Evolution of Bergman glia in developing human fetal cerebellum: A Golgi, electron microscopic and immunofluorescent study

Astrogliogenesis in the human fetal cerebellum was examined in 46 cerebella obtained from hysterotomy specimens ranging between 9 and 20 weeks of ovulation age. By correlating the results obtained by rapid Golgi and Golgi-Cox methods, the indirect immunofluorescence technique for glial fibrillary acidic protein, and electron microscopy, it was possible to ensure identification of cells and obtain a comprehensive view of the ontogenesis of cerebellar astroglia, in particular Bergmann fibers. Radial fibers were present at 9 weeks of ovulation age, with features of astroglial differentiation. In the cerebellar hemisphere radial fibers arising near the ventricular zone did not reach all the way to the pial surface but terminated in vascular walls of the intermediate zone. A second set of glial cells located in the intermediate zone gave rise to long, tapering processes oriented radially to the pia, some reaching to the pial surface and terminating there in conical swellings. Radial glia with these features were observed in cerebella at all fetal ages examined, indicating their availability for guidance of external granular cells as they migrate inward.

With advancing fetal age, the segment of those radial glia traversing the molecular layer demonstrated an increasing resemblance to Bergmann fibers, though the cell bodies giving rise to these processes were still located below the Purkinje cells. Transitional forms between radial glial processes and fibers beginning to resemble Bergmann fibers were observed in numerous specimens impregnated with the Golgi methods. Astrogliogenesis in human fetal cerebellum occurs earlier than formerly believed, and Bergmann fibers are a final stage in the development of a defined group of radial glia in the cerebellum.     

Glial fibrillary acidic protein in radial glia of early human fetal cerebrum: a light and electron microscopic immunoperoxidase study

In order to assess the nature of glial fibrillary acidic protein (GFAP) immunoreactivity in the radial glia of early human fetal cerebrum, full thickness blocks from the midconvexity of the frontoparietal region of the cerebrum of 25 human fetuses ranging from ten to 20 weeks of ovulation age were studied by light and electron microscopic (EM) immunoperoxidase methods. The presence of GFAP within radial glia was demonstrated in vibratome sections, in de-eponized 1 micron sections and in paraffin-embedded sections both at light and EM levels in suitably fixed human fetal cerebral tissue. The results indicate that the pattern of GFAP immunoreactivity observed in "routinely" processed autopsy brains, in which fixation is suboptimal, must be interpreted with care.     

Palisading pattern of subpial astroglial processes in the adult rodent brain: relationship between the glial palisading pattern and the axonal and astroglial organization

The architectural organization of the subpial astrocyte processes was examined near the brain surface by single immunostaining methods. The astroglial processes were stained on brain sections made parallel to the pial surface. The astroglial glial fibrillary acid protein (GFAP) antigen was used as a specific marker. We show that these subpial astrocyte processes present a well organized palisading pattern in the adult mouse and rat spinal cord, medulla and pons. This adult astrocyte palisading pattern is compared to the palisading radial glia organization we previously demonstrated in the fetal mouse brain. The observed analogies afford a new and strong argument in favor of a derivation of the subpial astrocytes from radial glia. Double immunostaining methods, using GFAP and neurofilament antigens as glial and neuronal markers respectively, show the close relationship existing between the trajectories of axonal and glial processes. Beside the colinearity already observed between the axon trajectories and the glial palisades we demonstrate a new kind of axon/glia relationship. Axons are closely intermingled, within the palisading glial tufts, with the peripheral processes of the subpial astrocytes progressing to the pial surface. The findings suggest that fetal radial glia organization has a direct and indirect influence on the adult astroglial and perhaps the axonal pattern.     

The history of radial glia.

Radial glial cells are now recognized as a transient population that serves as scaffolding for neuronal migration. The recognition of the existence and role of radial glia has not been smooth, and here we provide a brief historical overview on the pioneering studies on this subject. The histologists and embryologists Albert K?lliker and Wilhelm His performed seminal investigations on cortical morphogenesis in the last decades of the 19th century. However, the introduction of the silver impregnation Golgi technique, and its diffusion in the late 1880s, played a crucial role in the detection of radial glial processes. The radial arrangement of fibers emerging from the neuroepithelium lining the central canal was initially detected in the embryonic spinal cord by Camillo Golgi himself. The first Golgi impregnation of the cerebral cortex of mammalian fetuses was performed by Giuseppe Magini, who detected radial fibers extending from the ventricular neuroepithelium, and observed cells intercalated along these processes. Radial fibers, regarded as epithelial or ependymal processes, were then observed in the developing spinal cord and cerebral cortex by several investigators. Santiago Ramón y Cajal was the first to suggest that radial fibers were modified astrocytic processes functioning as a support during cortical histogenesis. Cajal acknowledged Magini's findings, but he criticized Magini's observations on the existence of neurons along radial fibers. With the advent of electron microscopy, the existence of radially arranged glial processes along which young neurons migrate was finally ascertained in the early 1970s by Pasko Rakic, thus opening a new era in the cellular and molecular biology of radial glia.     

Expression of glial fibrillary acidic protein by immature oligodendroglia and its implications

Correlative immunocytochemical and electron-microscopic studies of the subpial region of the human fetal spinal cord between 12-18 weeks of ovulation age revealed immature oligodendroglial cells showing immunoreactive GFAP within both the cytoplasm and its processes. By 17-18 weeks, however, GFAP immunoreactivity is no longer evident within such cells. The expression of GFAP by immature oligodendroglial cells in the developing human fetal spinal cord prior to the formation of compact myelin sheaths supports the hypothesis that oligodendrocytes, along with astrocytes, are ultimately derived from radial glial cells.     

The distribution of glial fibrillary acidic protein and vimentin in postnatal marmoset (Callithrix jacchus) brain

Antisera to glial fibrillary acidic protein (GFAP) and vimentin were used to elucidate the distribution of these intermediate filament proteins in postnatal marmoset brains of various ages. The ependyma of the lateral ventricles was unique in being equally immunoreactive for both GFAP and vimentin at all ages. Vimentin alone was consistently demonstrated in endothelial and leptomeningeal cells at all ages. In neonates, vimentin immunoreactivity greatly exceeded that of GFAP and was located primarily in radial glia in the subependymal plate of the anterior cerebrum. Their vimentin-positive processes formed thick fascicles in the corpus callosum but separated into fine fibres on entering the cortex. GFAP immunoreactivity in these cells and processes was very limited. With age, GFAP-positive cells increased in number and displayed the typical stellate appearance of astroglia. The vimentin-positive radial glial population decreased considerably during this period and by 6 months had virtually disappeared. The GFAP reaction in adult brain was even more widespread, largely due to the increased number of positive astrocytes in the white matter. Vimentin immunoreactivity in the adult was greatly diminished and positive radial glia were not detectable. A major change in intermediate filament protein expression, therefore, occurs in the early postnatal period and probably reflects phases in the differentiation of radial glial precursors into astrocytes.     

The topographical distribution of S-100 and GFA proteins in the adult rat brain: an immunohistochemical study using horseradish peroxidase-labelled antibodies.

The cytological and topographical distribution of S-100 and glial fibrillary acidic (GFA) proteins in the adult rat brain has been compared using the horseradish peroxidase-labelled antibody technique. Both proteins are present in astrocytes and structures composed of astrocytic processes, namely the glial limitans and the perivascular membranes, but the cytological localization varies between the two proteins. S-100 is found in the nucleus and cytoplasm whereas GFA protein is confined to the cytoplasm. Neither is found in neurons, but S-100 is present in some oligodendroglia, suggesting a general regulatory role in glia. Although GFA protein in present in both protoplasmic and fibrous astrocytes, it is more prominent in the latter, confirming its association with astrocytic filaments.     

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.     

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.     

Expression of glial cell line-derived neurotrophic factor (GDNF) in the developing human fetal brain

GDNF expression was examined immunocytochemically in developing human fetal brains obtained from aborted fetuses ranging from 7 to 39 weeks in gestational age. At 7-8 weeks, strong immunoreactivity was noted within radial glial processes, glia limitans and choroid plexus of the telencephalic vesicle. By 10 weeks, ependymal cells, primitive matrix cells and early developing cortical plate neurons showed positive staining. By 15-16 weeks, migrating neurons in the subventricular and intermediate zones and in the cortical plate were strongly positive for GDNF. The glia limitans of the cerebral cortex and subependymal astrocytes remained positive at this time. As fetal age increased, GDNF expression shifted to neurons and glial cells in the deeper structures of the brain. The most prominent GDNF staining was observed in the cytoplasm and dendrites of Purkinje cells of the cerebellum by 25 weeks and thereafter. Pyramidal neurons of the CA1 region and granule cells of the dentate fascia of the hippocampus, neurons of the entorhinal cortex, and scattered neurons within the brain stem, medulla and spinal cord all showed strong GDNF staining by 25-35 weeks. Widespread GDNF expression in neuronal and non-neuronal cells with distinct developmental shifts suggests that GDNF may play a critical role in the survival, differentiation and maintenance of neurons at different stages of development in the developing human fetal brain.     

Cortical dysplasia in a 23-week fetus with Fukuyama congenital muscular dystrophy (FCMD)

Summary A 23-week fetus who is thought to be affected with Fukuyama congenital muscular dystrophy (FCMD) is reported. Cortical dysplasia of the cerebrum was extensive and could be categorized into three major types. The cerebral cortex was thoroughly covered by glio-mesenchymal tissue (extracortical glial layer), in which neuronal clusters were irregularly scattered. Radial bundles of neuroglial tissue frequently extended from the cortex into the extra-cortical glial layer through the focally defective molecular layer and pia mater. The deep cerebral structures, such as basal ganglia, thalamus and white matter, appeared normal in contrast with extensive malformation in the cortex. Glial fibrillary acidic protein-immunoperoxidase stain revealed: (1) presence of abundant radial glial fibers in the ventricular, subventricular and intermediate zones; (2) focal or diffuse lack of glia limitans; (3) focal derangement of radial glial fibers; and (4) proliferation of stellate glial cells in the extra-cortical layer. It is suggested that ectopic accumulation of neurons into the extra-cortical glial layer seems a cardinal pathogenetic process to generate cortical dysplasia in FCMD. Early development of superficial glio-mesenchymal tissue seems essential for upward displacement of migrating neurons.Supported by Grant Nos. 85-04 and 86-02 from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare of Japan     

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.     

THE ASTROGLIAL RESPONSE TO STABBING. IMMUNOFLUORESCENCE STUDIES WITH ANTIBODIES TO ASTROCYTE-SPECIFIC PROTEIN (GFA) IN MAMMALIAN AND SUBMAMMALIAN VERTEBRATES

The astroglial response t o stabbing. Immunofluorescence studies with antibodies to astrocyte-specific protein (GFA) in mammalian and sub-mammalian vertebrates
The astroglial response to stabbing was studied by immunofluorescence with GFA protein antisera in adult and newborn rats, chickens and goldfish. In the adult normal rat most astrocytes of the isocortex and corpus striatum are protoplasmic and do not stain by immunofluorescence. Two days after injury many astrocytes became brightly fluorescent in the stabbed hemisphere and were still fluorescent 2 months later. In the newborn rat the astroglial response was more limited. Reactive glial cells in the medial frontal cortex and pyramidal layer of the hippocampus had a radial appearance with thin immunofluorescent processes crossing at right angles to the surface of the cortex. In rats stabbed at birth and killed 1 month later many immunofluorescent astrocytes were present in the frontal cortex of both cerebral hemispheres. Radial glia were no longer observed. In the normal adult rat radial glial processes were seen by immunofluorescence extending at right angles from the lateral wall of the third ventricle into the hypothalamus. In the chicken cerebellum the astroglial response to stabbing was limited, with few immunofluorescent fibers in the vicinity of the wound. No changes were observed in the goldfish optic tectum by immunofluorescence.     

Role of radial fibers in controlling the onset of myelination

Recent in vitro study showed that astrocytes induce oligodendrocyte processes to adhere to axons. However, the role of astrocytes in myelination in vivo remains unknown. We have, therefore, conducted a study to clarify the possible involvement of astrocytes during the initial myelination process. In newborn mice, the expression of glial fibrillary acidic protein (GFAP), a marker for astrocytes, was restricted to a few fibrous architectures in the subventricular zone (SVZ), but we did not observe any GFAP-positive astrocytes. Prior to the onset of myelination, GFAP became transiently expressed in the cells with radial fibers elongating from the SVZ to the pia of cerebral cortex, and myelin-associated glycoprotein (MAG)-positive premyelinating oligodendrocytes appeared as neighbors to them, with the processes attaching to radial fibers, but not to axons. These GFAP-positive "radial" cells lost their fibrous architecture and became typical GFAP-positive astrocytes at about 10 days postnatally, when myelination set in, indicating that the disappearance of radial fibers coordinates with the initiation of myelination. From these results, we propose that premyelinating oligodendrocytes are in contact with radial fibers rather than axons and that the cytoarchitectural transformation of radial fibers into astrocytes is involved substantially in controlling the onset of initial myelination. Our proposal was further confirmed by GFAP-deficient mice, in which the disappearance of these radial fibers and the initiation of myelination were delayed in parallel. Our findings together suggest that myelination in vivo is in concert with astrocytic differentiation, involving radial fibers therein, rather than being a mere axon-oligodendrocyte interaction.     

Astrocytic tau pathologies in aged human brain

Argyrophilic and tau‐positive abnormal structures in astrocytes are frequent in aged brains, with a new nomenclature of aging‐related tau astrogliopathy (ARTAG) proposed. The two major cytomorphologies of ARTAG are thorn‐shaped astrocytes (TSA) and granular or fuzzy tau immunoreactivity in processes of astrocytes (GFA). We selected 28 cases in which many AT8‐identified astrocytic tauopathies were observed in the central nervous system from 330 routine aged autopsied cases, including Alzheimer's disease. AT8‐identified and Gallyas silver staining‐positive TSA were observed in subpial, subependymal, perivascular areas as well as white matter. These TSA were 4‐repeat (4R) tau‐positive. In contrast, 3‐repeat (3R)‐tau was negative in TSA, but positive in short thick cell processes, likely neuropil threads, in subpial and subependymal areas. The frequency of 3R‐tau‐positive processes was variable. Small dot‐like AT8‐identified astrocytic processes surrounding vessels in the neuropil were also positive for 4R‐tau, but negative for 3R‐tau. GFA in cerebral gray matter were AT8‐identified and Gallyas‐positive, and positive for 4R‐tau but negative for 3R‐tau. In this study, we did not identify 3R‐tau+/4R‐tau+ or 3R‐tau+/4R‐tau– astrocytes. Further studies are needed to clarify the nature and progression of glial tau‐positive structures in ARTAG.     

Prenatal development of fibrous (white matter), protoplasmic (gray matter), and layer I astrocytes in the human cerebral cortex: A Golgi study

The prenatal developmental histories of layer I, fibrous (white matter), and protoplasmic (gray matter) astrocytes have been studied in the human neocortex by the rapid Golgi method. The developmental route followed by each of these astrocytes is a distinct process which evolves from a specific precursor, occurs at a different time, and is linked to a specific event. The differentiation of layer I astrocytes is linked to the neocortex external glial limiting membrane (EGLM), that of fibrous astrocytes to the early white matter vascularization and maturation, and that of protoplasmic astrocytes to the late gray matter ascending vascularization and maturation. At the start of development, three glial precursors are established in the neocortex: 1) original radial neuroectodermal cells with nuclei above the primordial plexiform layer (PPL) by losing their ependymal and retaining their pial attachments become early astrocytes of layer I and EGLM components; 2) neuroectodermal cells with nuclei below the PPL that retain their pial and ependymal attachments become type I radial glial cells which are committed to the guidance of neurons and the early EGLM maintenance; and, 3) neuroectodermal cells that lose their pial but retain their ependymal attachment are transformed into type II radial glial precursors. By progressively losing their ependymal attachment, type II radial glia precursors become freely migrating cells, establish vascular contacts, and differentiate into fibrous astrocytes (and into oligodendrocytes?) throughout the subplate, developing white matter, and paraventricular regions. After the formation of the gray matter, additional layer I astrocytes are needed for the EGLM late prenatal and postnatal maintenance because type I radial glia cells start to regress and to reabsorb their EGLM endfeet. A late ependyma-to-pia migration of glial precursors progressively repopulates layer I with additional astrocytes and establishes the ephemeral subpial granular layer (SGL) of Ranke. From the 15th week of gestation to the time of birth, late astrocytes of layer I lose their EGLM attachments, migrate freely into the maturing gray matter, establish vascular contacts, and differentiate into protoplasmic astrocytes. The protoplasmic astrocytes of the gray matter evolve from transformation of layer I astrocytes rather than from radial glia cells as is generally believed. © 1995 Wiley-Liss, Inc.     

The development of induced cerebrocortical microgyria in the rat.

Placement of a freezing probe on the skull of neonatal rats produces four-layered microgyria, complete with a lamina dissecans and microsulcus. We studied the developmental course of this induced microgyria under light microscopy by examining changes in neurons, glia, and macrophages following a focal freezing insult on the day of birth (postnatal day [P]0). The destruction of neurons and glia induced by the freezing probe extends through the cortical plate and occasionally through the subplate, but the pial membrane appears undamaged and radial glial cells, while damaged, are not eliminated. Reactive astrocytes and macrophages arrive in the damaged area within 24 hours of the injury, and repair of the damaged tissue peaks within the first week. Damaged radial glial fibers regrow, and supragranular neurons migrate through this damaged area, also within the first week. The newly formed supragranular layer overlies the cell-free area. The damaged cortex begins to assume its adult-like microgyric appearance from P5 to P10. On P15 and P32, long glial fibers, resembling radial glia, are present and are immunoreactive for glial fibrillary acidic protein and radial glial fiber antibodies (vimentin and Rat-401). No such fibers appear at this age in the non-microgyric areas or in normal brains. We conclude that microgyria formation may be the consequence of brain repair mechanisms occurring during neuronal migration to the neocortex, and that it appears to preserve primitive features characteristic of the developing cortex.     

Filament proteins in rat optic nerves undergoing Wallerian degeneration: Localization of vimentin,the fibroblastic 100-Å filament protein,in normal and reactive astrocytes

A major polypeptide in the gliosed optic nerves of blinded rats was identified as vimentin, the fibroblastic, 100-Å filament protein typical of mesenchymal cells, by comigration experiments with purified vimentin on SDS-polyacrylamide gel electrophoresis. The localization of vimentin to astrocytes was confirmed by immunofluorescence microscopy with vimentin antisera. The distribution of vimentin and of astrocyte-specific glial fibrillary acidic (GFA) protein in cryostat sections of rat brain and spinal cord was different. Vimentin was confined to the main processes of fibrous astrocytes and could not be identified in the delicate framework of glial fibrils demonstrated with GFA antisera. Bergmann radial glia in the molecular layer of the cerebellum were exceptional in this respect, because they were equally well stained with vimentin and GFA antisera.     

Dorsal-ventral differences in the glia limitans of the spinal cord: an ultrastructural study in developing normal and irradiated rats

The dorsal and ventral surfaces of the lumbosacral spinal cord were examined in normal and irradiated postnatal rats. In normal rats between three and 13 days postnatal (DP), the glia limitans (GL) of the ventral surface was a more complex structure than the dorsal GL. This greater degree of complexity was manifested in a greater number of subpial astrocytes, a greater number of radial glial processes and a more advanced state in differentiation of its constituents. In rats irradiated at three DP and examined at 13 DP, the ventral GL remained intact and relatively unaffected by the radiation. In contrast, the dorsal GL was disrupted, and Schwann cells were seen within the dorsal funiculus. The ventral GL of the rat lumbosacral spinal cord is a more substantial structure than the dorsal GL during normal development. This factor alone may account for the integrity of the barrier properties of the ventral GL following radiation. However, our observations suggest that subpial astrocytes of the dorsal GL are more susceptible to radiation damage at three DP than the subpial astrocytes and radial glia of the ventral GL.     

Developmental and aging changes in the expression patterns of beta-amyloid in the brains of normal and Down syndrome cases

Immunohistochemical staining with polyclonal antibodies to synthetic amyloid (residues 1-28 of A4) was performed on normal and Down syndrome brains from fetuses to adults. Positive staining appeared in the cytoplasmic processes of astrocytes in the subpial layer and white matter of developing brains, and reappeared in astrocytic fibers of the subpial layer as well as in cerebrovascular and plaque core amyloid in elderly brains. The reappearance of positively stained astrocytes and amyloid occurred earlier in adult Down syndrome patients. The results indicate that the A4 protein is a developmental protein, and its reappearance in Alzheimer and adult Down syndrome brains may be related to the regeneration process.