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.

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.

Dystroglycan has an important role in binding of perivascular glial end-feet tothe basal lamina. Its β-subunit is localized in the glial end-feet. The investigation period lasted from E(embryonic day)12 to E20. Laminin and β-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 β-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 β-dystroglycan in such immature cells may promote their differentiation to perivascular astrocytes and influence the formation of the glio-vascular processes.

The glial fibrillary acidic protein (GFAP) is the principal intermediate filament protein and histochemical marker for astroglia. It appears contradictory that there are extended GFAP-poor or even GFAP-free areas in the brains of various vertebrate clades: cartilaginous and ray-finned fishes, and amniotes. The “Relevant Subsections: Extended GFAP-free areas in various vertebrates” section in this study reviews our GFAP mapping studies on the brains of 58 species within these clades, as well as mappings from other authors, and demonstrates that these areas appeared independently from one another in the more advanced groups of different clades; it raises the supposition that the lack of GFAP is an apomorphic phenomenon. The GFAP expression has withdrawn mainly relatively: the GFAP-immunonegative areas increased more than the immunopositive ones. Primarily, regions that expanded and increased in complexity during evolution lack GFAP immunopositivity (except for their perivascular glia). The absence of GFAP expression, however, does not indicate the lack of astroglia. In the areas immunonegative to GFAP, astrocytes were visualized using other markers, such as glutamine synthetase or S-100 protein. In birds and mammals, lesions induced GFAP expression in these areas. It shows that the ability to express GFAP is not lost but has become facultative. These data suggest that the lack of GFAP production may provide an evolutionary advantage. The “Discussion” section relates the GFAP “withdrawal” to other steps of evolution: the increasing complexity and thickening of the brain wall, as well as the appearance of the astrocytes, particularly protoplasmic astrocytes, and then examines the proposed evolutionary advantages and disadvantages of the absence of GFAP. The role of the relative “withdrawal” of GFAP expression in brain evolution remains to be definitively answered. The most probable candidates may include the absence of synthesizing an unnecessary protein, improved adaptation of astrocytes to the demands of neurons, and an increased capacity for synaptic plasticity. In contrast, one must consider that the withdrawal of GFAP may not be a primary phenomenon but rather a consequence of the evolution of neural networks.

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 β‐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.

This study demonstrates glial and glio‐vascular markers of OVLT (organon vasculosum laminae terminalis) in 3 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 β‐dystroglycan immunolabelings altered along the vessels such as in the subfornical organ indicating altering glio‐vascular 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 3^rd ventricle. The lateral periventricular zone received forceps‐like process systems from the anterolateral part of the 3^rd ventricle. Most interestingly, the ’dorsal cap’ received a mixed group of long GFAP‐ and vimentin‐immunopositive processes from a distant part of the 3^rd 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 3^rd ventricle and OVLT.