Entopallium Lost GFAP Immunoreactivity during Avian Evolution: Is GFAP a "Condition Sine Qua Non"?

INTRODUCTION: The present study demonstrates that in the same brain area the astroglia can express GFAP (the main cytoskeletal protein of astroglia) in some species but not in the others of the same vertebrate class. It contrasts the former opinions that the distribution of GFAP found in a species is characteristic of the entire class. The present study investigated birds in different phylogenetic positions: duck (Cairina moschata domestica), chicken (Gallus gallus domesticus), and quails (Coturnix japonica and Excalfactoria chinensis) of Galloanserae; pigeon (Columba livia domestica) of a group of Neoaves, in comparison with representatives of other Neoaves lineages, which emerged more recently in evolution: finches (Taeniopygia guttata and Erythrura gouldiae), magpie (Pica pica), and parrots (Melopsittacus undulatus and Nymphicus hollandicus). METHODS: Following a perfusion with 4% buffered paraformaldehyde, immunoperoxidase reactions were performed with two types of anti-GFAP: monoclonal and polyclonal, on floating sections. RESULTS: The entopallium (formerly "ectostriatum," a telencephalic area in birds) was GFAP-immunopositive in pigeon and in the representatives of Galloanserae but not in songbirds and parrots, which emerged more recently in evolution. The lack of GFAP expression of a brain area, however, does not mean the lack of astroglia. Lesions induced GFAP expression in the territory of GFAP-immunonegative entopallia. It proved that the GFAP immunonegativity is not due to the lack of capability, but rather the suppression of GFAP production of the astrocytes in this territory. In the other areas investigated besides the entopallium (optic tectum and cerebellum), no considerable interspecific differences of GFAP immunopositivity were found. It proved that the immunonegativity of entopallium is due to neither the general lack of GFAP expression nor the incapability of our reagents to detect GFAP in these species. CONCLUSION: The data are congruent with our proposal that a lack of GFAP expression has evolved in different brain areas in vertebrate evolution, typically in lineages that emerged more recently. Comparative studies on GFAP-immunopositive and GFAP-immunonegative entopallia may promote understanding the role of GFAP in neural networks.

Postmortem immunohistochemical alterations following cerebral lesions: A possible pathohistological importance of the ß-dystroglycan immunoreactivity.

The frequency of cerebrovascular injuries raises the importance of their immunohistological investigation in postmortem materials. Most injuries involve the impairment of the blood-brain barrier. The barrier is maintained by the glio-vascular connections which break up following injuries. Some immunohistochemical alterations may refer to the impairment of the gliovascular connections. Laminin and the components of the dystroglycan complex show characteristic immunohistochemical alterations following various experimental injuries (stab wound, cryogenic lesion, arterial occlusions): immunoreactivity of ß-dystroglycan, α-dystrobrevin and aquaporin 4 disappeared while that of utrophin and laminin appeared along the vessels, whereas α-syntrophin visualized the reactive astrocytes but not the resting ones. The aims of the present study were to investigate whether these post-lesion alterations: (i) are reproducible with immersive fixation, which is used in postmortem histology; (ii) are resistant to a postmortem delay before fixation; and (iii) are to be attributed to a direct effect of the lesion, or are mediated by processes occurring only in the living brain. Three models were investigated: (i) following lesions, some brains were fixed by transcardial perfusion, others by immersion; (ii) following lesions, the animals were decapitated and stored at room temperature for 8 or 16 h before fixation; and (iii) the lesions were performed after decapitation. Cryogenic lesions were performed by applying a dry ice cooled copper rod to the brain surface of ketamine-xylazine anesthetized rats. The immunohistochemical reactions were performed on free-floating sections cut with vibratome. Both immunoperoxidase and immunofluorescence methods were used. The fixation method - perfusive or immersive - did not change the post-lesion phenomena investigated. The postmortem delay did not influence the ß-dystroglycan immunoreactivity, that is its lack delineated the area of the lesion. However, in the case of the other substances, various lengths of postmortem delay rendered the immunohistochemistry uninterpretable. The results suggest ß-dystroglycan immunostaining could be applied in the neuropathology to detect cerebrovascular impairments.

Phases of intermediate filament composition in Bergmann glia following cerebellar injury in adult rat.

In contrast to other astroglial populations, Bergmann glia (BG) form a strictly arranged system where each cell contacts the pia, with an architecture and function resembling that of immature radial glia. As a consequence, a post-lesion glial reaction is expected to differ from that observed in other parts of the brain. The present study describes the characteristic phases of intermediate filament protein formation during the different stages of BG response following injury and compares them with reactive glial patterns of other brain areas and patterns of glial development. The progress of Bergmann glial repair shares similar features with glial development. Following injury, BG developed nestin immunopositivity; then, colocalization of nestin and GFAP was observed. Finally, exclusively GFAP-immunopositive BG were restituted, denser, and thicker than before. The changes of intermediate filament composition appeared at first at the proximal and distal ends of BG fibers, i.e., at the perikaryal "root" and in the pial endfeet. No astrocytic invasion was present in the molecular layer, nor any distinct rearrangement of BG. These results demonstrate the role of the resident glia in glial reactions and refer to the priority of gliomeningeal connections.

Three-plane description of astroglial architecture and gliovascular connections of area postrema in rat: Long tanycyte connections to other parts of brainstem.

The study demonstrates the astroglial and gliovascular structures of the area postrema (AP) in three planes, and compares them to our former findings on the subfornical organ (SFO) and the organon vasculosum laminae terminalis (OVLT). The results revealed long glial processes interconnecting the AP with deeper areas of brain stem. The laminin and ß-dystroglycan immunolabeling altered along the vessels indicating alterations of the gliovascular relations. These and the distributions of glial markers displayed similarities to the SFO and OVLT. In every organ, there was a central area with vimentin- and nestin-immunopositive glia, whereas GFAP and the water-channel aquaporin 4 were found at the periphery. This separation supports different functions of the two regions. The presence of nestin may indicate stem cell capabilities, whereas aquaporin 4 has been suggested by other studies to be a possible participant of osmoperception. Numerous S100-immunopositive glial cells were found approximately evenly distributed in both parts of the AP. Frequency of glutamine synthetase-immunoreactive cells was similar in the surrounding brain tissue in contrast to that found in the OVLT and SFO. Our findings on the three sensory circumventricular organs (AP, OVLT, and SFO) are compared in parallel.

Evolutionary Modifications Are Moderate in the Astroglial System of Actinopterygii as Revealed by GFAP Immunohistochemistry.

The present paper is the first comparative study on the astroglia of several actinopterygian species at different phylogenetical positions, teleosts (16 species), and non-teleosts (3 species), based on the immunohistochemical staining of GFAP (glial fibrillary acidic protein), the characteristic cytoskeletal intermediary filament protein, and immunohistochemical marker of astroglia. The question was, how the astroglial architecture reflexes the high diversity of this largest vertebrate group. The actinopterygian telencephalon has a so-called 'eversive' development in contrast to the 'evagination' found in sarcopterygii (including tetrapods). Several brain parts either have no equivalents in tetrapod vertebrates (e.g., torus longitudinalis, lobus inferior, lobus nervi vagi), or have rather different shapes (e.g., the cerebellum). GFAP was visualized applying DAKO polyclonal anti-GFAP serum. The study was focused mainly on the telencephalon (eversion), tectum (visual orientation), and cerebellum (motor coordination) where the evolutionary changes were most expected, but the other areas were also investigated. The predominant astroglial elements were tanycytes (long, thin, fiber-like cells). In the teleost telencephala a 'fan-shape' re-arrangement of radial glia reflects the eversion. In bichir, starlet, and gar, in which the eversion is less pronounced, the 'fan-shape' re-arrangement did not form. In the tectum the radial glial processes were immunostained, but in Ostariophysi and Euteleostei it did not extend into their deep segments. In the cerebellum Bergmann-like glia was found in each group, including non-teleosts, except for Cyprinidae. The vagal lobe was uniquely enlarged and layered in Cyprininae, and had a corresponding layered astroglial system, which left almost free of GFAP the zones of sensory and motor neurons. In conclusion, despite the diversity and evolutionary alterations of Actinopterygii brains, the diversity of the astroglial architecture is moderate. In contrast to Chondrichthyes and Amniotes; in Actinopterygii true astrocytes (stellate-shaped extraependymal cells) did not appear during evolution, and the expansion of GFAP-free areas was limited.

No rapid and demarcating astroglial reaction to stab wounds in Agama and Gecko lizards and the caiman Paleosuchus - it is confined to birds and mammals.

The present study proves that rapid and demarcating astroglial reactions are confined to birds and mammals. To understand the function of post-lesion astroglial reaction, the phylogenetical aspects are also to be investigated. Considering the regenerative capabilities, reptiles represent an intermediate position between the brain regeneration-permissive fishes and amphibians and the almost non-permissive birds and mammals. Damage is followed by a rapid astroglial reaction in the mammalian and avian brain, which is held as an impediment of regeneration. In other vertebrates the reactions were usually observed following long survival periods together with signs of regeneration, therefore they can be regarded as concomitant phenomena of regeneration. The present study applies short post-lesion periods comparable to those seen in mammals and birds for astroglial reactions. Two species of lizards were used: gecko (leopard gecko, Eublepharis macularius, Blyth, 1854) and agama (bearded dragon, Pogona vitticeps, Ahl, 1926). The gecko brain is rich in GFAP whereas the agama brain is quite poor in this. Crocodilia, the closest extant relatives of birds were represented in this study by Cuvier's dwarf caiman (Paleosuchus palpebrosus, Cuvier, 1807). The post-lesion astroglial reactions of crocodilians have never been investigated. The injuries were stab wounds in the telencephalon. The survival periods lasted 3, 7, 10 or 14 days. Immunoperoxidase reactions were performed applying anti-GFAP, anti-vimentin and anti-nestin reagents. No rapid and demarcating astroglial reaction resembling that of mammalian or avian brains was found. Alterations of the perivascular immunoreactivities of laminin and ß-dystroglycan as indicators of glio-vascular decoupling proved that the lesions were effective on astroglia. The capability of rapid and demarcating astroglial reaction seems to be confined to mammals and birds and to appear by separate, parallel evolution in them.

Distribution of GFAP in Squamata: Extended Immunonegative Areas, Astrocytes, High Diversity, and Their Bearing on Evolution.

Squamata is one of the richest and most diverse extant groups. The present study investigates the glial fibrillary acidic protein (GFAP)-immunopositive elements of five lizard and three snake species; each represents a different family. The study continues our former studies on bird, turtle, and caiman brains. Although several studies have been published on lizards, they usually only investigated one species. Almost no data are available on snakes. The animals were transcardially perfused. Immunoperoxidase reactions were performed with a mouse monoclonal anti-GFAP (Novocastra). The original radial ependymoglia is enmeshed by secondary, non-radial processes almost beyond recognition in several brain areas like in other reptiles. Astrocytes occur but only as complementary elements like in caiman but unlike in turtles, where astrocytes are absent. In most species, extended areas are free of GFAP-a meaningful difference from other reptiles. The predominance of astrocytes and the presence of areas free of GFAP immunopositivity are characteristic of birds and mammals; therefore, they must be apomorphic features of Squamata, which appeared independently from the evolution of avian glia. However, these features show a high diversity; in some lizards, they are even absent. There was no principal difference between the glial structures of snakes and lizards. In conclusion, the glial structure of Squamata seems to be the most apomorphic one among reptiles. The high diversity suggests that its evolution is still intense. The comparison of identical brain areas with different GFAP contents in different species may promote understanding the role of GFAP in neuronal networks. Our findings are in accordance with the supposal based on our previous studies that the GFAP-free areas expand during evolution.

Cerebrovascular ß-dystroglycan immunoreactivity in vertebrates: not detected in anurans and in the teleosts Ostariophysi and Euteleostei.

The aim of the present paper was to check for the presence of cerebrovascular dystroglycan in vertebrates, because dystroglycan, which is localized in the vascular astroglial end-feet, has a pivotal function in glio-vascular connections. In mammalian brains, the immunoreactivity of ß-dystroglycan subunit delineates the vessels. The results of the present study demonstrate similar patterns in other vertebrates, except for anurans and the teleost groups Ostariophysi and Euteleostei. In this study, we investigated 1 or 2 representative species of the main groups of Chondrichthyes, teleost and non-teleost ray-finned fishes, urodeles, anurans, and reptiles. We also investigated 5 mammalian and 3 bird species. Animals were obtained from breeders or fishermen. The presence of ß-dystroglycan was investigated immunohistochemically in free-floating sections. Pre-embedding electron microscopical immunohistochemistry on Heterodontus japonicus shark brains demonstrated that in Elasmobranchii, ß-dystroglycan is also localized in the perivascular glial end-feet despite the different construction of their blood-brain barrier. The results indicated that the cerebrovascular ß-dystroglycan immunoreactivity disappeared separately in anurans, and in teleosts, in the latter group before its division to Ostariophysi and Euteleostei. Immunohistochemistry in muscles and western blots from brain homogenates, however, detected the presence of ß-dystroglycan, even in anurans and all teleosts. A possible explanation is that in the glial end-feet, ß-dystroglycan is masked in these animals, or disappeared during adaptation to the freshwater habitat.

Distribution of beta-dystroglycan immunopositive globules in the subventricular zone of rat brain.

Present study demonstrates a novel localization of beta-dystroglycan in rat brain. Beside the meningeal surface and the cerebral vessels where beta-dystroglycan immunopositivity has been described, now immunopositive solid bodies were found at the basis of ependymocytes. Since they proved to be round in either coronal, sagittal, and horizontal section, revealing their globular shape, they received the term 'globules.' Double immunofluorescent labeling of GFAP (glial fibrillary acidic protein) and beta-dystroglycan revealed that 'globules' are within the 'cordon' of subventricular astrocytes. The 'globules' were found throughout the ventricular system, except the ventral part and floor of the third ventricle. The latter one comprised the median eminence and extended caudally to the inframamillary recess. The area where the 'globules' were missing, corresponded to that where ependymocytes were replaced by GFAP-immunopositive tanycytes. Utrophin and alpha-dystrobrevin were co-localized with dystroglycan, whereas alpha1-syntrophin was detected along the borders of the ependymal cells. Comparing our results to former data, the 'globules' seem to be the anchoring places of the 'fractons' formed by laminin [Mercier et al. (2003) J Comp Neurol 455:324-340], which interconnect the vascular basal lamina and the ependymal layer. The ependymal localization of the 'globules' was proved by pre-embedding electron microscopic immunohistochemistry. The 'globules' proved to be a labyrinthine structure, which seemed to be identical with the 'basal membrane labyrinth' described by Leonhardt [Leonhardt (1970) Z Zellforsch Mikrosk Anat 105:395-404]. The description of the beta-dystroglycan-immunopositive 'globules' contributes to the better understanding of the structure of the subventricular zone where neurogenesis can occur during adulthood and provides an important link to the meningeo-glial network.

Three-plane description of astroglial populations of OVLT subdivisions in rat: Tanycyte connections to distant parts of third ventricle.

This study demonstrates glial and gliovascular markers of organon vasculosum laminae terminalis (OVLT) in three planes. The distribution of glial markers displayed similarities to the subfornical organ. There was an inner part with vimentin- and nestin-immunopositive glia whereas GFAP and the water-channel aquaporin 4 were found at the periphery. This separation indicates different functions of the two regions. The presence of nestin may indicate stem cell-capabilities whereas aquaporin 4 has been reported to promote the osmoreceptor function. Glutamine synthetase immunoreactivity was sparse like in the area postrema and subfornical organ. The laminin and ß-dystroglycan immunolabelings altered along the vessels such as in the subfornical organ indicating altering gliovascular relations. The different subdivisions of OVLT received glial processes of different origins. The posterior periventricular zone contained short vimentin-immunopositive processes from the ependyma of the adjacent surface of the third ventricle. The lateral periventricular zone received forceps-like process systems from the anterolateral part of the third ventricle. Most interestingly, the "dorsal cap" received a mixed group of long GFAP- and vimentin-immunopositive processes from a distant part of the third ventricle. The processes may have two functions: a guidance for newly produced cells like radial glia in immature brain and/or a connection between distant parts of the third ventricle and OVLT.

The First Postlesion Minutes: An In Vivo Study of Extravasation and Perivascular Astrocytes Following Cerebral Lesions in Various Experimental Mouse Models.

The immediate alterations following lesions cannot be investigated by using fixed tissues. Here, we employed two-photon microscopy to study the alterations to the permeability of blood-brain barrier and to glio-vascular connections in vivo during the first minutes following cortical lesions in mice. Four models were used: (1) cryogenic lesion, (2) photodisruption using laser pulses, (3) photothrombosis, and (4) bilateral carotid ligation. Sulforhodamine101 was used for supravital labeling of astrocytes and dextran-bound fluorescein isothiocyanate for the assessment of extravasation. Transgenic mice, in which the endothelium and astrocytes expressed a yellow fluorescent protein, were also used. Astrocytic labeling in vivo was verified with postmortem immunostaining against glial fibrillary acidic protein (GFAP). Summary of results: (1) the glio-vascular connections were stable in the intact brain with no sign of spontaneous dynamic attachment/detachment of glial end-feet; (2) only direct vascular damage (photodisruption or cryogenic) resulted in prompt extravasation; (3) even direct damage failed to provoke a prompt astroglial response. In conclusion, the results indicate that a detachment of the astrocytic end-feet does not precede the breakdown of blood-brain barrier following lesions. Whereas vasogenic edema develops immediately after the lesions, this is not the case with cytotoxic edemas. Time-lapse recordings and three-dimensional reconstructions are presented as supplemental materials.

Glial architecture of the ghost shark (Callorhinchus milii, Holocephali, Chondrichthyes) as revealed by different immunohistochemical markers.

This article presents the first study on the glial architecture of a representative species of Holocephali, Callorhinchus milii (ghost shark). Holocephali are a small subclass of Chondrichthyes, with only a few extant genera, and those are considered to have a brain organization more similar to squalomorph sharks than to galeomorph sharks, skates, and rays. Three different astroglial markers--glial fibrillary acidic protein, S-100 protein, and glutamine synthetase (GS)--were investigated by immunohistochemical methods, applying both diaminobenzidine (DAB) and fluorescent techniques. They revealed similar glial structures, although most of them were detected by immunohistochemical reaction against GS and visualized by DAB. The predominant elements were radial ependymoglia spanning the area between the ventricular and meningeal surfaces, as in squalomorph sharks. Other similar features were the light appearance of myelinated neural tracts devoid of immunoreactivity, and the glial architecture of the reticular formation of the brain stem, cerebellum, and tectum, the latter with recognizable layers. The immunoreactivity of the vascular walls was similar; however, it is believed that different cell types form the blood-brain barrier in chimeras and in elasmobranchs. Some glial structures, however, resembled those of skates, rays, and galeomorph sharks. In C. milii astrocyte-like elements were observed in the telencephalon, using GS and S-100, although typical astrocyte-rich regions were not found. In some areas, especially the telencephalon, not only endfeet but also cell bodies were observed to be attached to the meningeal surface, with processes extending into the brain substance.

Evolutionary changes of astroglia in Elasmobranchii comparing to amniotes: a study based on three immunohistochemical markers (GFAP, S-100, and glutamine synthetase).

This paper supplements former studies on elasmobranch species with an immunohistochemical investigation into glutamine synthetase and S-100 protein, in addition to GFAP, and extends its scope to the representatives of almost every group of Elasmobranchii: squalomorph sharks, galeomorph sharks, skates (Rajiformes) and rays (Torpediniformes and Myliobatifomes). More glial elements were labeled by S-100 protein, and even more so by using glutamine synthetase immunostaining than by GFAP: more astrocytes (mainly non-perivascular ones) were detected in the telencephalon of sharks, skates and rays. Only the markers S-100 and glutamine synthetase, but not GFAP, characterized the Bergmann-glia of skates and rays and astrocyte-like non-ependymal cells in Squalus acanthias. Another squalomorph shark species, Pristiophorus cirratus, however, had GFAP immunopositive astrocytes. Of all the species studied, the greatest number of GFAP positive astrocytes could be observed in Mobula japanica (order Myliobatiformes), in each major brain part. According to anatomical location, perivascular glia comprised varied types, including even a location in Mobula, which can also be found in mammals. Remnants of radial glia were found in confined areas of skates, less so in rays. In the rhombencephalon and in the spinal cord modified ependymoglia predominated in every group. In conclusion, there was no meaningful difference between the astroglial architectures of squalomorph and galeomorph sharks. The difference in the astroglial structure between sharks and batoids, however, was confined to the telencephalon and mesencephalon, and did not take place in the rhombencephalon, the latter structure being quite similar in all the species studied. The appearance of astrocytes in the relatively thin-walled shark telencephalon, however, indicates that the brain thickening promoted the preponderance of astrocytes rather than their appearance itself. Although the evolutionary changes of astroglia had some similarities in Elasmobranchii and Amniota, there was one meaningful difference: in Elasmobranchii astrocytes did not prevail in conservative brain regions as they did in the progressive brain regions.

Appearance of ß-dystroglycan precedes the formation of glio-vascular end-feet in developing rat brain.

Dystroglycan has an important role in binding of perivascular glial end-feet tothe basal lamina. Its ß-subunit is localized in the glial end-feet. The investigation period lasted from E(embryonic day)12 to E20. Laminin and ß-dystroglycan were detected by immunohistochemistry, the glial localization of the latter one was supported  by electron microscopy. The immatureglial structures were visualized by the immunostaining of nestin. The ß-dystroglycan immunoreactivity appeared at E16 following the laminin of basal lamina but preceding the perivascular processes of radial glia (E18) and astrocyte-like cells (E20). It occurred in cell bodies which attached to the vessels directly but not with vascular processes and end-feet. The presence of ß-dystroglycan in such immature cells may promote their differentiation to perivascular astrocytes and influence the formation of the glio-vascular processes.

Disappearance of cerebrovascular laminin immunoreactivity as related to the maturation of astroglia in rat brain.

The present paper provides novel findings on the temporo-spatial correlation of perivascular laminin immunoreactivity with the early postnatal astrocyte development. The cerebrovascular laminin immunoreactivity gradually disappears during development. The fusion of the glial and vascular basal laminae during development makes the laminin epitopes inaccessible for antibody molecules (Krum et al., 1991, Exp Neurol 111:151). The fusion is supposed to correlate with the maturation of the glio-vascular connections. Glial development was followed by immunostaining for GFAP (glial fibrillary acidic protein), S100 protein, glutamine synthetase as glial markers and for nestin to visualize the immature glial structures. Our investigation focused on the period from postnatal day (P)2 to P16, on the dorso-parietal pallium. In the wall of the telencephalon the laminin immunoreactivity disappeared between P5 and P10; in subcortical structures it persisted to P12 or even to P16. Its disappearance overlapped the period when GFAP-immunopositive astrocytes were taking the place of radial glia. Despite the parallel time courses, however, the spatial patterns of the two processes were just the opposite: disappearance of the laminin immunoreactivity progressed from the middle zone whereas the appearance of GFAP from the pial surface and the corpus callosum. Rather, the regression of the vascular laminin immunoreactivity followed the progression of the immunoreactivities of glutamine synthetase and S100 protein. Therefore, the regression really correlates with a 'maturation' of astrocytes which, however, affects other astrocyte functions rather than cytoskeleton.

Correlation Between Extravasation and Alterations of Cerebrovascular Laminin and ß-Dystroglycan Immunoreactivity Following Cryogenic Lesions in Rats.

The blood-brain barrier becomes "leaky" following lesions. Former studies revealed that following lesions the immunoreactivity of cerebrovascular laminin becomes detectable whereas that of ß-dystroglycan disappears. These alterations may be indicators of glio-vascular decoupling that may result in the impairment of the blood-brain-barrier. This study investigates correlation between the post-lesion extravasation and the above-mentioned immunohistochemical alterations. Following cryogenic lesions, the survival periods lasted 5, 10, 30 minutes, 1 or 12 hours, or 1 day. Some brains were fixed immediately post-lesion. Immunofluorescent reactions were performed in floating sections. The extravasation was detected with immunostaining for plasma fibronectin and rat immunoglobulins. When the survival period was 30 minutes or longer, the area of extravasation corresponded to the area of altered laminin and ß-dystroglycan immunoreactivities. Following immediate fixation some laminin immunoreactivity was already detected. The extravasation seemed to precede this early appearance of laminin immunoreactivity. The ß-dystroglycan immunoreactivity disappeared later. When the extravasation spread into the corpus callosum, vascular laminin immunoreactivity appeared but the ß-dystroglycan immunoreactivity persisted. It seems that extravasation separates the glial and vascular basal laminae, which results in the appearance of laminin immunoreactivity. The disappearance of ß-dystroglycan immunoreactivity is neither a condition nor an inevitable consequence of the 2 other phenomena.

Heterogeneous occurrence of aquaporin-4 in the ependyma and in the circumventricular organs in rat and chicken.

Aquaporins are selective water channel proteins critical in volume homeostasis. In the CNS AQP4 predominates, localized mainly in the glia limitans, the perivascular endfeet and ependyma. The present immunofluorescent study reveals the distribution of aquaporin-4 in the circumventricular organs in rat and chicken brains. The ventricular ependyma (especially in the third one), the subfornical organ, the area postrema, the rat pineal body (in part), and the vascular organ of lamina terminalis were marked by intense immunopositivity. Several areas, however, proved to be immunonegative: the central canal, the subcommissural organ, the ependymal zone of the median eminence in rat but its whole thickness in chicken, the subtrochlear organ, and the paraventricular organ. The immunostaining of the lateral septal and subseptal organs were similar to their environment. Results on developing rats suggested that the aquaporin-4 immunonegativity is a secondary phenomenon. Surveying other structural and functional features, no clear explanation of the heterogeneous occurrence of aquaporin-4 was found. The absence of aquaporin-4 seems to correlate with some features of the "ependymal organs" (thickened, pseudostratified ependyma, presence of blood-brain barrier) and with the avoidance of GFAP. On the other hand, the organs rich in aquaporin-4 have features of the "hypendymal organs" (glial and vascular plexus but no blood-brain barrier). There are organs, however, which do not fit into either group completely, i.e. the lateral septal and subseptal organs. Presence of tight junctions coincides with the absence of aquaporin-4 in the ependyma of spinal cord, the subcommissural organ and the ependyma of median eminence.

Glial and perivascular structures in the subfornical organ: distinguishing the shell and core.

The subfornical organ (SFO) is a circumventricular organ with a chemosensitive function, and its vessels have no blood-brain barrier. Our study investigated the glial and vascular components in the SFO to determine whether their distributions indicate subdivisions, how to characterize the vessels and how to demarcate the SFO. To this end, we investigated glial markers (GFAP, glutamine synthetase, S100) and other markers, including vimentin and nestin (immature glia), laminin (basal lamina), ß-dystroglycan (glio-vascular connections), and aquaporin 4 (glial water channels). We determined that the 'shell' of the SFO was marked by immunoreactivity for S100, GFAP and aquaporin 4. Nestin immunoreactivity was characteristic of the 'core'. Vimentin was almost evenly distributed. Glutamine synthetase immunoreactivity occurred in the shell but its expression was sparse. Vessels in the core were decorated with laminin but showed a discontinuous expression of aquaporin 4. Vimentin and GFAP staining was usually in separate glial elements, which may be related to their functional differences. Similar to other vessels in the brain, ß-dystroglycan was detected along the shell vessels but laminin was not. The gradual disappearance of the laminin immunopositivity was attributed to the gradual disappearance of the perivascular space. Thus, our findings suggest that the shell and core glio-vascular structures are adapted to different sensory functions: osmoperception and the perception of circulating peptides, respectively.