Detection of glial fibrillary acidic protein (GFAP) and vimentin (Vim) by immunoelectron microscopy of the glial cells in the central nervous system of the snail Megalobulimus abbreviatus

When examined under an electron microscope, the central nervous system of Megalobulimus abbreviatus showed two types of glial cells: firstly, protoplasmic glial cells which displayed a nucleus with peripheral heterochromatin, scanty or no intermediate filaments, a developed Golgi complex, rough and smooth endoplasmic reticula, mitochondria and polymorphic lysosomes that indicate phagocytic activity of debris from the extracellular space; and, secondly, fibrous glial cells which showed numerous glial fibrillary acidic protein (GFAP) and vimentin immunoreactive intermediate filament bundles, a discrete Golgi complex, mitochondria, endoplasmic reticulum, lipid droplets and lysosomes. The contacts between the glial cells consisted of desmosomes and puncta adherentia, while those between the glial cells and the basal lamina consisted of hemidesmosomes. Both glial cell types were located in the cortex and medullary regions, however, the protoplasmic glial cells prevailed in the cortical region, while the fibrous glial cells prevailed in the medullar region. As the nervous tissue is avascular, the passage of nutrients and waste products may be facilitated by the glial labyrinthic system which is located in the cortical region. Glial processes adjacent to large and giant neurones formed a trophospongium, which seemed to be involved in a metabolic exchange between these cells. Thus, this evidence suggests that glial cells of M. abbreviatus are involved in structural support, isolation of different ganglionic areas, the formation of a microcirculatory system and an intimate metabolic relationship with neurones.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage

The developing central nervous system of vertebrates contains an abundant cell type designated radial glial cells. These cells are known as guiding cables for migrating neurons, while their role as precursor cells is less clear. Since radial glial cells express a variety of astroglial characteristics and differentiate as astrocytes after completing their guidance function, they have been considered as part of the glial lineage. Using fluorescence-activated cell sorting, we show here that radial glial cells also are neuronal precursors and only later, after neurogenesis, do they shift towards an exclusive generation of astrocytes. These results thus demonstrate a novel function for radial glial cells, namely their ability to generate two major cell types found in the nervous system, neurons and astrocytes.     

Glial cells

The nervous system is built from two broad categories of cells, neurones and glial cells. The glial cells outnumber the neurones and the two cell types occupy a comparable amount of space in nervous tissue. The main glial cell types are, in the central nervous system, astrocytes and oligodendrocytes and, in the peripheral nervous system, Schwann cells, enteric glial cells and satellite cells. In the embryo, glial cells form a cellular framework that permits the development of the rest of the nervous system, and regulate neuronal survival and differentiation. The best known function of glia in the adult is the formation of myelin sheaths around axons thus allowing the fast conduction of signalling essential for nervous system function. Glia also maintain appropriate concentrations of ions and neurotransmitters in the neuronal environment. Increasing body of evidence indicates that glial cells are essential regulators of the formation, maintenance and function of synapses, the key functional unit of the nervous system.     

The appearance of neural and glial cell markers during early development of the nervous system in the amphibian embryo

Cell-type-specific antibodies have been used to follow the appearance of neurones and glia in the developing nervous system of the amphibian embryo. Differentiated neurones were recognized with antibodies against neurofilament protein while glial cells were identified with antibodies against glial fibrillary acidic protein (GFAP). The appearance of neurones containing the neurotransmitters 5-hydroxytryptamine and dopamine has been charted also. In Xenopus, neurofilament protein in developing neurones was observed occasionally at NF stage 21 and was present reliably in the neural tube and in caudal regions of the brain at stage 23. Antibodies to the low molecular weight fragment of the neurofilament triplet recognized early neurones most reliably. Radial glial cells, identified with GFAP antibody, were identified from stage 23 onwards in the neural tube and caudal regions of the brain. In the developing spinal cord, GFAP staining was apparent throughout the cytoplasm of each radial glial cell. In the brain, the peripheral region only of each glial cell contained GFAP. By stage 36, immunohistochemically recognizable neurones and glia were present throughout the nervous system. In the axolotl, by stage 36 the pattern of neural and glial staining was identical to that observed in Xenopus. GFAP staining of glial cells was obvious at stage 23, although neuronal staining was clearly absent. This implies that glial cells differentiate before neurones. 5-HT-containing cell bodies were first observed in caudal regions of the developing brain on either side of the midline at stage 26. An extensive network of 5-HT neurones appeared gradually, with a substantial subset crossing to the opposite side of the brain through the developing optic chiasma. 5,7-dihydroxytryptamine prevented the appearance of 5-HT. Depletion of 5-HT had little effect on development or swimming behaviour. Dopamine-containing neurones in the brain first differentiated at stage 35-36 and gradually increased in number up to stage 45-47, the latest stage examined. The functional role of 5-HT- or dopamine-containing neurones remains to be elucidated. We conclude that cell-type-specific antibodies can be used to identify neurones and glial cells at early times during neural development and may be useful tools in circumstances where functional identification is difficult.     

Neural repair in an insect central nervous system: cell kinetics and proliferation after selective glial disruption

Summary Autoradiographs of tritiated thymidine uptake and subsequent light- and electron-microscopical examination revealed an onset of perineurial glial cell proliferation 3 days after injury to the CNS. The number of cells labelled increased rapidly until 7 days post-lesioning. At 2 weeks, the labelled cells equalled the number of nuclei present in the perineurium. No label was seen in the subperineurial cells, possibly because of the inability of the label to penetrate into a region where localised division is taking place.Prior to the onset of thymidine uptake, the damaged nerve cord was invaded by an exogenous reactive cell. The number of these cells increased rapidly in the first 48 h, then decreased as a negative exponential, very few remaining after 7 days. We suggest that this cell type must either return to the haemocoel or transform into a functional glial cell class.The repair of the insect central nervous system can be divided into three phases which show striking similarities to vertebrate repair sequences. These include: initial invasion of the lesion by exogenous cells, subsequent proliferation of glial cells, the longer term flux of cell numbers, their distribution and the time scale of events. This suggests that the insect CNS might provide a system for examining common cellular mechanisms and events.     

Neural repair and glial proliferation: parallels with gliogenesis in insects

There is a growing recognition, stemming from work with both vertebrates and invertebrates, that the capacity for neuronal regeneration is critically dependent on the local microenvironment. That environment is largely created by the non-neuronal elements of the nervous system, the neuroglia. Therefore an understanding of how glial cells respond to injury is crucial to understanding neuronal regeneration. Here we examine the process of repair in a relatively simple nervous system, that of the insect, in which it is possible to define precisely the cellular events of the repair process. This repair is rapid and well organised; it involves the recruitment of blood cells, the division of endogenous glial elements and, possibly, migration from pre-existing glial pools in adjacent ganglia. There are clear parallels between the events of repair and those of normal glial development. It seems likely that the ability of the insect central nervous system to repair resides in the retention of developmental capacities throughout its life and that damage results in the activation of this potential.     

On the structure of the pulmonate osphradium

Summary The osphradium of Planorbarius consists of a blindly-ending ciliated canal, formed by an infolding of the mantle epithelium, and a basal ganglion of nerve cells which is comparable in complexity with ganglia of the central nervous system. The distribution of cell types in the osphradial epithelium is specialised so that three regions can be recognised; the ciliated, the secretory and the sensory regions. The basal sensory region of the canal epithelium consists of ciliated cells and is innervated by sensory neurones of the osphradial ganglion. The middle secretory region contains mainly of mucus-secreting cells and the epithelium adjacent to the osphradial aperture of ciliated cells and secretory cells of a second type. The sensory neurones of the osphradial ganglion are bipolar or of a modified monopolar type. Other monopolar neurones, similar to those common in the central nervous system are of non-sensory function. The osphradium of Paludina, although of typical prosobranch form, possesses ciliated pits similar to the single canal of Planorbarius, which may indicate a shared modality of receptor function. A definite function cannot be ascribed to the pulmonate osphradium based on morphological evidence alone.     

Phenotypical peculiarities and species-specific differences of canine and murine satellite glial cells of spinal ganglia

Satellite glial cells (SGCs) are located in the spinal ganglia (SG) of the peripheral nervous system and tightly envelop each neuron. They preserve tissue homeostasis, protect neurons and react in response to injury. This study comparatively characterizes the phenotype of murine (mSGCs) and canine SGCs (cSGCs). Immunohistochemistry and immunofluorescence as well as 2D and 3D imaging techniques were performed to describe a SGC-specific marker panel, identify potential functional subsets and other phenotypical, species-specific peculiarities. Glutamine synthetase (GS) and the potassium channel Kir 4.1 are SGC-specific markers in murine and canine SG. Furthermore, a subset of mSGCs showed CD45 immunoreactivity and the majority of mSGCs were immunopositive for neural/glial antigen 2 (NG2), indicating an immune and a progenitor cell character. The majority of cSGCs were immunopositive for glial fibrillary acidic protein (GFAP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and Sox2. Therefore, cSGCs resemble central nervous system glial cells and progenitor cells. SGCs lacked expression of macrophage markers CD107b, Iba1 and CD204. Double labelling with GS/Kir 4.1 highlights the unique anatomy of SGC-neuron units and emphasizes the indispensability of further staining and imaging techniques for closer insights into the specific distribution of markers and potential colocalizations.     

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.     

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na(+) and Ca(2+) currents as well as delayed-rectifier K(+) currents. Immunohistochemical staining confirmed that most neurons have Na(+) channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K(+) inward currents and depolarization-induced K(+) outward currents. Glia showed large "passive" currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.     

Shiga toxin-2 enhances heat-shock-induced apoptotic cell death in cultured and primary glial cells

The blood–brain barrier (BBB) selectively controls the homeostasis of the central nervous system (CNS) environment using specific structural and biochemical features of the endothelial cells, pericytes, and glial limitans. Glial cells, which represent the cellular components of the mature BBB, are the most numerous cells in the brain and are indispensable for neuronal functioning. We investigated the effects of Shiga toxin on glial cells in vitro. Shiga toxin failed to inhibit cell proliferation but attenuated expression of heat shock protein 70, which is one of the chaperone proteins, in cultured and primary glial cells. Furthermore, the combination of Shiga toxin and a heat shock procedure induced cell apoptosis and decreased cell proliferation in both cells. Thus, we speculate that glial cell death in response to the combination of Shiga toxin and heat shock might weaken the BBB and induce central nervous system complications.     

Fine structure of glial cells in the central nervous system of the tapeworm Grillotia erinaceus (Cestoda: Trypanorhyncha)

The problem of glial cells existing in parasitic and free living flatworms is correlated with organization of parenchyma in platyhelmintes. In the contrary to the widespread opinion that myelin-like envelopes and glial cells do not exist in the nervous system of parasitic flatworms, it has been shown by ultrastructural researches that Amphilina foliacea (Cestoda, Amphilinidea) has well developed glial cells and myelin-like envelopes in the ganglia and main cords, which include both glial cells and intercellular components. The aim of our research was to reveal and investigate in details structural components corresponding to the concept of the glial cell in the CNS of Grillotia erinaceus (Cestoda: Trypanorhyncha). Three types of glial cells have been found. The first type is the fibroblast-like glial cells; cells locate in the cerebral ganglion, contain in cytoplasm and extract out fibrillar matrix, form desmosomes and have supporting function. The glial cells of the second type form myeline-like envelope of the giant axons and bulbar nerves in scolex and have laminar cytoplasm. These cells are numerous and exceed in number the neurons bodies into the nerve. The glial cells of the third type form multilayer envelopes in the main nerve cords; extra cellular fibers and gap-junctions take place between the layers. There are contacts between the glial cells of the third type and excretory epithelium but specialized contacts with neurons have been not found. The existing of glial cells in free living and parasitic flatworms is discussed.     

Calcium signalling in sensory neurones and peripheral glia in the context of diabetic neuropathies

Peripheral sensory nervous system is comprised of neurones with their axons and neuroglia that includes satellite glial cells in sensory ganglia, myelinating, non-myelinating and perisynaptic Schwann cells. Pathogenesis of peripheral diabetic polyneuropathies is associated with aberrant function of both neurones and glia. Deregulated Ca²⁺ homoeostasis and aberrant Ca²⁺ signalling in neuronal and glial elements contributes to many forms of neuropathology and is fundamental to neurodegenerative diseases. In diabetes both neurones and glia experience metabolic stress and mitochondrial dysfunction which lead to deregulation of Ca²⁺ homeostasis and Ca²⁺ signalling, which in their turn lead to pathological cellular reactions contributing to development of diabetic neuropathies. Molecular cascades responsible for Ca²⁺ homeostasis and signalling, therefore, can be regarded as potential therapeutic targets.     

Sculpting the nervous system: glial control of neuronal development

Glial cells are not passive spectators during nervous system assembly, rather they are active participants that exert significant control over neuronal development. Well-established roles for glia in shaping the developing nervous system include providing trophic support to neurons, modulating axon pathfinding, and driving nerve fasciculation. Exciting recent studies have revealed additional ways in which glial cells also modulate neurodevelopment. Glial cells regulate the number of neurons at early developmental stages by dynamically influencing neural precursor divisions, and at later stages by promoting neuronal cell death through engulfment. Glia also participate in the fine sculpting of neuronal connections by pruning excess axonal projections, shaping dendritic spines, and secreting multiple factors that promote synapse formation and functional maturation. These recent insights provide further compelling evidence that glial cells, through their diverse cellular actions, are essential contributors to the construction of a functionally mature nervous system.     

Histochemical survey of transmitters in the central ganglia of the gastropod mollusc Phestilla sibogae

The aeolid nudibranch Phestilla sibogae is well studied in terms of its larval nervous system and neuronal involvement in metamorphosis. Central neurones in the adult have also been identified anatomically and electrophysiologically. We describe the neurotransmitter contents of these neurones and provide details of neuritic projections and developmental changes during growth (3 to 18 mm body length). Central ganglia from specimens of all sizes contained 100-115 serotonin-immunoreactive neurones, some of which appeared to be homologues of cells identified in other gastropods. Tyrosine hydroxylase immunoreactivity and aldehyde-induced fluorescence marked a common set of 28-30 catecholaminergic neurones located anteriorly in the cerebropleural ganglia and laterally in the pedal ganglia. Ganglionic neuropile and nerve trunks also contained many catecholaminergic fibres. About 65-100 intensely labelled FMRFamide-immunoreactive neurones were located symmetrically throughout the central ganglia, although one population was located only in the right pedal ganglion. Another 40-45 FMRFamide-immunoreactive neurones were weakly or variably stained. Central ganglia also contained 27-29 intensely labelled pedalpeptide-immunoreactive neurones, including those that were apparently homologues of cells previously described in Tritonia diomedea, and 16-19 weakly labelled pedal-peptide-immunoreactive neurones, including giant cerebropleural neurones coexhibiting FMRFamide immunoreactivity. Little cell addition involving any transmitter phenotype occurred as animals grew in body length, body growth being accommodated by growth in the size of individual cells, consistent with an approximate doubling in the size of the ganglia themselves.     

Distribution and ontogeny of glial fibrillary acidic protein in the snail Megalobulimus abbreviatus

Glial fibrillary acidic protein (GFAP) is the major component of intermediate glial filaments in the central nervous system of many vertebrates and invertebrates. In vertebrates, this protein is mainly expressed in mature astrocytes and provides structural cell stability. The highly conserved structure and glial specificity of this protein have allowed studies of ontogeny and phylogeny using antibodies. The present study investigated the ontogenetic profile and molecular weight of GFAP in the snail, Megalobulimus abbreviatus, particularly in cerebral ganglia and subesophageal mass, by immunohistochemistry and immunoblotting. Our results confirm and extend previous studies about glial intermediate filaments in snails, showing: (i) a higher GFAP content in cerebral ganglia than in subesophageal mass; (ii) a developmental increase of GFAP immunocontent in cerebral ganglia, as described in Vertebrates; and (iii) an electrophoretic band for GFAP of approximately 55 kDa.     

Immunoblot identification of glial fibrillary acidic protein in rat sciatic nerve,brain, and spinal cord during development

The appearance of the glial fibrillary acidic protein (GFAP) during embryonic and postnatal development of the rat brain and spinal cord and in rat sciatic nerve during postnatal development was examined by the immunoblot technique. Cytoskeletal proteins were isolated from the central and peripheral nervous system and separated by SDS slab gel electrophoresis or two-dimensional gel electrophoresis. Proteins from the acrylamide gels were transferred to nitrocellulose sheets which were treated with anti-bovine GFAP serum and GFAP was identified by the immunoblot technique. GFAP was present in the embryonic rat brain and spinal cord at 14 and 16 days of gestation respectively. The appearance of GFAP at this stage of neural development suggests that the synthesis of GFAP may be related to the proliferation of radial glial cells from which astrocytes are derived. It is also feasible that GFAP provides structural support for the radial glial cell processes analogous to its role in differentiated astrocytes. GFAP was found to be present in rat sciatic nerves at birth and at all subsequent stages of development. These results indicate that some cellular elements in the rat sciatic nerve, such as Schwann cells, are capable of synthesizing GFAP which is immunochemically indistinguishable from its counterpart in the central nervous system. Thus it appears that GFAP is present both in the central and peripheral nervous system of the rat when the glial cells synthesizing GFAP are still undergoing differentiation.     

Calcium and glial cell death

Calcium (Ca2+) homeostasis is crucial for development and survival of virtually all types of cells including glia of the central nervous system (CNS). Astrocytes, oligodendrocytes and microglia, the major glial cell types in the CNS, are endowed with a rather sophisticated array of Ca2+-permeable receptors and channels, as well as store-operated channels and pumps, all of which determine Ca2+ homeostasis. In addition, glial cells detect functional activity in neighbouring neurons and respond to it by means of Ca2+ signals that can modulate synaptic interactions. Like in neurons, Ca2+ overload resulting from dysregulation of channels and pumps can be deleterious to glia. In this review, we summarize recent advances in the understanding Ca2+ homeostasis in glial cells, the consequences of its alteration in cell demise as well as in neurological and psychiatric disorders that experience glial cell loss.