cluster 9 brain cells. These results suggest that as in other invertebrates and vertebrates, glial cells could be involved in the circadian system of P. clarkii; however, thus far we cannot know whether the glial cells are only effectors, participate in afferent pathways, or are part of the circadian clock.     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

Circadian rhythms and glial cells of the central nervous system

Glial cells are the most abundant cells in the central nervous system and play crucial roles in neural development, homeostasis, immunity, and conductivity. Over the past few decades, glial cell activity in mammals has been linked to circadian rhythms, the 24-h chronobiological clocks that regulate many physiological processes. Indeed, glial cells rhythmically express clock genes that cell-autonomously regulate glial function. In addition, recent findings in rodents have revealed that disruption of the glial molecular clock could impact the entire organism. In this review, we discuss the impact of circadian rhythms on the function of the three major glial cell types – astrocytes, microglia, and oligodendrocytes – across different locations within the central nervous system. We also review recent evidence uncovering the impact of glial cells on the body's circadian rhythm. Together, this sheds new light on the involvement of glial clock machinery in various diseases.     

Fluorescence marking of neuropile glial cells in the central nervous system of the leech Hirudo medicinalis

Summary Neuropile glial (NG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis L., were studied by histological and intracellular electrophysiological methods. Potential profiles of single leech ganglia were mapped by advancing an electrolyte-filled microelectrode into the ganglion as far as the NG cell. A small negative potential usually appeared during or immediately after penetration of the ganglion sheath. Most of the ganglia in the chain (ganglia 1–4 and 7–21) have Retzius-cell-bodies of normal size; in these, the potential associated with the ganglion sheath was followed by a jump to a more negative potential. Superimposed action potentials were associated with entry of the electrode into a Retzius cell. When the electrode tip passed out of the cell into the center of the ganglion, another potential change was observed, namely that to the membrane potential of the anterior NG cell. This membrane potential averaged -60.2 mV and ranged from -50 to -73 mV. In ganglia 5 and 6 the Retzius-cell-bodies are particularly small, and no changes of potential associated with these cells were observed; the first potential to appear after the electrode passed through the sheath of the ganglion was the membrane potential of the NG cell. Potential profiles like those of ganglia 5 and 6 are recorded in the posterior parts of all ganglia.Potential profiles of single leech ganglia were also recorded with microelectrodes filled with the fluorescent dye Procion Yellow M4-RAN. When the presumed membrane potential of an NG cell appeared, the dye was injected into the ganglion. Subsequent histological examination with the fluorescence microscope revealed that all of the dye was contained in NG cells.Supported by a Fellowship (Heisenberg-Stipendium, Schl 169/5) and grants (Schl 169/2, 4) to W.R.S. from the Deutsche ForschungsgemeinschaftThe authors thank Gisela Geiger for excellent assistance during this work     

Sexual dimorphism of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus

The intensity of immunostaining for the glial fibrillary acidic protein (GFAP) is outstandingly high in the interpeduncular nucleus. This nucleus was compared in males and females for its GFAP immunoreaction. Immunohistochemistry was carried out on free floating vibratome slices and evaluated by surface densitometry. While in males the reactions were similar, females showed individual variations. Since the interpeduncular nucleus is a hormonally inactive brain area where gonadal hormones do not induce plastic synaptic changes, it is concluded that concerning this astroglial marker a sexual dimorphism exists also outside the "endocrine brain".     

Seasonal fluctuations of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat suprachiasmatic nucleus

The pacemaker of the "biological clock", the suprachiasmatic nucleus (SCN) of the hypothalamus was studied in intact male rats for glial fibrillary acidic protein (GFAP) a specific marker for astrocytes. Immunohistochemical reactions were carried out in winter (January-February) and in summer (June-July). In winter the GFAP-immunoreactivity of the SCN was found low whereas in summer it was high. Gonadectomy reduced differences. Since photic stimuli that apparently trigger the observed differences reach the SCN through identified neuronal pathways we conluded that the reaction of astrocytes is an indicator of seasonally altered neuronal function in the SCN.     

Aging results in copper accumulations in glial fibrillary acidic protein‐positive cells in the subventricular zone

Analysis of rodent brains with X‐ray fluorescence (XRF) microscopy combined with immunohistochemistry allowed us to demonstrate that local Cu concentrations are thousands of times higher in the glia of the subventricular zone (SVZ) than in other cells. Using XRF microscopy with subcellular resolution and intracellular X‐ray absorption spectroscopy we determined the copper (I) oxidation state and the sulfur ligand environment. Cu K‐edge X‐ray absorption near edge spectroscopy is consistent with Cu being bound as a multimetallic Cu‐S cluster similar to one present in Cu‐metallothionein. Analysis of age‐related changes show that Cu content in astrocytes of the SVZ increases fourfold from 3 weeks to 9 months, while Cu concentration in other brain areas remain essentially constant. This increase in Cu correlates with a decrease in adult neurogenesis assessed using the Ki67 marker (both, however, can be age‐related effects). We demonstrate that the Cu distribution and age‐related concentration changes in the brain are highly cell specific.     

Expression of GFAP (glial fibrillary acidic protein) antigenicity and differentiation of glioma tumor cells of astrocytic origin

Polyclonal antibodies were used in the diagnostic procedure of brain tumors of astrocytic origin. Investigations were undertaken to determine the percentage of GFAP positive and negative cells in arbitrary selected fields of the tumor specimen taking into account the number of mitotic figures and the diameter of the cell nucleus. It has been found that 18.0% cells were GFAP positive in fields with mitotic figures and in 29.8% cells in fields without mitoses. If the differences in nucleus diameter are concerned the GFAP positive cells constituted 64.2% of cells with diameter between 6 to 10 microns. Instead 78% of GFAP negative cells had a diameter of 6.4 to 7.2 microns. The results indicate that cells with a smaller diameter of the nucleus and localized in fields with mitotic figures are considerably less frequently GFAP positive as compared with cells of larger diameter of the nucleus and localized outside the fields with mitoses. It is suggested that cells of higher differentiation show an undisturbed organization of the cytoskeleton as compared with their counterparts. Thus the determination of GFAP antigenicity can be considered as a tool in the evaluation of differentiation of the tumor and as an indicator of its heterogeneity.