Astroglial Elements Research Articles

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.

Functional retinal input stimulates expression of astroglial elements in the suprachiasmatic nucleus of postnatal developing rat

Astrocytes are abundant in the hypothalamic suprachiasmatic nucleus (SCN), particularly in the retinorecipient region. Using glial fibrillary acidic protein (GFAP) immunocytochemistry, we investigated the effect of light on the development of astrocytes in the SCN housing under light–dark (LD) or constant dark (DD) conditions after birth. GFAP immunoreactivity in the DD group showed lower levels than those in the LD group at P50. However, there was no difference in density of retinohypothalamic tract (RHT) terminals in the SCN between the DD and LD groups. After the adult pattern of GFAP immunoreactivity was established at P30, transferring rats to different LD conditions produced changes in GFAP immunoreactivity evident when rats were sacrificed at P50. We next examined, using a primary culture of hypothalamic astrocytes, whether neurotransmitters of RHT such as glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) can stimulate GFAP expression directly. PACAP-38 increased the length and number of astrocytic processes but glutamate did not. These findings indicate that the functional aspects of RHT such as the light stimulated release of neurotransmitters is important for the development of astrocytes in rat SCN. Dynamic plasticity of astroglial elements in the SCN occurs even after GFAP shows an adult pattern.

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.

Lesions do not provoke GFAP-expression in the GFAP-immunonegative areas of the teleost brain

In the mammalian and avian brains the predominant astroglial elements are astrocytes, and the distribution of GFAP-immunopositivity is rather uneven, some large brain areas being almost devoid of GFAP-immunopositivity. In these areas however, an intense GFAP-immunopositivity appears following injury. In the teleost brain most of the areas are GFAP-immunopositive and ependymoglia predominates. However, a large area, the layer of the sensory neurons in the vagal lobe, is devoid of GFAP. The question arises, whether the lack of GFAP-immunopositivity in the teleost brain is also due to the repression of the GFAP-production, as in birds and mammals, or due to the lack of cells capable of expressing GFAP. To answer this question, stab wounds were made in the vagal lobe of goldfish, as well as in the tectum, in which moderately dense but highly organized GFAP-immunopositive glia has been detected in intact animals. In the layer of the sensory neurons in the vagal lobe no GFAP-immunopositivity appeared even after lesions had been introduced. In the tectum, a rather slight increase of the intensity of the immunostaining was observed in the glial fibers near the lesions but no typical reactive glia similar to that found in mammals or birds, was observed. The results suggest that a lesion does not provoke GFAP-expression in GFAP-immunonegative brain areas in teleosts, in contrast to what is observed in mammals and birds.

Astroglial Elements in the Suprachiasmatic Nucleus of the Golden Hamster and the Rat. Comparative Morphological Study Using Immunocytochemistry.

Astroglial elements in the suprachiasmatic nucleus (SCN) in golden hamsters and rats were investigated by GFAP immunocytochemistry and a computer−assisted analyzing system. Serial frontal sections, including the SCN region, were prepared and GFAP immunoreactivity in the following areas were measured as optical density (O.D.) per area (O.D./μm2); (1) the whole SCN region, (2) the ventrolateral portion (VLSCN), where vasoactive intestinal peptide (VIP) neurons were mainly distributed, (3) the dorsomedial portion, where arginine vasopressin (AVP) neurons were mainly distributed, and (4) as determined by unilateral intraocular injection of cholera toxin B subunit, the area where retinohypothalamic tract (RHT) fibers were distributed. In the rat, GFAP immunoreactivity was observed throughout the SCN, particularly in the VLSCN, while in the golden hamster, astroglial elements enclosed the whole region of the SCN. Inaddition, quantitative analysis revealed that the O. D. of the GFAP immunoreactivity in the VLSCN, where VIP neurons and the RHT fibers were distributed, were significantly higher than those in other regions of the SCN. These results strongly suggested that the astroglial elements, VIP neurons and RHT fibers have close morphological interactions, although delicate differences were demonstrated in both species.

Patterned distribution of immunoreactive astroglial processes in the striate (V1) cortex of new world monkeys

The possibility of a modular organization of non-neuronal elements was analyzed in the opercular region of the striate neocortex in adult New World monkeys. For this purpose, and in order to follow possible correlations in the general organization of neuronal and astroglial elements, immunocytochemical procedures for Glial Fibrillary Acid Protein (GFAP) and Microtubule Associated Protein 2 (MAP-2), in addition to cytochrome oxidase (COX) histochemistry, were applied to tangential and coronal sections and analyzed by using computer-assisted procedures. Astroglial interlaminar processes stemming from superficial laminae did not traverse lamina IVA, and thus did not appear in deeper layers. Clearly definable interlaminar processes were predominantly concentrated in laminae II-III. A honeycomb- like lattice was observed in tangential sections, with a "cell" size distribution similar to the MAP-2-IR lattice, suggesting an intimate association with the pyramidal columns. Additionally, analysis of similar sections disclosed the periodic appearance of large patches with high density of interlaminar processes, indicating a nonhomogeneous distribution of GFAP-IR processes in the striate cortex. COX "blobs" appeared frequently to coincide with areas expressing high density of GFAP-IR elements. These findings add a new perspective to current concepts of astroglial organization in the striate cortex of primates and reveal the existence of a non-neuronal modular organization in the primate striate cerebral cortex, and suggest that possible correlations between relative distributions of neuronal and astroglial elements should be further analyzed in cortical areas with a clear modular organization such as the striate cortex.

Neuron–Glia Interaction in the Suprachiasmatic Nucleus: A Double Labeling Light and Electron Microscopic Immunocytochemical Study in the Rat

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.

Associations between neuropeptide Y nerve terminals and intraparenchymal microvessels in rat and human cerebral cortex

Neuropeptide Y (NPY) can influence local brain perfusion, possibly via direct relationships with the microvascular bed. To evaluate this possibility, the authors quantitatively analyzed by light and electron microscopy the morphological associations between immunostained NPY neuronal elements and intraparenchymal microvessels in the rat and human cerebral cortex. At the light microscopic level in the rat frontoparietal cortex, about 16% of NPY neurons and large proximal processes as well as a subset of nerve terminals not affected by double sympathectomy were associated with penetrating arterioles and local microvessels. In human temporal cortex, a dense network of NPY nerve fibers was observed, many of which approached and/or contacted intracortical vessels. At the ultrastructural level, 14% of NPY axonal varicosities in the rat cerebral cortex were considered perivascular and associated with capillaries (approximately 70%) or microarterioles (approximately 30%). They were particularly enriched in the immediate vicinity (< 0.25 micron) of the microvessels, where the perivascular astrocytic leaflets represented a frequent target. In human cerebral cortex, NPY varicosities were observed in proximity to microvessels corresponding primarily to capillaries. Perivascular NPY varicosities never established synaptic junctions with vascular or astroglial elements. The results show that central NPY nerve terminals associate with microvessels and perivascular astroglial cells in the rat and human cerebral cortex. Thus, NPY released from these nerves could possibly influence (via a parasynaptic mode of action) vascular and/or astrocytic functions depending on the distribution of NPY receptors in these cellular compartments. These results provide morphological support for the effects of NPY on brain perfusion and homeostasis.

A rapid replacement of vimentin-containing radial glia by glial fibrillary acidic protein-containing astrocytes in transplanted telencephalon.

The present study follows the early events in the development of astroglia in rat embryonic (El5) tissue grafted into the cortex of adult rats. Astroglial elements (radial glia and astrocytes) were studied by glial fibrillary acidic protein (GFAP) and vimentin immunohistochemistry on post-transplantation (PT) days 7, 11, 14, 17, and 21. At PT7, GFAP-immunopositive elements were only scarce fibers in the transplants. At PTll, a dense network of long, GFAP-immunopositive fibers enmeshed the entire transplant, and astrocytes were already recognized. The fibers also showed vimentin immunoreactivity. By PT14, astrocytes became the predominant GFAP-labeled elements, although a few long fibers persisted. When compared with in situ development, the grafts showed earlier GFAP-immunoreactivity and earlier appearance of astrocytes, as well as a more rapid transition from the immature to the mature form of the glial system.

Differential Abundance of Glutamate Transporter Subtypes in Amyotrophic Lateral Sclerosis (ALS)-Vulnerable versus ALS-Resistant Brain Stem Motor Cell Groups

Previous studies have suggested that defective high-affinity glutamate uptake, due mainly to a major loss of the astroglial-specific GLT-1 glutamate transporter, underlies the selective motoneuron degeneration observed in sporadic ALS (24, 28). If a defect in glutamate transport underlies the pathogenesis of sporadic ALS, the glutamate transporter subtype found to be lost in sporadic ALS should be present in abundance in the affected motor nuclei under normal conditions. To investigate this, we used immunohistochemical methods to analyze the localization of two subtypes of high-affinity glutamate transporters in the cranial motor nuclei of normal monkey brain stem: GLT-1, localized to astroglia; and EAAC1, localized to neurons. Our results indicated that all motor cell groups of monkey brain stem are rich in the GLT-1 glutamate transporter, which is localized to astroglial cells and processes that surround and envelop motoneuron cell bodies and dendrites. Image analysis indicated that the abundance of GLT-1 immunoreactive astroglial elements in ALS-vulnerable motor cell groups (i.e., the trigeminal, facial, and hypoglossal motor cell groups) is higher than in ALS-resistant motor cell groups (i.e., the oculomotor, trochlear, and abducens motor cell groups), and statistical analysis showed that this difference is significant. Our results also indicated that both ALS-vulnerable and ALS-resistant motor cell groups of monkey brain stem are relatively poor in EAAC1 immunoreactivity. Therefore, in the case of a loss in the GLT-1 glutamate transporter in sporadic ALS, glutamate may increase in the vicinity of motoneurons in all brain-stem motor cell groups, but especially in the ALS-vulnerable motor cell groups, which are normally richer in GLT-1. Increased extracellular glutamate could lead to excess entry of Ca2+into motoneurons via glutamate-gated or voltage-activated Ca2+channels and produce degeneration of those motoneurons unable to resist the insult. Since motoneurons in the ALS-resistant motor cell groups of the brain stem are enriched in the Ca2+buffering protein parvalbumin, they should be better able to resist the damage than the majority of motoneurons in the ALS-vulnerable motor cell groups, which lack parvalbumin (20).

Temporal patterns of human immunodeficiency virus type 1 transcripts in human fetal astrocytes

Human immunodeficiency virus type 1 (HIV-1) infection of the developing central nervous system results in a dementing process in children, termed HIV-1-associated encephalopathy. Infection of astroglial elements of the pediatric nervous system has been demonstrated and suggests that direct infection of some astrocytes may contribute to the neurologic deficit. In this model, HIV-1 establishes a persistent state of infection in astrocytes, which can be reactivated by the cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta). To better understand the natural history of viral persistence in astroglial cells, we characterized infection at the transcriptional level. The most abundant viral transcript during the establishment of persistence was the subgenomic multiply spliced 2-kb message, similar to mononuclear cell models of HIV-1 latency. Following reactivation with TNF-alpha or IL-1 beta the multiply spliced 2-kb message remained the most abundant viral transcript, in contrast to infected mononuclear cells in which reactivation leads to the reemergence of the 9- and 4-kb transcripts. Further characterization of the persistent 2-kb transcript by PCR amplification of in vitro-synthesized viral cDNA showed that, in the absence of cytokine stimulation, the most abundant multiply spliced transcripts were the Nef- and Rev-specific messages. However, following cytokine stimulation, double- and triple-spliced Tat-, Rev-, and Nef-specific messages could be identified. Immunohistochemical staining demonstrated that, during viral persistence, astrocytes expressed Nef protein but few or no viral structural proteins. These results demonstrate that viral persistence in astrocytes at the transcriptional level is fundamentally different from that seen in mononuclear cells and could account for the virtual absence of astroglial expression of viral structural antigens in vivo.

Differential effects of platelet-derived growth factors on fetal hippocampal and cortical grafts: Evidence from intraocular transplantation in rats

Effects of platelet-derived growth factor-AA (PDGF-AA) and platelet-derived growth factor-BB (PDGF-BB) on developing parietal cortex (E16) and hippocampal (E18–E19) grafts were studied using the in vivo method of intraocular transplantation. Survival and growth of grafts in the anterior eye chamber of adult host rats under the influence of regular treatments with 0.5 ng (in a 100 ng/ml concentration) PDGF-AA or PDGF-BB was followed and compared to those receiving vehicle solution alone (0.5 mg HSA/ml Hanks). Both PDGF-AA and PDGF-BB increased the volume of transplanted cortical grafts. PDGF-BB also exerted trophic effects on grafted hippocampal tissue whereas PDGF-AA seemed to inhibit hippocampal growth. Histological and immunohistochemical studies revealed an increase in the density of astroglial elements in PDGF-AA- and PDGF-BB-treated cortical grafts whereas the PDGF-AA- and PDGF-BB-treated hippocampal grafts maintained a cytoarchitecture closely resembling that of control grafts. These findings support in vitro experiments showing that developing glial cells are stimulated by PDGFs and we further propose regional differences of action of PDGFs in the developing central nervous system.

Leptomeningeal and adventitial gliosis as a consequence of chronic inflammation.

Astroglial cell bodies and processes identified ultrastructurally and immunocytochemically are described within the subarachnoid and Virchow-Robin spaces of white matter throughout the CNS of Strain 13 guinea-pigs with chronic relapsing experimental allergic encephalomyelitis. These heterotopic astroglial elements, invariably in close proximity to subpial chronically-demyelinated lesions, were derived from subpial astrocytes and extended for great distances perpendicular and circumferential to the underlying white matter. Subpial astrocytes overlying adjacent grey matter regions appeared unaffected. This phenomenon is probably a subpial astrocyte response to inflammation and demyelination and may be related to the protection of damaged white matter against subsequent inflammatory events.

Immunohistochemical distribution of glial fibrillary acidic protein, neurofilament polypeptides and neuronal specific enolase in the human cerebellum

The human normal cerebellar cortex has been investigated using paraffin-embedded sections and a panel of antibodies against GFAP, NSE, NF 70 Kd and 200 Kd triplet proteins. The study has shown that: 1. (1) antiserum to GFAP labelled all and only astroglial elements; 2. (2) antibodies to NSE, NF 70 Kd and 200 Kd stained the perikaryon of all neurons, with two noticeable exceptions: Purkinje cells, which remained unresponsive to all 3 reagents, and basket cells which only became labelled with the NSE antiserum; 3. (3) the staining of dendrites and axons was exquisitely selective with both the NF and the NSE antibodies, each of which displayed unique binding patterns. These immunohistochemical features, first to be described in man, are compared with those so far reported in rodents.

Evidence that mouse astrocytes may be derived from the radial glia: An immunohistochemical study of the cerebellum in the normal and reeler mouse

Astrocytic cells of unusual aspect can be detected in the cerebellum of normal mice during the first 4 weeks of life. They are visualized with anti-GFAP (glial fibrillary acidic protein), anti-S100 and anti-vimentin immune sera. Their perikaryons, located in the white matter or in the granular layer, extend long processes which are inserted onto the pial surface. These cells may be transitional forms between the radial glial cells and some of the differentiated astroglial elements. These unusual astrocytes are more numerous and heavily stained in the reeler mutant than in the normal mouse and it is suggested that our observations signify some degree of glial immaturity in the cerebellum of the mutant.

Early postnatal development of entopeduncular neurons and their neostriatal connections

The early postnatal development of dendrites and axodendritic connectivity in the entopeduncular nucleus of kittens was studied by light and electron microscopy. We focused on the fine structure of neuropil and the organization of synaptic elements at several ages during the first weeks of life. We were able to define, in newborn to 1-week-old kittens, several of the distinct types of nerve endings seen previously by us in adult animals. They were found singly or in small clusters widely spaced along surfaces of dendrites and cell bodies. Most endings were small, contained 45 to 50-nm synaptic vesicles, and formed symmetrical synaptic junctions. Fewer large endings, filled with 30- to 35-nm vesicles, formed asymmetrical synapses. By the end of the second week, most dendrites and much of the cell body surfaces were ensheathed by these endings. Astroglial elements apposed nonsynaptic surfaces and enveloped arrays of axodendritic synapses. We used Fink and Heimer staining of degenerating axons to show that much of this early connectivity originated in the neostriatum. These morphologic studies support physiologic data that striopallidal pathways are functional at birth and suggest that many of the age-related changes in response parameters reflect a quantitative increase in axodendritic connectivity. To assess this notion, we initiated a computer-assisted study of Golgi-impregnated neurons and measured dendritic growth during this period. Preliminary data suggested dendritic fields are extensive in 3-day-old kittens (mean total dendritic length of 2800 μm) and individual dendrites radiate for long distances and branch sparsely (mean total branch length of 700 μm). By 18 days of age, these parameters had increased by about 40% to 4800 and 1200 μm, respectively.

CYTOLOGIC AND CYTOCHEMICAL STUDIES OF NEUROGLIA. II. THE OCCURRENCE OF TWO DNA CLASSES AMONG GLIAL NUCLEI IN THE PURKINJE CELL LAYER OF NORMAL ADULT HUMAN CEREBELLAR CORTEX.

The neuroglia located in the Purkinje cell layer of the cerebellum constitute one of the most notable concentration of these cells in the central nervous system. The astroglial elements in this region and in the remainder of the cerebellar cortex have been the subject of many writings. Following the pioneering studies of Bergmann, Golgi, Weigert and others in the last century, a number of investigations were reported in the early part of this century, based upon the use of metallic impregnation methods, in which morphologic details of the cerebellar astrocytes were described. Outstanding contributions were those of Cajal,1Fananas,2and later Schroeder.3Schroeder's study, which was carried out after Hortega had described and characterized the oligodendroglia, provides a detailed account of the features of oligodendrocytes as well as astrocytes in cerebellar cortex. Following Penfield's summation in 19324of information then available concerning the neuroglia, little attention