Primary dissociated cultures of human brainstem cells: a useful tool for their characterization and neuroprotection study

Dissociated cell cultures were prepared from brainstems of 5- to 10-week-old human fetuses. Catecholamine- as well as indolamine-containing cells were visualized using respectively dopamine (DA), noradrenaline (NA) and serotonin (5HT) as immunocytochemical markers. NA-, DA-, and 5HT-stained cells were characterized in the rhombencephalic cultures, representing respectively the fetal localization of the locus coeruleus and raphe nuclei. DA-stained cells were characterized in the mesencephalic cultures; these DA-cells originating from the substantia nigra presented morphological aspects different from the DA-rhombencephalic cells. Two types of GABA neurons and glial cells presenting glial fibrillary acidic protein (GFA-P) reactivity were also found in all the cultures. Two non-competitiveN-methyl-D-aspartate antagonists, 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) andcis-Pip/Me 1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine (GK11) in enantiomeric form (–), have been investigated for survival on rhombencephalic cultured cells. The number of 5HT-cells was found to be greater in the treated cultures than in the control ones. Thisin vitro system appears to be a useful tool for the investigation of the development of central nervous system (CNS) cells as well as the study of neuroprotection.Abbreviations CA catecholamine - DA dopamine - CNS central nervous system - GFA-P glial fibrillary acidic protein - IR immunoreactivity - NMDA N-methyl-d-aspartate - NA noradrenaline - 5HT serotonin - TCP 1-[1-(2-thienyl)cyclohexyl]piperidine - GK11 cis-Pip/Me 1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine     

Enzyme activities of monoamine oxidase,cathechol-o-methyltransferase and γ-aminoubutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions

The activities of monoamine oxidase (MAO), cathechol-O-methyltransferase (COMT) and -aminobutyric acid transaminase (GABA-T) were measured in primary cultures from newborn rat cultivated from 6 different brain regions. These primary cultures contained mostly astroglial cells, evaluated by the presence of the glial fibrillary acidic protein (GFAp, -albumin) and the S-100 protein. The enzyme activities in the corresponding brain areas from adult rat were also quantified. MAO activities were on the same level in 14-day old cultures and in adult rat brain homogenates, with significantly lower values in brain stem as compared to the other brain regions examined. COMT activities were on a higher level in the cultures than in adult rat brain homogenates. Astroglial cells from hippocampus were found to have the highest and those from brain stem the lowest COMT-activities. GABA-T activities were lower in the cultures than in adult rat homogenates. No significant differences were seen in the various astroglial cultures. Accumulation of [³H]dopamine and [³H]-aminobutyric acid (GABA) visualized by autoradiography showed only a slight uptake of dopamine in comparison with the uptake of GABA. It is concluded that astroglial cells in culture have enzymatic properties similar to those of astroglial cells in different brain regions of adult rat brain. Studies are in progress to evaluate if the regional heterogeneity observed among cultivated astroglial cells is affected by in vivo differentiation until cultivation and/or time in culture.     

Neurotrophic and neuroprotective effects of the neuregulin glial growth factor-2 on dopaminergic neurons in rat primary midbrain cultures

Glial growth factor-2 (GGF2) and other neuregulin (NRG) isoforms have been shown to play important roles in survival, migration, and differentiation of certain neural and non-neural cells. Because midbrain dopamine (DA) cells express the NRG receptor, ErbB4, the present study examined the potential neurotrophic and/or neuroprotective effects of GGF2 on cultured primary dopaminergic neurons. Embryonic day 14 rat mesencephalic cell cultures were maintained in serum-free medium and treated with GGF2 or vehicle. The number of tyrosine hydroxylase-positive (TH+) neurons and high-affinity [3H]DA uptake were assessed at day in vitro (DIV) 9. Separate midbrain cultures were treated with 100 ng/mL GGF2 on DIV 0 and exposed to the catecholamine-specific neurotoxin 6-hydroxydopamine (6-OHDA) on DIV 4. GGF2 treatment significantly increased DA uptake, the number of TH+ neurons, and neurite outgrowth when compared to the controls in both the serum-free and the 6-OHDA-challenged cultures. Furthermore, three NRG receptors were detected in the midbrain cultures by western blot analysis. Immunostaining for glial fibrillary acidic protein revealed that GGF2 also weakly promoted mesencephalic glial proliferation in the midbrain cultures. These results indicate that GGF2 is neurotrophic and neuroprotective for developing dopaminergic neurons and suggest a role for NRGs in repair of the damaged nigrostriatal system that occurs in Parkinson's disease.     

Glial organization and chondroitin sulfate proteoglycan expression in the developing thalamus

This study examines the early organization of glial cells, together with the expression of chondroitin sulfate proteoglycans in the developing thalamus of ferrets. Glia were identified with antibodies against vimentin and glial fibrillary acidic protein and the chondroitin sulfate proteoglycans were identified by using an antibody against chondroitin sulfate side chains. Our results reveal three striking features of early thalamic development. First, there is a distinct population of glial fibrillary acidic protein-immunoreactive astrocytes (first seen at E30) that resides in the perireticular thalamic nucleus of the primordial internal capsule. These glial fibrillary acidic protein-immunoreactive astrocytes of the perireticular nucleus are transient and form a conspicuous feature of the early developing forebrain. They are first apparent well before any glial fibrillary acidic protein-immunoreactive astrocytes are seen in other regions of the thalamus (at about P8). Further, unlike in other thalamic regions, these peculiar perireticular astrocytes do not express vimentin before they express glial fibrillary acidic protein. Second, in the reticular thalamic nucleus, the radial glial cells express glial fibrillary acidic protein; they are the only ones to do so in the thalamus during development. The glial fibrillary acidic protein-immunoreactive radial glial cells of the reticular nucleus form a rather distinct band across the developing thalamus at these early stages (E30–P1). Finally, and preceding the expression of glial fibrillary acidic protein, the radial glial cells of the reticular nucleus, unlike those in other thalamic regions, are associated closely with the expression of chondroitin sulfate proteoglycans (E20–E30). Later (after E30), the expression of the chondroitin sulfate proteoglycans in the reticular nucleus declines sharply. The significance of this finding is related to the early organization of the cortico-fugal and cortico-petal pathways.     

Metabolic studies in vitro of the CNS cytoskeletal proteins: Synthesis and degradation

General aspects of metabolic features of the most prominent CNS intermediate filament proteins, the 200,000 (200K), 150,000 (150K), and 70,000 (70K) dalton proteins of the neuron, and the glial fibrillary acidic protein (GFAP) have been explored using the incubated spinal cord slice from the rat. Measurement of shortterm uptake of³H-labeled amino acids into the individual proteins separated on polyacrylamide gels revealed that of the three neurofilament proteins, 200K was most metabolically active, 150K was less active, and 70K contained very little incorporated radioactivity. Glial fibrillary acidic protein based on Coomassie blue stain affinity showed less metabolic activity than any of the neurofilament proteins. Those relationships were constant at all ages, but the metabolic activity of all CNS intermediate filaments decreased with age. When Ca²⁺ was present in the medium of the incubated slices, the intermediate filaments were rapidly destroyed, but GFAP was more resistant to degradation than the neurofilament proteins. GFAP and probably the neurofilament proteins also were relatively resistant to Ca²⁺-activated degradative mechanisms in spinal cords of rats at younger ages (15 day) than in those of older animals (10–18 months). It is likely that the Ca²⁺ activated protease is less active in developing animals in which the nerve tracts are still elongating, than in adults. These results suggest that GFAP is less active metabolically and more resistant to degradation than the neurofilament proteins at all stages of maturation, but that metabolic activity of all CNS intermediate filaments decreases with age while the susceptibility to degradation increases.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Tumor Necrosis Factor-α and Basic Fibroblast Growth Factor Decrease Glial Fibrillary Acidic Protein and Its Encoding mRNA in Astrocyte Cultures and Glioblastoma Cells

Abstract: Tumor necrosis factor-α is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor-α. Treatment with tumor necrosis factor-α for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor-α treatment increased the expression of the cytokine interleukin-6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor-α, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor-α induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor-α. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor-α treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.     

Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures

The transport of [³H]l-glutamate, [³H]l-aspartate, [³H]-aminobutyric acid ([³H]GABA), [³H]dopamine, [³H]norepinephrine and [³H]5-hydroxytryptamine (³H-5-HT) was measured in primary astroglial cultures from newborn rat cerebral hemispheres. There was a high-affinity uptake with aK _m of 69.0 M for L-glutamate, 12.3 M forl-aspartate and 3.1 M for GABA. The uptake showed properties of high capacity with aV _max of 17.0 nmol·mg prot^–1·min^–1 forl-glutamate, 1.1 nmol·mg prot^–1·min^–1 forl-aspartate and 0.04 nmol·mg prot^–1·min^–1 for GABA. No high-affinity high capacity transport system was found for the monoamines studies. Autoradiographic examination demonstrated a heavy deposit of grains suggesting a prominent accumulation of [³H]l-glutamate and [³H]l-aspartate in the astroglial-like cells of the cultures, while the [³H]GABA accumulation was less intense. On the other hand, there was only a weak accumulation of grains after incubating the cultures with [³H]dopamine, [³H]norepinephrine or [³H]5-HT. Thus, astroglial cells in culture accumulate amino acid neurotransmitters and monoamines in different ways with a high-affinity high-capacity uptake of glutamate, aspartate and GABA and a diffusion-uptake of dopamine, norepinephrine and 5-HT.     

Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells

The reported expression of the cell surface-associated, mainly mesenchymal glycoprotein fibronectin by cultured glial cells is in discrepancy with recent work on brain tissue failing to demonstrate any glial or neuronal fibronectin. We have investigated the expression of fibronectin in relation to glial fibrillary acidic protein in cultured human glial and glioma cell lines as well as in cultures derived from newborn rat brain. Using double immunofluorescence technique we found that cells containing glial fibrillary acidic protein do not express fibronectin, and vice versa. The only exception to this rule was the occasional finding of fibronectin at points of cell-to-cell adhesion also in relation to cells containing glial fibrillary acidic protein. The results were also tested by polyacrylamide gel electrophoresis of the culture media of the human cell lines, and by subcultures from the brain of newborn rat, cultures stimulated with dibutyryl cyclic AMP (db-cAMP), and by vinblastine treatment of the cells. The lack of expression of fibronectin in cells containing glial fibrillary acidic protein, a gliospecific cytoskeletal protein, is discussed with reference to glio-mesenchymal interactions and glial markers in vitro.     

The effect of MPTP on the neuronal uptake of monoamines]

The results of kinetic analysis of synaptosomal uptake of dopamine, noradrenaline, adrenaline and serotonin showed the presence of their own carrier systems with high or low affinity for each monoamine. The low affinity system of the uptake of monoamines by nerve endings differs from extraneuronal one by higher affinity. MPTP noncompetitively inhibits the system of highly effective uptake of the studied monoamines by nerve endings, competitively inhibiting synaptosomal uptake with low affinity of noradrenaline, adrenaline and noncompetitively serotonin and dopamine. The constant values of inhibition showed that MPTP most strongly blocks the system of synaptosomal uptake of low affinity serotonin and approximately 2-times weaker affects its system of high affinity. Carrier systems of high affinity of dopamine, adrenaline and noradrenaline block MPTP 150-500 times weaker than that of serotonin, and as for low affinity--in 2000-4000 times. It may be supposed that synaptosomal uptake of low affinity serotonin is most perceptible to the effect of MPTP and is of a particular importance in the development of Parkinson's disease symptoms.     

Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures

We describe the use of the neutral protease Dispase for the dissociation of neonatal rat brain tissue for the preparation of primary monolayer astrocyte cultures. The method involves 5 to 6 successive extractions with careful separation of sedimenting, undissociated tissue. This method gives an initial cell suspension of high viability (93.7±1.7% cells exclude trypan blue). In comparison trypsin (0.25%) dissociated tissue gave a cell suspension that showed a lower viability of 58.2±7.6%. Identical saturation densities of 1.1 to 1.2×10⁴ cells/cm² after two weeks in culture were obtained for a range of seeding densities from 1 to 4×10⁴ cells/cm² of the Dispase dissociated tissue. Staining for glial fibrillary acidic protein showed that 90–100% cells were positive for this astroglial marker. Thus, the use of Dispase for the initial dissociation of rat brain tissue seems to give primary astrocyte cultures which are very reproducible and homogeneous.     

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0–12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin⁺ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN⁺ neurons were best preserved in the dorsal horn whereas large NeuN⁺ and choline acetyltransferase⁺ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein⁺ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B₄, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.     

Combined immunostaining of neurofilaments, neuron specific enolase, GFAP and S-100. A possible means for assessing the morphological and functional status of the enteric nervous system

Neurofilaments, part of the cytoskeletal network, and neuron specific enolase, a major enzyme in glycolysis, are both present in central and peripheral neurons. Glial fibrillary acidic protein and S-100, on the other hand, are soluble proteins which are found exclusively in the supportive cells of the nervous system, i.e. the glial cells. Examination was made, using immunocytochemistry, of all main areas of the gastrointestinal tract of three mammalian species, rat, pig and man. By applying serial tissue sectioning, it was possible to study the relative occurrences of the two neuronal markers in the same cell bodies and to examine the relationships of the neurons with the glial cells as revealed by the antibodies to glial fibrillary acidic protein and S-100. Both neurofilaments and neuron specific enolase were localised to an extensive system of enteric nerves, with the level of neuron specific enolase-immunoreactivity showing greater variability than that observed using antibodies to neurofilaments. Comparison of the occurrence of neuron specific enolase and neurofilament immunoreactivity in serially sectioned neuronal cell bodies revealed that a minor population stained only with antibodies to neurofilaments. The equivocal or absent neuron specific enolase-immunoreactivity in some perikarya may reflect variations in functional status within the nervous system. Glial fibrillary acidic protein- and S-100-immunoreactivities were confined to glial cells which, in this normal tissue, were always in close association with the neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Changes in mitotic rate and GFAP expression in the primary olfactory axis of Streptozotocin-induced diabetic rats

Many diabetic individuals develop anosmia but the mechanism(s) causing the dysfunction in the olfactory system is (are) unknown. Glial fibrillary acidic protein expression is reduced in diabetic retinopathy and is also reduced, with unknown consequences, in other brain regions of diabetic rats. We used immunohistochemistry and immunoblotting from untreated control and streptozotocin-induced type 1 (insulin dependent) diabetic rats to investigate main olfactory epithelial mitotic rate and glial fibrillary acidic protein expression in the lamina propria of the sensory epithelium and in the olfactory bulb. Numbers of bromodeoxyuridine-positive cells were significantly lower in the diabetic sensory epithelium compared to non-diabetic controls. Immunohistochemical observations suggested a qualitative difference in glial fibrillary acidic protein expression in both regions examined especially in the olfactory bulb external plexiform layer and the lamina propria. Immunoblot analysis confirmed that the diabetic olfactory bulb and lamina propria expressed less glial fibrillary acidic protein compared to the non-diabetic control group. The lower expression levels in the olfactory bulb external plexiform layer suggested by immunohistochemistry do not reflect a change in the number of astrocytes since the numbers of S100B(+) cells were not different between the two groups.     

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.     

Toxoplasma gondii Migration within and Infection of Human Retina

Toxoplasmic retinochoroiditis is a common blinding retinal infection caused by the parasite, Toxoplasma gondii. Basic processes relating to establishment of infection in the human eye by T. gondii tachyzoites have not been investigated. To evaluate the ability of tachyzoites to navigate the human retina, we developed an ex vivo assay, in which a suspension containing 1.5×10⁷ parasites replaced vitreous in a posterior eyecup. After 8 hours, the retina was formalin-fixed and paraffin-embedded, and sections were immunostained to identify tachyzoites. To determine the preference of tachyzoites for human retinal neuronal versus glial populations, we infected dissociated retinal cultures, subsequently characterized by neuron-specific enolase or glial fibrillary acidic protein expression, and retinal cell lines, with YFP-expressing tachyzoites. In migration assays, retinas contained 110–250 live tachyzoites; 64.5–95.2% (mean  = 79.6%) were localized to the nerve fiber layer, but some were detected in the outer retina. Epifluorescence imaging of dissociated retinal cultures 24 hours after infection indicated preferential infection of glia. This observation was confirmed in growth assays, with significantly higher (p≤0.005) numbers of tachyzoites measured in glial verus neuronal cell lines. Our translational studies indicate that, after entering retina, tachyzoites may navigate multiple tissue layers. Tachyzoites preferentially infect glial cells, which exist throughout the retina. These properties may contribute to the success of T. gondii as a human pathogen.     

Comparative atomic force and scanning electron microscopy: An investigation of structural differentiation of hepatic stellate cells

The molecular mechanism leading to the transdifferentiation of hepatic stellate cells (HSC) into myofibroblast-like cells following liver injury is not well understood. The state of cultured rat HSCs was determined using primarily fluorescence microscopy (UV), immunofluorescence (IF) (Glial fibrillary acidic protein (GFAP), Desmin, alpha-smooth muscle actin (alpha-SMA), F-actin) and immunocytochemistry (ICC) (GFAP, Desmin, alpha-SMA, Fibulin-2). Additionally, tapping-mode atomic force microscopy (TM-AFM) and field-emission scanning electron microscopy (FE-SEM) with low-resistivity indium-tin-oxide (ITO) thin-film were performed to observe the micro-morphological character of cells during HSC differentiation. Quiescent HSCs changed to the activated state were identified via UV, IF, and ICC observations. Normal rat HSCs (NHSCs) and thioacetamide-induced rat HSCs (THSCs) were demonstrated to be UV⁻, GFAP⁺, Desmin⁺, alpha-SMA⁺ and Fibulin-2⁻. After F-actin staining, lamellipodia and filopodia were found in both NHSCs and THSCs, but membrane ruffles were only seen in THSCs. The micro-structures of lamellipodia and filopodia in both NHSCs and THSCs were confirmed using FE-SEM and TM-AFM with ITO; in contrast, the micro-projection was not found. Moreover, “aerial root” structures were observed for the first time in the filopodia of THSCs using TM-AFM. These results reveal that HSC transdifferentiation to a myofibroblastic-like cell (activated HSC) from thioacetamide-induced rat HSC induces extensive changes in the cytoskeleton.     

Neurochemical characterization of the release and uptake of dopamine in ventral tegmental area and serotonin in substantia nigra of the mouse

In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.