Radial astrocytes and ependymocytes in the spinal cord of the adult toad (Bufo bufo L.)

Summary Immunohistochemical and ultrastructural techniques have been used to demonstrate glial fibrillary acidic protein (GFAP) immuno-positive cells in the adult toad spinal cord. Two types of GFAP-immunoreactive cells were observed: ependymocytes and radial astrocytes. GFAP-positive ependymocytes were scarce and contained the immunoreactive product in their processes. They showed intermediate filaments in the basal pole and in their processes when studied with the electron microscope. These immuno-positive ependymocytes represent the tanycytic form of ependymal cells because their processes ended at the subpial zone. The radial astrocytes showed a more intensive immunoreactive product in somata and processes when they were located far away from the ependymal layer. Cell bodies and processes were also associated with blood vessels, but most of the processes ended at the subpial zone forming a continuous subpial glia limitans. The GFAP-positive processes, which form this subpial glia limitans in the toad spinal cord, belong to both tanycytic ependymocytes and radial astrocytes, whose somata are located in the grey matter. These findings lead us to suggest that both types of GFAP-immunopositive cells might be the functional equivalents of mammalian astrocytes.     

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.     

Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord

Following tail amputation in urodele amphibians, an ependymal tube, that resembles a developing neural tube, forms from ependymal cells that migrate from the cord stump and elongates by cell proliferation. Expression of the keratin pair 8 and 18 has been observed in the developing urodele nervous system and is maintained in the ependymal cells of the mature cord. We show here that expression of these keratins is not unique to urodeles, but is also observed in the radial glia of the human spinal cord, suggesting that these proteins might play a role both in neural development and regeneration. Analysis of their expression in the regenerating spinal cord following tail amputation shows that their expression, as well as that of glial fibrillary acidic protein (GFAP), is maintained in the ependymal tube during regeneration, though differences in their levels of expression are observed along the anteroposterior axis and appear to be related to the progression of morphogenesis. In addition, we show that following tail amputation the ependymal tube expresses the neural stem cell markers nestin and vimentin, which are undetectable in normal urodele spinal cord. This up-regulation of neural stem cell markers shows that the ependymal cells undergo a phenotypic change. Whereas maintenance of keratin and GFAP expression in the adult ependyma may reflect a higher plasticity of these cells in adult urodeles than in other vertebrates, re-expression of markers of early neural development suggests the occurrence of a dedifferentiation process in the spinal cord in response to injury.Edited by J. Campos-Ortega     

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.     

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species

Summary The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.     

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.     

Spinal cord regeneration in a tail autotomizing urodele

Adult urodele amphibians possess extensive regenerative abilities, including lens, jaws, limbs, and tails. In this study, we examined the cellular events and time course of spinal cord regeneration in a species, Plethodon cinereus, that has the ability to autotomize its tail as an antipredator strategy. We propose that this species may have enhanced regenerative abilities as further coadaptations with this antipredator strategy. We examined the expression of nestin, vimentin, and glial fibrillary acidic protein (GFAP) after autotomy as markers of neural precursor cells and astroglia; we also traced the appearance of new neurons using 5‐bromo‐2′‐deoxyuridine/neuronal nuclei (BrdU/NeuN) double labeling. As expected, the regenerating ependymal tube was a major source of new neurons; however, the spinal cord cranial to the plane of autotomy showed significant mitotic activity, more extensive than what is reported for other urodeles that cannot autotomize their tails. In addition, this species shows upregulation of nestin, vimentin, and GFAP within days after tail autotomy; further, this expression is upregulated within the spinal cord cranial to the plane of autotomy, not just within the extending ependymal tube, as reported in other urodeles. We suggest that enhanced survival of the spinal cord cranial to autotomy allows this portion to participate in the enhanced recovery and regeneration of the spinal cord. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

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.     

Primary culture of axolotl spinal cord ependymal cells

In order to examine the role of ependymal cells in the spinal cord regeneration of urodele amphibians, procedures were established to identify and culture these cells. Cell isolation and culture conditions were determined for ependymal cells from larval and adult axolotls (Ambystoma mexicanum). Dissociated cells prepared from intact spinal cords were cultured on fibronectin- or laminin-coated dishes. Dissociated cells attached more rapidly to fibronectin, but attached and spread on both fibronectin and laminin. Essentially pure populations of ependymal cells were obtained by removing 2 week old ependymal outgrowth from lesion sites of adult spinal cords. These ependymal outgrowths attached and grew only on fibronectin-coated dishes. Growth and trophic factors were tested to formulate a medium that would support ependymal cell proliferation. The necessary peptide hormones were PDGF, EGF, and insulin. TGF-beta(1) affected the organization of cell outgrowth. Initially, longterm culture required the presence of high levels of axolotl serum. Addition of purified bovine hemaglobin in the culture medium reduced the serum requirement. Outgrowth from expiants was subcultured by transferring groups of cells. Intrinsic markers were used to identify ependymal cells in culture. The ependymal cells have characteristic ring-shaped nucleoli in both intact axolotl spinal cords and in culture. Indirect immunofluorescence examination of intermediate filaments showed that ependymal cells were glial fibrillary acidic protein (GFAP) negative and vimentin positive in culture. Identification of dividing cells was made using (3)H-thymidine incorporation and autoradiography, and by the presence of mitotic figures in the cultured cells.     

A new protein (J1-31 antigen, 30 kD) is expressed by astrocytes,Müller glia and ependyma

Monoclonal antibody (MAb) J1-31 raised using human brain homogenate as immunogen in mice can be used as a cell type marker for certain types of CNS macroglia, namely astrocytes, Müller cells and tanycytes as well as ciliated ependymal cells. Except for the ciliated ependymal cells, these types of macroglia express glial fibrillary acidic protein (GFAP). J1-31 antigen is an intracellular protein which has a MW of 30 kD under reducing conditions for gel electrophoresis (Singhet al., 1986). This protein is distinct from GFAP (MW 50 kD) and vimentin (MW 55 kD), the two core proteins of 10 nm IFs known to be expressed in the above types ofmacroglia. This conclusion is based on several criteria including temporal differences in the onset of expression of GFAP and J1-31 antigen during development of the rat cerebellum. Also, there is no detectable (by immunofluorescence microscopy) expression of J1-31 antigen in the prenatal CNS or outside the CNS where vimentin has been reported to be abundant. The most direct evidence that J 1-31 antigen and GFAP are distinct proteins comes from studies on the mature ciliated ependymal cells which do not express GFAP and yet show intense immunostaining for J1-31 antigen.     

Ependyma and meninges of the spinal cord of the mouse

Summary In addition to ependymal epithelial cells, numerous tanycytes are found along the entire central canal of the mouse. These tanycytes are arranged in clusters in the cervical, thoracic and lumbar segments of the spinal cord. In the conus medullaris, tanycytes separate and ensheath bundles of myelinated and unmyelinated axons; their processes take part in the formation of the stratum marginale gliae. In the caudal part of the spinal cord, the ventral wall of the central canal is thin and some areas are reduced to a single-cell thickness. In this region, ependymal cells participate directly in the formation of the stratum marginale gliae.The meninges consist of the intima piae, the pia mater, the arachnoid, a subdural neurothelium and the dura mater. The subarachnoid space appears occluded and opens only around the spinal roots. In the vicinity of the spinal ganglia, the dura mater, the subdural neurothelium and the arachnoid form a cellular reticulum.     

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.     

Glial fibrillary acidic protein-like immunoreactive ependymal elements in the third ventricle of the rat. A study at different stages of development.

Using the peroxidase-antiperoxidase method a study was made of the cells immunoreactive to glial fibrillary acidic protein (GFAP) anti-serum in the ependyma of the third ventricle of the rat at different stages of growth. Most of the ependymal cells of the third ventricle were seen to be unreactive to this protein; however, it was sometimes possible to observe some GFAP-immunoreactive ependymocytes and occasionally other immunoreactive cellular types, such as tanycytes and supraependymal cells. Despite this, the most frequent localization of the elements immunoreactive to the protein adopted the shape of an immunoreactive subependymal band situated parallel to the ventricular wall. As the weights of the animals increased an increase in the elements immunoreactive to this protein could be observed in all the zones considered, there being no differences between the male and female animals.     

Co-localization of serine/threonine kinase 33 (Stk33) and vimentin in the hypothalamus

We investigate the immunoreactivity of serine/threonine kinase 33 (Stk33) and of vimentin in the brain of mouse, rat and hamster. Using a Stk33-specific polyclonal antibody, we show by immunofluorescence staining that Stk33 is present in a variety of brain regions. We found a strong staining in the ependymal lining of all cerebral ventricles and the central canal of the spinal cord as well as in hypothalamic tanycytes. Stk33 immunoreactivity was also found in circumventricular organs such as the area postrema, subfornical organ and pituitary and pineal glands. Double-immunostaining experiments with antibodies against Stk33 and vimentin showed a striking colocalization of Stk33 and vimentin. As shown previously, Stk33 phosphorylates recombinant vimentin in vitro. Co-immunoprecipitation experiments and co-sedimentation assays indicate that Stk33 and vimentin are associated in vivo and that this association does not depend on further interacting partners (Brauksiepe et al. in BMC Biochem 9:25, 2008). This indicates that Stk33 is involved in the dynamics of vimentin polymerization/depolymerization. Since in tanycytes the vimentin expression is regulated by the photoperiod (Kameda et al. in Cell Tissue Res 314:251–262, 2003), we determine whether this also holds true for Stk33. We study hypothalamic sections from adult Djungarian hamsters (Phodopus sungorus) held under either long photoperiods (L:D 16:8 h) or short photoperiods (L:D 8:16 h) for 2 months. In addition, we examine whether age-dependent changes in Stk33 protein content exist. Our results show that Stk33 in tanycytes is regulated by the photoperiod as is the case for vimentin. Stk33 may participate in photoperiodic regulation of the endocrine system.     

The cytoskeleton of chondrocytes of Sepia officinalis (Mollusca, Cephalopoda): an immunocytochemical study

Our previous electron microscope study showed that chondrocytes from cephalopod cartilage possess a highly developed cytoskeleton and numerous cytoplasmic processes that ramify extensively through the tissue. We have now carried out a light microscope immunocytochemical study of chondrocytes from the orbital cartilage of Sepia officinalis to obtain indications as to the nature of the cytoskeletal components. We found clear positivity to antibodies against mammalian tubulin, vimentin, GFAP, and actin, but not keratin. The simultaneous presence of several cytoskeletal components is consistent with the hypothesis that cephalopod chondrocytes have the characteristics of both chondrocytes and osteocytes of vertebrates, which endow the tissue as a whole with some of the properties of vertebrate bone. We confirm, therefore, the presence in molluscs of the ubiquitous cytoskeletal proteins of metazoan cells that have remained highly conserved throughout phylogenetic evolution.     

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.     

Colocalization of prostaglandin F2�� receptor FP and prostaglandin F synthase-I in the spinal cord

Prostaglandin F_2α is synthesized by prostaglandin F synthase, which exists in two types, prostaglandin F synthase I (PGFS I) and prostaglandin F synthase II (PGFS II). Prostaglandin F_2α binds to its specific receptor, FP. Our previous immunohistochemical study showed the distinct localization of prostaglandin F synthases in rat spinal cord. PGFS I exists in neuronal somata and dendrites in the gray substance, and PGFS II exists in ependymal cells and tanycytes surrounding the central canal. Both enzymes are also present in endothelial cells of blood vessels in the white and gray substances of the spinal cord. In this study, we found that FP localizes in neuronal somata and dendrites but not in ependymal cells, tanycytes, or endothelial cells. Immunohistochemical analysis of serial sections showed the colocalization of FP and PGFS I. FP immunoreactivity was intense in spinal laminae I and II of the dorsal horn, a connection site of pain transmission, and was similar to that of PGFS I in neuronal elements. These findings suggest that prostaglandin F_2α synthesized in the neuronal somata and dendrites exert an autocrine action there.—Suzuki-Yamamoto, T., K. Toida, Y. Sugimoto, and K. Ishimura. Colocalization of prostaglandin F_2α receptor FP and prostaglandin F synthase-I in the spinal cord.     

Differential immunocytochemical staining for glial fibrillary acidic (GFA) protein,S-100 protein and glutamine synthetase in the rat subcommissural organ,nonspecialized ventricular ependyma and adjacent neuropil

Summary Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.     

Transmission electron microscopic studies of the organization of the ependymocytes in the central canal of the spinal cord of Cercopithecus nigroviridis (Pocock 1907), (Platyrrhina, Cercopithecoidea)

The ependyma of the central canal of the spinal cord of the monkey Cercopithecus nigroviridis was examined by transmission electron microscopy. In the lumbar region and in the filum terminale, many cytoplasmatic protrusions are visible. They are irregular in size and shape and display many microvilli. They are extending into the lumen of the central canal. The basal parts of the ependymocytes occasionally have a very close association with the ependymal blood vessels. The pericapillary space, the pericapillary structures like pericytes and collagen fibrils, and the basal lamina are absent. Opposite branches of the ependymocytes growing together could be observed in the central canal, eventually forming a cytoplasmic unit. Cytoplasmatic extensions of the ependymocytes bridge the lumen of the central canal and melt into each another. Lacunae, such as described by LEONHARDT (1980) in the apical cytoplasm of the ependyma in the rabbit, do also exist in the ependyma coating the central canal of the spinal cord of the monkey Cercopithecus nigroviridis. Some of these lacunae have direct contact to the luminar surface of the central canal, others are separated. Cilia and short microvilli are coating the lacunae. Adjacent ependymal cells form complex interdigitations with each other. Close to their surface on the central canal, there are numerous zonulae adhaerentes. Profiles of the granular and agranular endoplasmatic reticulum are in very close contact to the fine filaments of the zonulae.