Changing glial organization relates to changing fiber order in the developing optic nerve of ferrets

The structures of the developing eye-stalk and the relationships of early retinofugal fibers as they pass through the stalk, chiasm, and tract have been studied by light and electron microscopical methods in fetal ferrets aged 23–27 days. The early eye-stalk can be divided into two parts: a narrow extracranial part has a narrow lumen and is lined by few cells, whereas a thicker intracranial part has a wider lumen and is lined by several rows of cells. At the earliest stages no axon bundles are recognizable in the stalk, but fibers of the supraoptic commissure are already beginning to cross the midline in the diencephalon. Subsequently, as retinofugal axons invade the stalk, the glia of the extracranial part of the stalk have an interfascicular distribution and axon bundles are separately encircled by glial cytoplasm. In the intracranial part, as in the chiasm and tract, the glial cells occupy a periventricular position and send slender radial cytoplasmic processes to the subpial surface; these pass between groups of axons that here lie immediately deep to the subpial glia. Whereas axonal growth cones have no evident preferred distribution in the extracranial stalk, they tend to accumulate near the pial surface intracranially. The boundary between the two types of organization shifts as development proceeds so that the interfascicular glial structure of the early extracranial stalk first encroaches upon the intracranial parts and later appears in the chiasm. The characteristic adult arrangement of fibers in an age-related order in the optic chiasm and tract, but not in the optic nerve, can be understood if axonal growth cones are guided toward the pial surface by radial glia but not by interfascicular glia. From the distribution of the growth cones, this is what appears to happen.     

Development of astroglial elements in the suprachiasmatic nucleus of the rat: with special reference to the involvement of the optic nerve

The development of astroglial cells and the effect of the retinohypothalamic tract on it were studied by vimentin and glial fibrillary acidic protein (GFAP) immunocytochemistry in the suprachiasmatic nucleus (SCN) of the rat. At the embryonic stage, vimentin-immunoreactive (VIM-IR) radial glia, precursors of astrocytes, were dominant. However, their filaments vanished in the first few postnatal days. Instead of VIM-IR glial filaments, GFAP-immunoreactive (GFAP-IR) astrocytes appeared at E20 and grew rapidly from the P3 stage. GFAP immunoreactivity in the ventrolateral portion of the SCN (VLSCN) was measured using a computer-assisted image analyzing system. In normal rats, GFAP immunoreactivity showed a stepwise pattern with two slopes at P3-P4 and P20-P25. Bilaterally eye-enucleated rats operated on the day of birth showed lower GFAP immunoreactivity than normal rats and the GFAP immunoreactivity did not increase between P20 and P25 when GFAP-IR glial processes rapidly expand. Electron microscopic investigation at P50 (adult stage) revealed that neurons in the VLSCN had often direct apposition without astroglial processes and the frequency of this finding was significantly higher in eye-enucleated rats than in the control rats. These findings strongly suggest that the postnatal development of astroglial elements, particularly the expansion of GFAP-IR processes in the SCN, is regulated by retinohypothalamic projection.     

Developmental pattern of GFAP and vimentin gene expression in rat brain and in radial glial cultures

In the present study we analyze the events which occur during the early stages of astrogliogenesis by examining the pattern of both GFAP and vimentin gene expression and their corresponding immunoreactive proteins during rat brain development. This study was carried out “in vivo” (whole brain) and “in vitro” (primary culture of radial glia) using immunofluorescence, immunoblotting, and Northern blot analysis. Our results demonstrate that although GFAP immunostaining appeared late in gestation and at day 5 in radial glia cultures, GFAP mRNA expression was first detected, at very low levels, on fetal (F) day 15 and increased to F21. During postnatal development a striking increase in GFAP and its encoding messenger occurs. In contrast, the levels of vimentin and its mRNA expression were very high during the fetal stage (F15 to F21). Thereafter vimentin expression declined during postnatal (P) development until P21 and then remained constant at adult levels. In contrast, an increase in vimentin expression was observed in glial cells throughout the entire culture period. The biological significance of the developmental patterns of GFAP and vimentin expression in astroglial cells during brain development is discussed. © 1995 Wiley-Liss, Inc.     

Radial glia in the human fetal cerebrum: A combined golgi, immunofluorescent and electron microscopic study

Golgi techniques, immunofluorescence for glial fibrillary acidic (GFA) protein, and electron microscopy (EM) were used to determine the nature of radial glia in the cerebrum of human fetuses ranging from 7 to 20 weeks of ovulation age. Successful Golgi impregnation of radial fibers was achieved in fetuses 12 weeks of age and older. These fibers spanned the entire thickness of the hemisphere. At the pial surface many of them branched and terminated in pyramidal end feet expansions. Indirect immunofluorescent preparations utilizing antiserum to GFA protein, a protein specific for astrocytes, demonstrated numerous radially oriented nearly parallel fluorescent fibres between the ventricular zone and pia mater. GFA protein-positive fibers were demonstrated in all fetal specimens examined with this technique (10 weeks of age and older). Along the outer border of the marginal zone they formed a horizontal GFA protein-containing subpial membrane. By EM there were numerous linear electron lucent astrocytic processes containing 8-9 nm filaments and occasional glycogen granules at all levels of the cerebrum. They were interspersed among smaller and darker neuronal processes containing 20-25 nm neurotubules, and were demonstrable at all fetal ages between 7 and 18 weeks. They formed pericapillary investments and subpial terminal expansions closely abutting basal lamina of pia mater in every specimen examined. On the basis of these combined analyses, we conclude that radial glial fibers in early human fetal cerebrum represent processes of immature astrocytes. Although subsequently undergoing further maturation, radial glia already possess fundamental immunocytochemical and morphological characteristics indicative of astrocytic differentiation. A significant implication of our findings is that the development of astrocytes in the human fetal brain occurs much earlier than formerly believed.     

Mab22C11 antibody to amyloid precursor protein recognizes a protein associated with specific astroglial cells of the rat central nervous system characterized by their capacity to support axonal outgrowth

Amyloid precursor protein (APP) is a transmembrane glycoprotein which is believed to promote neural cell adhesion, neural survival, and neuritogenesis. The present study was undertaken to determine whether APP could be detected within different types of astroglial cells present in the central nervous system (CNS) of neonatal or adult rats. The localization of this protein within glial cells was studied by using a monoclonal antibody (Mab22C11) that recognizes all APP isoforms and in addition cross-reacts with APP-like proteins. In the brain of neonatal rats, Mab22C11 immunostaining was associated with numerous elongated radial glia-like structures. In the intact brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with (i) numerous neuron-like structures and (ii) glial structures immunostained for glial fibrillary acidic protein (GFAP) and/or vimentin, including tanycytes mostly located in the mediobasal hypothalamus, fibrous astrocytes located in the white matter and ependymocytes bordering the ventricles. On the other hand, all the GFAP-immunostained astrocytes located in the grey matter were Mab22C11 negative. In the lesioned brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with intensely GFAP-immunostained reactive astrocytes located close to a surgical lesion, but not with those induced by Wallerian degeneration that appear at a distance from a lesion. Electron microscopic observations further indicated that in all these labeled astroglial cells, Mab22C11 immunostaining was mainly localized to the limiting plasma membrane and the membrane of intracytoplasmic cisternae and vesicles. These data indicate that Mab22C11 antibody induces strong immunostaining of specific astroglial cells of the neonatal and adult rat CNS that support axonal outgrowth, therefore suggesting that an APP-like protein associated with these cells participates in their axonal outgrowth promoting properties. J Comp Neurol 377:550–564, 1997. © 1997 Wiley-Liss, Inc.     

Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats

In the development of the CNS, radial glial cells are among the first cells derived from neuroepithelial cells. Recent studies have reported that radial glia possess properties of neural stem cells. We analyzed the antigen expression and distribution of radial glia after spinal cord injury (SCI). Sprague-Dawley rats had a laminectomy at Th11-12, and spinal cord contusion was created by compression with 30 g of force for 10 min. In the injury group, rats were examined at 24 h and 1, 4, and 12 weeks after injury. Frozen sections of 20-microm thickness were prepared from regions 5 and 10 mm rostral and caudal to the injury epicenter. Immunohistochemical staining was performed using antibodies to 3CB2 (a specific marker for radial glia), nestin, and glial fibrillary acidic protein (GFAP). At 1 week after injury, radial glia that bound anti-3CB2 MAb had spread throughout the white matter from below the pial surface. From 4 weeks after injury, 3CB2 expression was also observed in the gray matter around the central canal, and was especially strong around the ependymal cells and around blood vessels. In double-immunohistochemical assays for 3CB2 and GFAP or 3CB2 and nestin, coexpression was observed in subpial structures that extended into the white matter as arborizing processes and around blood vessels in the gray matter. The present study demonstrated the emergence of radial glia after SCI in adult mammals. Radial glia derived from subpial astrocytes most likely play an important role in neural repair and regeneration after SCI.     

Development of astroglial cells in the proliferative matrices, the granule cell layer, and the hippocampal fissure of the hamster dentate gyrus.

The histogenesis of the hamster dentate gyrus was studied with light and electron microscopy and antisera against the astrocyte-associated antigens vimentin and GFAP, in order to follow the differentiation of radial glial cells and astrocytes. The formation of the stratum granulosum is preceded by the establishment of successive dentate matrices, which are formed by cells that leave the ventricular neuroepithelium and occupy positions above the fimbria (suprafimbrial), below the pial surface (subpial), and within the dentate hilus (hilar dentate matrix). The subpial dentate matrix invades the marginal zone of that region of the cerebral wall, where the stratum granulosum will later develop. From the beginning of its existence on embryonal day 13 (E13) up to its disappearance about postnatal day 7 (P7), it is characterized by a high content of GFAP-positive cells and mitoses. This indicates early gliogenesis in the dentate anlage, long before the appearance of the stratum granulosum. Many of the bipolar GFAP-positive cells are oriented parallel to the pial surface and have focal contacts to the pial basement membrane. The establishment of the subpial dentate matrix splits the primordial radial glial scaffold of the hippocampal/dentate anlage into two bundles: 1) the suprafimbrial bundle that retains its original radial position between ventricle and pial surface; and 2) the dorsal glial bundle that traverses the ventral tip of the pyramidal cell layer of future CA3. The latter is pushed dorsolaterally, away from the pial surface, by the enlargement of the subpial dentate matrix and, later, by the suprapyramidal blade. The latter emerges around birth as small radial columns of granule cells located between the bent basal parts of the ventralmost fibers of the dorsal glial bundle and the subpial dentate matrix. From the beginning of its existence it is traversed by unipolar "secondary" radial glial fibers that appear to originate from the subpial dentate matrix. Both the supra- and the infrapyramidal blades seem to elongate by the addition of postmitotic granule cells and "secondary" radial glial cells from the subpial dentate matrix to the growing end of the primordial stratum granulosum. The hilar dentate matrix that is localized in the prospective hilar region, inside the growing stratum granulosum, also contains glial cells that seem to be incorporated into the stratum granulosum. The dentate gyrus is demarcated from the CA1 region of the hippocampus proper by GFAP-positive cells that populate the hippocampal fissure, and that also originate from the subpial dentate matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphological evidence for direct interaction between gonadotrophin-releasing hormone neurones and astroglial cells in the human hypothalamus

In rodents, there is compelling evidence indicating that dynamic cell-to-cell communications involving cross talk between astroglial cells (such as astrocytes and specialised ependymoglial cells known as tanycytes) and neurones are important in regulating the secretion of gonadotrophin-releasing hormone (GnRH), the neurohormone that controls both sexual maturation and adult reproductive function. However, whether such astroglial cell-GnRH neurone interactions occur in the human brain is not known. In the present study, we used immunofluorescence to examine the anatomical relationship between GnRH neurones and glial cells within the hypothalamus of five women. Double-staining experiments demonstrated the ensheathment of GnRH neurone perikarya by glial fibrillary acidic protein (GFAP)-immunoreactive astrocyte processes in the periventricular zone of the tuberal region of the hypothalamus. GFAP immunoreactivity did not overlap that of GnRH at the GnRH neurone's projection site (i.e. the median eminence of the hypothalamus). Rather, human GnRH neuroendocrine fibres were found to be closely associated with vimentin or nestin-immunopositive radial glial processes likely belonging to tanycytes. In line with these light microscopy data, ultrastructural examination of GnRH-immunoreactive neurones showed numerous glial cells in direct apposition to pre-embedding-labelled GnRH cell bodies and/or dendrites in the infundibular nucleus, whereas postembedding immunogold-labelled GnRH nerve terminals were often seen to be enwrapped by glial cell processes in the median eminence. GnRH nerve button were sometimes visualised in close proximity to fenestrated pituitary portal blood capillaries and/or evaginations of the basal lamina that delineate the pericapillary space. In summary, these data demonstrate that GnRH neurones morphologically interact with astrocytes and tanycytes in the human brain and provide evidence that glial cells may contribute physiologically to the process by which the neuroendocrine brain controls the function of GnRH neurones in humans.     

Development of glial cells in the cerebral wall of ferrets: direct tracing of their transformation from radial glia into astrocytes

Coronal sections of the cerebral wall from developing ferrets (newborn to adult) were double-stained with antibodies to vimentin and glial fibrillary acidic protein (GFAP). At birth, the dominant glial population was radial glia and these cells labeled only for vimentin. A small population of immature astrocytes in the cortical plate was double labeled for GFAP and vimentin. In successive days, the number of vimentin-positive radial glia gradually decreased and they disappeared entirely at about 21 days. During this same period, the double-stained astrocytes increased in number and were distributed throughout the cortical plate and intermediate zone. After 6 weeks of age the astrocytes were mostly confined to the developing white matter. Around this time they gradually lost their vimentin staining, and in the adult no vimentin-positive elements were seen except at the ependymal surface. In newborn ferrets single radial glial cells were also visualized by applying the carbocyanine dye DiI onto the pial surface of fixed brains. While most radial glia extended from the ventricular zone to the pial surface, a substantial fraction of them had lost their contact to the ventricular zone. Their somata were displaced into the subventricular zone and lower portion of the intermediate zone. The possibility that radial glia transform into astrocytes was directly tested by injecting fluorescent dyes under the pial surface of newborn ferrets at a time when virtually no GFAP-positive astrocytes are present. The tracer, which was taken up in the upper portion of the cortical plate, stained the radial glial cell somata in the ventricular zone in a similar way as the dye DiI did in the fixed brains. As the radial glial cells disappeared at successively longer survival times, the tracer was ultimately found within newly formed GFAP-positive astrocytes. These results provide strong support for the hypothesis that radial glia cells are the immature form of astrocytes (Choi and Lapham: Brain Res. 148:295-311, '78; Schmechel and Rakic: Anat. Embryol. (Berl.) 156:115-152, '79), and also show that, at least in the ferret cortex, the transformation is accompanied by a change in the expression of intermediate filament protein.     

Prenatal exposure to ethanol alters the postnatal development and transformation of radial glia to astrocytes in the cortex

Postmitotic neurons migrate from a zone(s) near the ventricles to the neocortex. During this migration, neurons associate with radial glia. After serving their role as guides for neuronal migration, the radial glia transform into astrocytes. Prenatal exposure to ethanol causes abnormal neuronal migration. We examined the effects of gestational exposure to ethanol on radial glia and astrocytes. Radial glia were stained immunohistochemically with the antibody RAT-401, and astrocytes were labeled with an antibody directed against glial-fibrillary acidic protein (GFAP). The subjects were the offspring of rats fed an ethanol-containing liquid. diet (Et), pair-fed a liquid control diet (Ct), or fed chow and water (Ch). During the first postnatal week, radial glial fibers (in Et-treated rats and controls) stretched from the ventricular surface through the developing. cerebral wall to the pial surface. In the Et-treated rats, the radial processes were less dense and more poorly fasciculated than they were in the Ch-and Ct-treated rats. Moreover, by postnatal day (P) 5, there was a significant reduction in RAT-401 immunostaining in the Et-treated rats, particularly in the superficial cortex. A similar reduction in control rats did not begin until P10. In all three treatment groups, GFAP-immunoreactive astrocytes were in the cortex throughout the period from P1 to P45. In neonates, GFAP-positive cells were distributed in the marginal zone (layer I) and the intermediate zone (the white matter). The number of GFAP-positive cells in the cortical plate increased steadily with time so that, by P26, GFAP-immunoreactive astrocytes were distributed evenly through all cortical laminae. Interestingly, between P5 and P12, the number of astrocytes was significantly greater in Et-treated rats than in controls. Thus prenatal exposure to ethanol induces the premature loss of RAT-401-positive processes and the precocious increase in GFAP immunostaining. These ethanol-induced changes in glial development indicate that ethanol accelerates the transformation of radial glia into astrocytes. Moreover, the ethanol-induced premature degradation of the network of radial glial fibers may underlie the migration of late-generated neurons to ectopic sites. © 1993 Wiley-Liss, Inc.     

Ganglioside GD3 in radial glia and astrocytes in situ in brains of young and adult mice

Ganglioside GD3 occurs in immature cells in the neuroectoderm. However, with regard to particular cellular locations of GD3, rat brain has received more attention than mouse brain. In brains from neonatal mice the most intense GD3 immunostaining appears to occur in structures that differ from those that immunostain the most intensely in brains from neonatal rats (Cammer and Zhang: J Histochem Cytochem 44: 143–149, 1996). In the present study epifluorescence and confocal microscopy were used for the purpose of identifying the types of GD3-immunopositive structures in brains of neonatal, 2-week-old, and adult mice. Vibratome sections from mouse brains were double immunostained for GD3 and respective markers for macrophages, microglia, and cells belonging to the oligodendrocyte lineage. Surprisingly, none of those marker antigens immunostained intensely in the same respective structures as GD3. The GD3-positive structures, however, did resemble protoplasmic astrocytes and radial glia, some with GD3-positive end-feet at the glia limitans; however, we did not rule out the possibility that there might be some GD3 on the surfaces of prooligodendroblasts. The scarcity of glial fibrillary acidic protein (GFAP)-positive cells in brains of neonatal mice made it impractical to look for GD3+/GFAP+ structures that might belong to the astrocyte lineage. The Mu subunit of glutathione-S-transferase (Mu) was shown to label radial glia and the few GFAP-positive cells in brains of neonatal mice. Subsequently, confocal microscopy showed Mu and GD3 to be colocalized in radial glia and protoplasmic astrocytes in the neonate. In brains from mice ≥2 weeks of age GD3 immunostaining was demonstrated in GFAP-positive astrocytes, including reactive astrocytes. Much of the GD3 appeared to occur at the tips of astrocyte processes. It is suggested that GD3 in radial glia and astrocytes may function as a ligand enabling recognition of those structures by neurons or as a precursor of more complex gangliosides in neurons. © 1996 Wiley-Liss, Inc.     

Differential patterns of glial fibrillary acidic protein-immunolabeling in the brain of adult lizards

The present study describes by means of immunohistochemistry the comparative distribution of glial fibrillary acidic protein (GFAP)-positive cells in the forebrain and midbrain of three species of lizards: Eumeces algeriensis, Scincoidae; Agama impalearis, Agamidae; Tarentola mauritanica, Gekkonidae. In the species studied, the different types and proportions of glial cells expressing GFAP showed considerable variation. These cells include radial glia, oval cells, tanycytes, ependymocytes, glia limitans, and astrocytes. In Eumeces, astrocytes are particularly abundant and their processes form numerous perivascular end-feet; in addition well-differentiated ependymal cells and glia limitans express GFAP. These mature glial features are concordant with the relatively advanced phylogenetic level of Eumeces. In Tarentola, relatively few GFAP-expressing glial cells are observed, consisting mainly of radial glia and tanycytes. These features indicate a relatively immature state of the glial cell populations in this species. In Agama, GFAP-immunostained cells are confined to the periventricular and subpial brain areas; the ventricular lining contains numerous GFAP-immunopositive tanycytes and well-differentiated glia limitans. This pattern indicates that the glial cell profile in Agama exhibits characteristics intermediate between Eumeces and Tarentola, a feature which is discordant with the relatively primitive phylogenetic level of Agamidae compared to Gekkonidae. Together, the results of the present study provide novel data on the characterization of GFAP-expressing cell populations in different species of lizards. We suggest that the different glial patterns observed in the lizard brain correlates with developmental and functional aspects.     

Multifocal pattern of postnatal development of the macroglial framework of the rat fimbria

The development of the rat fimbria over the first postnatal month is associated with an approximate doubling of the tract diameter, a large increase in the number of glial cells, and the transformation of the prenatal radial glial skeleton into the adult interfascicular glial rows of solitary astrocytes and contiguous myelinating oligodendrocytes. The ventricular zone is reduced from a heterogeneous germinal layer of three or more cells thick at birth to the mature adult unicellular ependyma of homogeneous pale, mitotically inactive cells by the end of the second postnatal week. Mitoses are present throughout the body of the tract at all times, and persist, at reduced levels, in the adult. At birth the interior of the fimbria has only few scattered glial cell nuclei, largely solitary, or at most in longitudinal pairs. Over the first two postnatal weeks, the numbers and density of the interfascicular glia increase continuously. The scattered cells and cell clusters become progressively transformed into longer unicellular rows, which are aligned along the longitudinal axis of the tract, and which finally coalesce to form the continuous regular astrocyte/oligodendrocyte units that make up the interfascicular glial rows of the adult fimbrial glial skeleton. The increased cell packing density of the developing fimbrial glia is associated with a substantial decrease in nuclear and cytoplasmic size. From the end of the second postnatal week, the characteristic, large pale solitary astrocytes, and the smaller, more numerous, densely stained, closely packed oligodendrocytes are recognisable. Immunostaining for glial fibrillary acidic protein shows that immediately after birth the characteristic embryonic pattern of regular parallel radial glial processes starts to be modified by the progressive accumulation of longitudinal astrocytic processes, so the prenatal radial glial framework is rapidly transformed into the adult type of rectilinear array of radial and longitudinal processes. The development of the oligodendrocytes is shown clearly by immunostaining for myelin basic protein in enlarged, cytoplasm-rich, symmetrically placed cell pairs first seen at around P7. At P8-P10, there is a characteristic pattern of simultaneous multifocal maturation in which a single oligodendrocyte in each cluster develops a full complement of parallel, rather varicose myelinating processes. By P14 myelination is becoming confluent, oligodendrocytes are smaller, darker, with little cytoplasm, and individual myelinating processes cannot be discerned. Even at the end of the first postnatal month there are still many immature glia of indeterminate morphology. Myelination tends at first to be concentrated in the region adjacent to the hippocampus, and only reaches strengthen the hope that it may in future become possible to devise some form of self-reconstruction of the damaged adult glial tract structure in traumatic lesions completion by the end of the second month. During the first postnatal week, the entire array of fimbrial axons is traversed by the entire population of dentate neuroblasts. The highly vascular connective tissue on the pial surface of the fimbria is occupied by a prominent but transient population of mast cells which disappear in the adult.     

GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes

Neural stem cells in the adult subventricular zone (SVZ) derive from radial glia and express the astroglial marker glial fibrillary acidic protein (GFAP). Thus, they have been termed astrocytes. However, it remains unknown whether these GFAP-expressing cells express the functional features common to astrocytes. Using immunostaining and patch clamp recordings in acute slices from transgenic mice expressing green fluorescent protein (GFP) driven by the promoter of human GFAP, we show that GFAP-expressing cells in the postnatal SVZ display typical glial properties shared by astrocytes and prenatal radial glia such as lack of action potentials, hyperpolarized resting potentials, gap junction coupling, connexin 43 expression, hemichannels, a passive current profile, and functional glutamate transporters. GFAP-expressing cells express both GLAST and GLT-1 glutamate transporters but lack AMPA-type glutamate receptors as reported for dye-coupled astrocytes. However, they lack 100 microM Ba2+-sensitive inwardly rectifying K+ (K(IR)) currents expressed by astrocytes, but display delayed rectifying K+ currents and 1 mM Ba2+-sensitive K+ currents. These currents contribute to K+ transport at rest and maintain hyperpolarized resting potentials. GFAP-expressing cells stained positive for both K(IR)2.1 and K(IR)4.1 channels, two major K(IR) channels in astrocytes. Ependymal cells, which also derive from radial glia and express GFAP, display typical glial properties and K(IR) currents consistent with their postmitotic nature. Our results suggest that GFAP-expressing cells in concert with ependymal cells can perform typical astrocytic functions such as K+ and glutamate buffering in the postnatal SVZ but display a unique set of functional characteristics intermediate between astrocytes and radial glia.     

Migration of astrocytes transplanted to the midbrain of neonatal rats.

Previous studies have indicated that transplanted astrocytes are able to survive, express glial fibrillary acidic protein (GFAP), and migrate in the host brain, and that the pattern and speed of astrocyte migration is largely determined by the location of the graft. We examine here the pattern of astrocyte migration in the midbrain by transplanting CD-1 mouse corpus callosum (P2-3) into the midbrain of neonatal rats. The location of the grafts and the distribution of donor astrocytes were assessed by using a monoclonal antibody (anti-M2) specific for mouse astrocytes. A characteristic donor astrocyte distribution was seen. The highest density of cells was in the region of the substantia nigra (SN); lower numbers were found in the medial geniculate nucleus (MGN). Donor astrocytes were also found in the superior colliculus (SC) and central gray region, but only when the body of a graft was located nearby. [3H]thymidine studies showed that the concentrations of donor astrocytes in the SN were not the result of high levels of mitotic activity: all indications were that the proportion of dividing donor cells closely matched that of host glia. The pattern of astrocyte migration in the midbrain did not follow the course established by radial glia and was not influenced by axonal degeneration in the SC after removal of eyes. Moreover, donor cells failed to migrate along the course of axonal outgrowth from co-grafted retinae. Reciprocally, axonal elongation from retinal grafts did not follow the pathway of astrocyte migration, thus suggesting that astrocyte migration and neuronal outgrowth follow different cues.     

Dynamic transformation of Bergmann glial fibers proceeds in correlation with dendritic outgrowth and synapse formation of cerebellar Purkinje cells

Bergmann glia (BG) are unipolar cerebellar astrocytes, whose radial (or Bergmann) fibers associate with developing granule cells and mature Purkinje cells (PCs). In the present study, we investigated the morphodifferentiation of BG by immunohistochemistry for glutamate transporter GLAST and electron microscopy. GLAST was expressed widely in cerebellar radial glia/astrocytes during fetal and neonatal periods and became concentrated in BG postnatally. During the second postnatal week when PC dendrites grow actively, GLAST immunostaining revealed dynamic cytologic changes in Bergmann fibers in a deep-to-superficial gradient; Bergmann fibers traversing the external granular layer were stained as rod-like fibers, whereas in the molecular layer, the rod-like pattern was gradually replaced with a reticular meshwork. At postnatal day 10, the superficial rod-like domain was composed of glial fibrillary acidic protein (GFAP)-positive/GLAST-positive straight fibers, forming cytoplasmic swellings and short filopodia. Along this domain, the tip of growing PC dendrites ascended vertically and entered the base of the external granular layer. The deeper reticular domain of Bergmann fibers was characterized by active expansion of GFAP-negative/GLAST-positive lamellate processes, which surrounded PC synapses almost completely. Therefore, the transformation of Bergmann fibers proceeds in correlation with dendritic differentiation of PCs. The intimate PC-BG relationships during cerebellar development raise the possibility that a preexisting glial shaft could serve as a structural substrate that directs dendritic outgrowth toward the pial surface, whereas the successive formation of a reticular glial meshwork should lead to structural maturation of newly formed PC synapses.     

Postnatal development of glial fibrillary acidic protein immunoreactivity in the hamster arcuate nucleus

The postnatal development (from 2 days to 1 year) of glial fibrillary acidic protein (GFAP) immunoreactive cells was studied in the arcuate nucleus of male hamsters. In the first postnatal week, GFAP immunoreactivity was observed in radial glial cells whose cell bodies were located in the ependymal layer. Cell processes of GFAP immunoreactive radial glia crossed the arcuate nucleus and reached the pial surface, where they formed a thin and incomplete external limiting membrane. During the second postnatal week, some immunoreactive cell bodies were also located far from the ependymal layer. Some of these cell bodies presented processes that made contact with the ependymal layer whereas others, probably corresponding to maturing astrocytes, did not show ventricular connections. In the third week, only astrocytes showed GFAP immunoreactive perikarya and their immunoreactive processes reached either the blood vessels to form end-feet, or the basal hypothalamic zone to form the glia limitans. In successive weeks, there was an increase of the amount of GFAP-immunoreactive profiles on the glia limitans and surrounding the arcuate nucleus blood vessels. After the 6th postnatal week we observed some GFAP-immunoreactive cells close to arcuate neurons. The number of these cells increased from the 8th postnatal week. From this age on GFAP immunoreactive astrocytic processes compartimentalized the arcuate nucleus defining several rows of aligned neurons. These results indicate that the cytoarchitectonic organization of GFAP immunoreactive elements and their relationship with neurons, blood vessels and pia is not completed until the first 8 weeks of postnatal life in the arcuate nucleus of the hamster.     

Distant microglial and astroglial activation secondary to experimental spinal cord lesion

This paper analysed whether glial responses following a spinal cord lesion is restricted to a scar formation close to the wound or they might be also related to widespread paracrine trophic events in the entire cord. Spinal cord hemitransection was performed in adult rats at the thoracic level. Seven days and three months later the spinal cords were removed and submitted to immunohistochemistry of glial fibrillary acidic protein (GFAP) and OX42, markers for astrocytes and microglia, as well as of basic fibroblast growth factor (bFGF), an astroglial neurotrophic factor. Computer assisted image analysis was employed in the quantification of the immunoreactivity changes. At the lesion site an increased number of GFAP positive astrocytes and OX42 positive phagocytic cells characterized a dense scar formation by seven days, which was further augmented after three months. Morphometric analysis of the area and microdensitometric analysis of the intensity of the GFAP and OX42 immunoreactivities showed reactive astrocytes and microglia in the entire spinal cord white and gray matters 7 days and 3 months after surgery. Double immunofluorescence demonstrated increased bFGF immunostaining in reactive astrocytes. The results indicated that glial reaction close to an injury site of the spinal cord is related to wounding and repair events. Although gliosis constitutes a barrier to axonal regeneration, glial activation far from the lesion may contribute to neuronal trophism and plasticity in the lesioned spinal cord favoring neuronal maintenance and fiber outgrowth.     

Radial Glia-Like Cells in the Supraoptic Nucleus of the Adult Rat

Conventional light and confocal microscopy of thick vibratome sections of the hypothalamus of adult male and female rats immunostained for the astrocytic marker glial fibrillary acidic protein (GFAP) revealed that the supraoptic nucleus (SON) contains two morphologically distinct types of astrocytes. One has a stellate form, similar to that of most astrocytes in the adult CMS. The other has a morphology reminiscent of radial glia in the developing CNS: from their cell bodies, located along the ventral glia lamina (VGL), arise one long thick process that spans the SON in the coronal plane, several horizontally-oriented processes that form a dense network in the VGL, and a short process oriented towards the pia. The latter astrocytes are immunoreactive for vimentin, an intermediate filament protein of immature glial cells and a marker for radial glia. The stellate astrocytes showed no vimentin immunoreactivity. The functional significance of each type of supraoptic astrocyte is at present unknown but the presence of radial glia-like cells in this hypothalamic region suggests that the SON retains a certain degree of immaturity during adulthood, that may be linked to its well known capacity to undergo neuronal-glial plasticity under physiological and experimental stimulation.     

Astroglial structures in the zebrafish brain

To understand components shaping the neuronal environment we studied the astroglial cells in the zebrafish brain using immunocytochemistry for structural and junctional markers, electron microscopy including freeze fracturing, and probed for the water channel protein aquaporin‐4. Glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) showed largely overlapping immunoreactivity: GFAP in the main glial processes and GS in main processes and smaller branches. Claudin‐3 immunoreactivity was spread in astroglial cells along their major processes. The ventricular lining was immunoreactive for the tight‐junction associated protein ZO‐1, in the telencephalon located on the dorsal, lateral, and medial surface due to the everting morphogenesis. In the tectum, subpial glial endfeet were also positive for ZO‐1. Correspondingly, electron microscopy revealed junctional complexes between subpial glial endfeet. However, in freeze‐fracture analysis tight junctional strands were not found between astroglial membranes, either in the optic tectum or in the telencephalon. Occurrence of aquaporin‐4, the major astrocytic water channel in mammals, was demonstrated by polymerase chain reaction (PCR) analysis and immunocytochemistry in tectum and telencephalon. Localization of aquaporin‐4 was not polarized but distributed along the entire radial extent of the cell. Interestingly, their membranes were devoid of the orthogonal arrays of particles formed by aquaporin‐4 in mammals. Finally, we investigated astroglial cells in proliferative areas. Brain lipid basic protein, a marker of early glial differentiation but not GS, were present in some proliferation zones, whereas cells lining the ventricle were positive for both markers. Thus, astroglial cells in the zebrafish differ in many aspects from mammalian astrocytes. J. Comp. Neurol. 518:4277–4287, 2010. © 2010 Wiley‐Liss, Inc.