An electron microscopic study of macroglia and microglia in the lateral funiculus of the developing spinal cord in the fetal monkey

Summary An electron-microscopic study has been made of the glial cells in the developing lateral funiculus of the cervical spinal cord in fetal rhesus monkeys. The various macroglial cell types, their precursor cells, and microglia are discussed in detail. An astrocytic lineage is proposed in which glioblasts present in the lateral funiculus give rise to astroblasts that then develop into mature astrocytes. Oligoblasts apparently migrate into the lateral funiculus as such and develop into active oligocytes. The active oligocytes become most predominant during the initial stages of myelinogenesis and are in direct continuity with developing myelin. The active oligocytes develop into mature oligocytes after myelination is completed. Microglia cells are present throughout development as three forms; resting microglia, globose microglia, and active microglia. The globose and active microglia predominates at specific times early in development when degeneration of apparent neuronal processes is taking place. The microglia cells are characterized by dense nuclear chromatin clumps, lipid inclusion bodies, dense vesicles, and, often, intracellular debris.Supported in part by a Parson Trust Endowment Research Grant at the University of South Dakota School of Medicine. The author gratefully acknowledges the help of Dr. Ronald DiGiacomo who was responsible for the surgery involved in the fetal deliveries.     

Postnatal development of glial fibrillary acidic protein (GFAP) immunoreactivity in pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

The postnatal development (day of birth up to the end of the third month) of neurohypophyseal pituicytes and tanycytes of the median eminence (ME) and the medial basal hypothalamus (MBH) was studied immunohistochemically in the Mongolian gerbil (Meriones unguiculatus) with antibodies directed against glial fibrillary acidic protein (GFAP; the major protein subunit of glial filaments). Weak GFAP-immunoreactivity (IR) was scattered in the neural lobe (NL), the ME and the lining of the ventral 3rd ventricle at the first postnatal days. By the end of the second postnatal week, the intensity of the IR had reached a level comparable to that of adult animals. Generally, in the whole neurohypophysis a cytoarchitectonic pattern, which essentially corresponded to adult conditions, was reached around the beginning of the second month. During the first week postnatum, solely perinuclear stainings, mostly unipolar pituicytes with short processes and isolated fibers were discernible in the NL. In the course of the second and third postnatal week, a growing number of the densely arranged pituicytes appeared in form of bi- and multipolar cells. Thickness and length of pituicyte processes, as well as their degree of branching, increased progressively in the first month. The number of GFAP-positive tanycytes in the ventral 3rd ventricle and in the ME most markedly augmented in the first week postnatum. In the MBH, long tanycyte processes emerged from the ventricular lining to cross the arcuate nucleus in large bows, delimiting groups of neurons. Ependymal and subependymal tanycytes in the ME gave rise to radial processes extending to the external zone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal development of glial fibrillary acidic protein (GFAP) immunoreactivity in pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

Summary The postnatal development (day of birth up to the end of the third month) of neurohypophyseal pituicytes and tanycytes of the median eminence (ME) and the medial basal hypothalamus (MBH) was studied immunohistochemically in the Mongolian gerbil (meriones unguiculatus) with antibodies directed against glial fibrillary acidic protein (GFAP; the major protein subunit of glial filaments). Weak GFAP-immunoreactivity (IR) was scattered in the neural lobe (NL), the ME and the lining of the ventral 3rd ventricle at the first postnatal days. By the end of the second postnatal week, the intensity of the IR had reached a level comparable to that of adult animals. Generally, in the whole neurohypophysis a cytoarchitectonic pattern, which essentially corresponded to adult conditions, was reached around the beginning of the second month. During the first week postnatum, solely perinuclear stainings, mostly unipolar pituicytes with short processes and isolated fibers were discernible in the NL. In the course of the second and third postnatal week, a growing number of the densely arranged pituicytes appeared in form of bi- and multipolar cells. Thickness and length of pituicyte processes, as well as their degree of branching, increased progressively in the first month. The number of GFAP-positive tanycytes in the ventral 3rd ventricle and in the ME most markedly augmented in the first week postnatum. In the MBH, long tanycyte processes emerged from the ventricular lining to cross the arcuate nucleus in large bows, delimiting groups of neurons. Ependymal and subependymal tanycytes in the ME gave rise to radial processes extending to the external zone. Moreover, in this zone several tanycyte-like cells, whose number increased considerably in the second postnatal week, contributed to the palisadal arrangement of glial processes. In the third month, the arcuate nucleus was still penetrated by many immunopositive tanycyte processes. It was characteristic of the adult staining pattern that the highly branched processes of tanycytes and tanycyte-like cells in the ME were thicker and more variable in size than the less densely arranged tanycyte processes of the MBH. The postnatal increase of GFAP-IR intensity was paralleled by a similar development of vasopressin-IR in the ME and NL, indicating that the maturation of neurohypophyseal glia is closely linked to the functional differentiation of the neurosecretory axons.     

Golgi-like immunostaining of pituicytes and tanycytes positive for glial fibrillary acidic protein in the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus)

Summary Glial cells that contain the glial fibrillary acidic protein (GFAP; the major protein constituent of glial filaments) were stained immunohistochemically in thick frozen sections of the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus). The resulting Golgi-like images provided informations on cytological features and distributional patterns of tanycytes and pituicytes. In the proximal median eminence, numerous bundled processes of tanycytes were revealed. They emerged from the ependymal and subependymal layer and mostly reached the brain surface. Several tanycytic processes extended into the anatomical neural stalk. In the whole neural lobe, a dense network of GFAP-immunoreactive pituicyte processes was visualized. Stained pituicytes were highly asymmetric and exhibited a great morphological variability. Immunopositive fibers which were encountered in the intermediate lobe might be derived from pituicytes. Electron-microscopically further evidence was obtained that GFAP-positive pituicytes correspond to filament-rich fibrous pituicytes at the ultrastructural level.     

Golgi-like immunostaining of pituicytes and tanycytes positive for glial fibrillary acidic protein in the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus)

Glial cells that contain the glial fibrillary acidic protein (GFAP; the major protein constituent of glial filaments) were stained immunohistochemically in thick frozen sections of the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus). The resulting Golgi-like images provided informations on cytological features and distributional patterns of tanycytes and pituicytes. In the proximal median eminence, numerous bundled processes of tanycytes were revealed. They emerged from the ependymal and sub-ependymal layer and mostly reached the brain surface. Several tanycytic processes extended into the anatomical neural stalk. In the whole neural lobe, a dense network of GFAP-immunoreactive pituicyte processes was visualized. Stained pituicytes were highly asymmetric and exhibited a great morphological variability. Immunopositive fibers which were encountered in the intermediate lobe might be derived from pituicytes. Electron-microscopically further evidence was obtained that GFAP-positive pituicytes correspond to filament-rich fibrous pituicytes at the ultrastructural level.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

Summary The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Ultrastructure of the subfornical organ of the chicken (Gallus domesticus)

Summary The SFO of the chicken is divided in half by a large central blood sinus; ventrally it is covered by a thin layer of ependyma (including tanycytes, dendrites, and axons) which connects the two lateral halves and protrudes as a midsagittal crest into the lumen of the third ventricle. The ependyma consists predominantly of tanycytes with long basal processes which terminate upon perivascular spaces. These cells have an extensive Golgi apparatus and abundant lysosomes; their cellular apices containing polyribosomes and a few vesicles frequently protrude into the ventricle. In addition to astrocytes, oligodendrocytes, and microglial cells, there is another glial cell population that is distinguished by the presence of parallel stacks or spherical to ovoid conglomerates of rough ER and their unique location, i.e., limited to areas ventral and ventral-lateral to the large blood sinus. Two types of neurons are present: neurons in which there is a paucity of granulated vesicles and occasional vacuoles in both the cytoplasm and nuclei, the second type of neuron elaborates many granulated vesicles. Numerous puncta adhaerentia are observed between adjacent neuronal perikarya and between glial processes and neuronal perikarya.Diverse axon types are found within the chicken SFO. Axo-dendritic and axo-somatic axon terminals and presynaptic axon dilations contain assorted combinations of electron-lucent and granulated vesicles of different maximal diameters. Based on the morphology of these axons, cholinergic, peptidergic, and serotoninergic fibers are described. There are two additional groups of axons whose classification awaits further investigation.The chicken SFO differs from the mammalian SFO in several respects: it possesses an ependyma with secretory and/or absorptive tanycytes predominating; it is divided midsagittally by a central blood sinus; its lateral and dorsal limits are nebulous; a previously undescribed peculiar type of glial cell is found in a limited portion of the organ; supraependymal neurons are lacking.Dedicated to Prof. H. Grau at the occasion of his 80th birthdayWe gratefully acknowledge the technical help of Susan Woroch and secretarial assistance of Diana Hapes and Debbie Harrison     

Physiological roles of microglia during development

In all the species examined thus far, the behavior of microglia during development appears to be highly stereotyped. This reproducibility supports the notion that these cells have a physiological role in development. Microglia are macrophages that migrate from the yolk sac and colonize the central nervous system early during development. The first invading yolk-sac macrophages are highly proliferative and their role has not yet been addressed. At later developmental stages, microglia can be found throughout the brain and tend to preferentially reside at specific locations that are often associated with known developmental processes. Thus, it appears that microglia concentrate in areas of cell death, in proximity of developing blood vessels, in the marginal layer, which contains developing axon fascicles, and in close association with radial glial cells. This review describes the main features of brain colonization by microglia and discusses the possible physiological roles of these cells during development.     

Glial cells positive for glial fibrillary acidic protein in the neurohypophysis of the Djungarian hamster (Phodopus sungorus)

Summary The presence and distribution of the glial fibrillary acidic protein (GFAP; an astrocytic marker protein associated with glial filaments) in the neurohypophysis of the Djungarian hamster (Phodopus sungorus) were investigated immunohistochemically. Our study revealed characteristic GFAP-staining patterns within the median eminence, infundibular stem and neural lobe. In the whole neurohypophysis, few glial cells showed immunoreactivity. In the neural lobe, immunopositive pituicytes appeared preferentially in the periphery. At the ultrastructural level, we found some pituicytes containing filaments, most notably in their processes. We thus demonstrated that, in contrast to the GFAP-immunoreactivity of cultured pituicytes, pituicytic GFAP-expression in vivo coincides with the presence of electron-microscopically detectable filaments.     

Glial fibrillary acidic protein and differentiation of neonatal rat pituicytes in vitro

Summary The appearance and intracellular localisation of glial fibrillary acidic protein (GFAP) in pituicytes in neural lobe cultures of newborn rats aged 7 to 30 days were investigated by use of the indirect immunofluorescence method. GFAP-immunoreactive cells were observed mostly in the outgrowth zone. GFAP was localised in the perikaryal cytoplasm as well as in pituicyte processes. GFAP-positive pituicytes showed considerable morphological polymorphism. The presence of GFAP — astrocytic marker — in pituicytes in vitro and the evident morphological similarity to cultured astrocytes suggest the astroglial character of these cells.     

Interrelations between neural elements and tanycytes during the perinatal period of the rat

Summary The present study has utilized a correlative scanning-transmission electron microscopic technique to examine interrelations between neural elements and differentiated tanycytes and to identify supraependymal cells in the ventral region of the 3rd ventricle during the perinatal period in the rat. From the 18th day of fetal life monoaminergic and/or peptidergic axons penetrate into the ventricle between the tanycytes. After birth, they form an extensive network covering the surface of the infundibular recess. The axons possess morphological characteristics suggestive of neurohormone secretion. From the 20th day of prenatal life subependymal axons begin to innervate the tanycytes. Supraependymal cells differ in their shape and ultrastructure, but all of them bear resemblance to macrophages, as they contain numerous lysosomes and phagosome-like bodies.     

Biological effects of bovine glia maturation factor on glial cells in culture

Optimal bioassay conditions for bovine glia maturation factor (GMF) were determined among glial cells from normal glioblasts to glioma cells. Rat glioblasts 4–8 days after subculture show the highest response to GMF with regard to morphological transformation and mitogenic activity. Bovine GMF enhances DNA synthesis of rat glioblasts at 12 hr after stimulation; maximum incorporation of [methyl-³H]thymidine was detected at 18 hr. GMF increases twofold the saturation density of rat glioblasts but does not alter that of C6 astrocytoma cells. The apparent inhibition of mitogenic activity of high doses of GMF is seen in both normal and malignant glial cells.     

Development of the various glial cell types in the cerebral cortex of postnatal rats

The present quantitative study in the postnatal rats showed the rapid growth of the various glial cell types in the cerebral cortex. Among them, the increase of microglia was most dramatic. The increase was about 15 times, covering a period of 15 days extending from 5 days of age to 20 days. The majority of the microglia observed were in the outer third of the cortex. During the same period, the number of oligodendrocytes and astrocytes also showed a steady but moderate increase. The increase of oligodendrocytes was most significant between 5 and 10 days. Their density was greater in the inner third of the cortex. Astrocytes were distributed uniformly throughout. Examination of the cerebral cortex in 1- to 3-day-old rats by electron microscopy showed sporadic ameboid microglia cells and glioblasts. The possibility that they served as the precursor cells of microglia and macroglia (astrocytes and oligodendrocytes), respectively, was considered.     

Cell migration in the rostral migratory stream

Adult neurogenesis in the olfactory bulb of rodents is provided by cells which migrate tangentially from their site of genesis into the forebrain subependymal layer (SEL). This migration involves 'chains' of neuroblasts sliding into a meshwork of astrocytic cells and processes (glial tubes). The analysis of this process in postnatal rodents and in adult rabbits reveals different types of relationships occurring both among the migrating cells and between these cells and the glial structures of the SEL.     

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.     

Role of the lesion scar in the response to damage and repair of the central nervous system

Traumatic damage to the central nervous system (CNS) destroys the blood-brain barrier (BBB) and provokes the invasion of hematogenous cells into the neural tissue. Invading leukocytes, macrophages and lymphocytes secrete various cytokines that induce an inflammatory reaction in the injured CNS and result in local neural degeneration, formation of a cystic cavity and activation of glial cells around the lesion site. As a consequence of these processes, two types of scarring tissue are formed in the lesion site. One is a glial scar that consists in reactive astrocytes, reactive microglia and glial precursor cells. The other is a fibrotic scar formed by fibroblasts, which have invaded the lesion site from adjacent meningeal and perivascular cells. At the interface, the reactive astrocytes and the fibroblasts interact to form an organized tissue, the glia limitans. The astrocytic reaction has a protective role by reconstituting the BBB, preventing neuronal degeneration and limiting the spread of damage. While much attention has been paid to the inhibitory effects of the astrocytic component of the scars on axon regeneration, this review will cover a number of recent studies in which manipulations of the fibroblastic component of the scar by reagents, such as blockers of collagen synthesis have been found to be beneficial for axon regeneration. To what extent these changes in the fibroblasts act via subsequent downstream actions on the astrocytes remains for future investigation.     

The origin of the dendritic reticulum cell

Summary ¹⁴C-5,6-DHT-Melanin was injected into the left lateral ventricle of adult rats and its fate followed by light and EM autoradiography and by TEM of structures identified as labeled in preceding light micrographs. Shortly after injection, melanin particles were seen ingested by supraependymal and epiplexus cells, by cells residing in the pia-arachnoid, i.e. free subarachnoidal cells and perivascular cells, and by subependymally located microglia-like cells with intraventricular processes. Up to day four, an increase in the number of labelled phagocytes in the CSF was noted which transformed into typical reactive macrophages. After this time, many intraventricular melanin-laden phagocytes formed rounded clusters; cells of such clusters were subsequently found to invade the brain parenchyma by penetrating the ependymal lining and to accumulate in the perivascular space of brain vessels. ¹⁴C-Melanin-storing macrophages were found in the marginal sinus of the deep jugular lymph nodes suggesting emigration of CNS-derived phagocytes via lymphatics or prelymphatics that contact the subarachnoidal space compartment. This does not exclude the possibility that some of the macrophages leave the brain via the systemic circulation by penetrating the vascular endothelium; these may be disposed of in peripheral organs other than the lymph nodes.The ability of supraependymal, epiplexus, free subarachnoidal and perivascular cells in the pia and of subependymal microglia cells to accumulate synthetic melanin by phagocytosis suggests that these cells are local variants of the same type of resting potential phagocytes of the mammalian brain. The present study shows that ¹⁴C-5,6-DHT-melanin is an ideal phagocytic stimulant and marker for phagocytosis.Supported by grants from the Deutsche Forschungsgemeinschaft     

Cytochrome c can be released into extracellular space and modulate functions of human astrocytes in a toll-like receptor 4-dependent manner

BackgroundChronic activation of glial cells contributes to neurodegenerative diseases. Cytochrome c (CytC) is a soluble mitochondrial protein that can act as a damage-associated molecular pattern (DAMP) when released into the extracellular space from damaged cells. CytC causes immune activation of microglia in a toll-like receptor (TLR) 4-dependent manner. The effects of extracellular CytC on astrocytes are unknown. Astrocytes, which are the most abundant glial cell type in the brain, express TLR 4 and secrete inflammatory mediators; therefore, we hypothesized that extracellular CytC can interact with the TLR 4 of astrocytes inducing their release of inflammatory molecules and cytotoxins.MethodExperiments were conducted using primary human astrocytes, U118 MG human astrocytic cells, BV-2 murine microglia, and SH-SY5Y human neuronal cells.ResultsExtracellularly applied CytC increased the secretion of interleukin (IL)-1β, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-12 p70 by cultured primary human astrocytes. Anti-TLR 4 antibodies blocked the CytC-induced secretion of IL-1β and GM-CSF by astrocytes. Supernatants from CytC-activated astrocytes were toxic to human SH-SY5Y neuronal cells. We also demonstrated CytC release from damaged glial cells by measuring CytC in the supernatants of BV-2 microglia after their exposure to cytotoxic concentrations of staurosporine, amyloid-β peptides (Aβ42) and tumor necrosis factor-α.ConclusionCytC can be released into the extracellular space from damaged glial cells causing immune activation of astrocytes in a TLR 4-dependent manner.General significanceAstrocyte activation by CytC may contribute to neuroinflammation and neuronal death in neurodegenerative diseases. Astrocyte TLR 4 could be a potential therapeutic target in these diseases.     

A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric divisions

In the Drosophila central nervous system (CNS) glial cells are known to be generated from glioblasts, which produce exclusively glia or neuroglioblasts that bifurcate to produce both neuronal and glial sublineages. We show that the genesis of a subset of glial cells, the subperineurial glia (SPGs), involves a new mechanism and requires Notch. We demonstrate that the SPGs share direct sibling relationships with neurones and are the products of asymmetric divisions. This mechanism of specifying glial cell fates within the CNS is novel and provides further insight into regulatory interactions leading to glial cell fate determination. Furthermore, we show that Notch signalling positively regulates glial cells missing (gcm) expression in the context of SPG development.