Astroglial cells provide a template for the positioning of developing cerebellar neurons in vitro

Indirect immunocytochemical staining with antisera raised against purified glial filament protein and a neurofilament polypeptide was used to study cell interactions between astrocytes and neurons dissociated from embryonic and early postnatal cerebellum. Staining with antibodies raised against purified glial filament protein revealed that greater than 99% of all processes present in cerebellar cultures during the 1st wk in vitro were glial in origin. After 1 wk in culture, unstained processes that were presumably neuronal were observed. Stained astroglial processes formed a dense network that served as a template for cerebellar neurons, identified by indirect immunocytochemical localization of tetanus toxin. More than 90% of neurons from postnatal days 1 or 7 were positioned within one cell diameter of a glial process. In contrast, less than 40% of the neurons dissociated from early embryonic cerebellum were located adjacent to a glial process. Staining with antibodies raised against purified glial filament protein also revealed differences in astroglial morphology that were under developmental regulation. Astroglial cells from embryonic cerebellum were fewer in number and had thick, unbranched processes. Those from postnatal day 1 were more slender, branched, and stellate. Those from postnatal day 7 were highly branched and stellate. Some veil-like astroglial processes were also observed in cells from postnatal animals. These morphological changes were also observed when cells from embryonic day 13 were maintained for a week in vitro. No specific staining of embryonic or postnatal cerebellum cells was observed with antibodies raised against purified neurofilament polypeptides.     

Plasticity of Astroglial Glutamate and γ-Aminobutyric Acid Uptake in Cell Cultures Derived from Postnatal Mouse Cerebellum

Abstract: The plasticity of astroglial glutamate and γ-aminobutyric acid (GABA) uptakes was investigated using mouse cerebellar cell cultures. The influence of external factors, such as different sera and/or the presence of neurons, was examined. Control autoradiography experiments showed that after short-term exposure to radioactive amino acids, granule cells took up neither glutamate nor GABA, and β-alanine predominantly inhibited astroglial GABA uptake. Astroglial uptake was quantified by measuring the radioactivity taken up by the cells in the culture and relating this measurement to the number of glial fibrillary acidic protein-positive cells present. Glutamate uptake was investigated in astroglial cultures and subcultures and in neuro-nal-astroglial cultures derived from postnatal day 4 mouse cerebella. In the absence of neurons, glutamate uptake increased during the first 9 days after plating and then leveled off. At 14 days in vitro in horse serum, which favors the differentiation of fibrous-like astrocytes, glutamate uptake related to astrocyte number was twice as high as in fetal calf serum. In the presence of cerebellar neurons, this rate was even higher. The specificity of the responsiveness of astrocytes to neurons with respect to glutamate uptake was investigated by comparing GABA uptake in the different culture conditions. Neurons also increased the rate of GABA uptake by astrocytes. Another component of the astroglial plasma membrane, the density of β-adrenergic receptors, was, however, not markedly affected by the presence of neurons. Hence, these results showed that in astrocytes plated from postnatal day 4 mouse cerebella, the level of neuro-transmitter uptake can be regulated in vitro by factors present in sera and by cerebellar neurons in the culture. However, this plasticity declined during development because astrocytes plated from postnatal day 8 cerebella and cultured under identical conditions were less active in glutamate uptake and were insensitive to the presence of horse serum. The latter observation suggested that the metabolic plasticity of astrocytes is restricted to a period defined early in cerebellar development and is no longer evident by postnatal day 8.     

Transient increase in the high affinity [3H]-L-glutamate uptake activity during in vitro development of hippocampal neurons in culture

The glial GLAST and GLT-1 glutamate transporters are transiently expressed in hippocampal neurons as shown by immunocytochemistry (Plachez et al., 2000. J. Neurosci. Res., 59, 587-593). In order to test if this transient expression is associated to a transient glutamate uptake activity, [3H]-glutamate uptake was studied during the in vitro development of embryonic hippocampal neurons cultured in a defined (serum free) medium. In these cultures, the ratio of the number of glial cells to the number of neurons increased from 1.7 to 11.3% during the first 10 days of culture, while 77% of the neurons died. The number of neurons then remains stable up to 23 days of culture. The initial glutamate uptake velocity at 20 and 200 microM [3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 microM [3H]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum being reached at 2 DIV and a delayed regular increase from 8 to 23 DIV. The concentration-dependent curves were best fitted with two saturable sites high and low affinities, at both 2 and 10 DIV. To pharmacologically characterize the transient increased glutamate uptake activity, four uptake inhibitors, L-threo-3-hydroxy-aspartic acid (THA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC), dihydrokainate (DHK), and DL-threo-beta-benzyloxyaspartate (TBOA) were tested. THA, L-trans-2,4-PDC and DL-TBOA inhibited glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake inhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. These data indicated that, besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glutamate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression of the glial GLAST transporter in neurons.     

The postnatal development of the hypoglossal nucleus in the rat]

Using light and electron microscopy the neurons, glial cells and capillaries in hypoglossal nucleus of the rats have been examined up to 20 days after birth. The neuronal nuclei are usually situated ecentrically. The mitochondria and extensively developed Golgi-zones occupy the perinuclear region. The microtubules and lysosomes become more numerous with aging. At the earliest periods rough endoplasmic reticulum (ER) occupies the neuronal periphery, whereas after 14th day it is extended to the perinuclear region also. The ER forms elongated and concentric lamellated bodies and subsurface cisternae. At this time nucleolus like bodies are also numerous in the cytoplasm. After 4th and 6th days the extensive growth of dendrites, containing many cell organelles, and axons rich in microtubules are observed. Only at the birthday do neurons contain glycogen deposit. After 1st day the glycogen leaves the pericaryon, but it persists a long time in the neuronal processes. The symmetrical and asymmetrical contacts are characteristic for the examined period. The axo-somatic and axo-dendritic synapses are more abundant, but "double synapses" are also established. More synaptic boutons possess besides synaptic vesicles dense-core vesicles at the earlier periods. The quantity of asymmetric synapses increases with differentiation. Extensive cell degeneration has been established between 8 and 18th days. At 4 and 6 days the glial cells penetrate from subependymal layer and they have satellite neuronal position. This is more pronounced between 14 and 18 days when the oligodendrocytes are more numerous and active. At the same time fibrous astrocyte like cells are appeared. Microglial cells were not observed. Capillary differentiation, expressed by changes of the endothelial cells, pericytes and connective tissue cells, continues after birth also.     

无血清原代培养大鼠海马神经元的形态与膜电位

分离新生Wistar鼠海马,采用添加B27的无血清培养液进行海马神经元原代培养,动态观察海马神经元形态学变化;通过免疫荧光细胞化学法检测神经纤丝(NF)的表达,进行神经元鉴定及纯度计算;采用电位敏感的荧光探针标记神经元,在激光扫描共聚焦显微镜上动态监测去极化剂KCl作用前后膜电位的变化,观察神经元电生理反应。结果表明:此方法培养的大鼠海马神经元可在体外存活20天以上,9～14天为发育最成熟阶段,培养7天神经元纯度达90%。KCl作用于细胞后胞内荧光强度增强,细胞迅速去极化。本培养方法在体外获得高纯度的海马神经元并延长体外存活时间,且显示出神经元的电生理反应特性。     

POTASSIUM ACCUMULATION BY BULK PREPARED NEURONAL AND GLIAL CELLS

Abstract— Neuronal and glial cell enriched fractions were prepared by density gradient centrifugation of suspensions from rabbit cerebral cortex. The two cell types were incubated separately in media of extracellular ionic composition. The potassium accumulation was determined from analysis of potassium content of the cells by ultramicro flame photometry. Both neuronal and glial cells were capable of active potassium transport which was inhibited by ouabain (2 × 10⁻⁴ m ). The glial cells could accumulate potassium up to four to five times the concentration of the incubation medium and neurons up to one and a half to two times the medium concentration. The respiration in low potassium media was stimulated 15 per cent for neurons and 85 per cent for glia when potassium was added to a final concentration of 50 m m . The uptake by both neurons and glia showed temperature and sodium dependence. There was a definite magnesium requirement for the potassium uptake, particularly demonstrable for glial cells. Calcium inhibited potassium uptake by glia but stimulated slightly that by neurons.     

Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence.

We have analyzed the appearance of neurons and glial cells in chick dorsal root ganglia during development. Neurons were identified by the presence of polysialogangliosides recognized by tetanus toxin (GD1b, GT1) or by the monoclonal antibody Q211 directed against polysialogangliosides containing four, five and six sialic acid residues. Glial cells were identified by the presence of 04 antigen. A population of undifferentiated cells, i.e., cells which express neither neuronal nor glial cell surface antigens, present in dorsal root ganglia until embryonic day 7, was separated from the neuronal and glial population. This cell population contains neuronal progenitor cells which differentiate to neurons within 1 day in culture. This differentiation process is characterized by the appearance of neuronal morphology, of neuron-specific gangliosides and by the appearance of voltage-dependent sodium and calcium channels.     

The effects of malnutrition on the prenatal development of rat brain]

Female rats were submitted to caloric and protein restriction 8 days before fecondation and during pregnancy. A reduction of the number of brain cells in the fetuses was evident only after the 20th day of pregnancy. This reduction appeared to be limited to glial cells, and not to affect neurons contrarily to what had previously been stated by some authors.     

Microheterogeneity of tubulin proteins in neuronal and glial cells from the mouse brain in culture

The microheterogeneity of the alpha and beta isoforms of tubulin in brain cells in culture was studied. The cells were prepared from two precise regions of the embryonic mouse brain (ED15), the striatum and the mesencephalon. It was possible to maintain virtually pure cultures of neuronal or glial cells up to 1 and 4 weeks in vitro, respectively. The tubulin heterogeneity of striatal and mesencephalic neurons was found to be very similar after a few days in culture. More precise examination of pure neurons from the striatum revealed that their tubulin content after 7 days in vitro exhibited the same degree of complexity as a control extract from a 4 day-old mouse brain. In fact, we could detect the presence of at least six alpha and nine beta tubulin isoforms. Among these isoforms a specific family of beta proteins (beta' tubulin) and the more acidic alpha proteins were present. Since these isoforms have, up to now, been found only in tubulin extracts prepared from the nervous system, our experiments suggest that they belong to the neuronal subpopulation of this tissue. This point is reinforced by their complete absence from the tubulin proteins extracted from pure glial cells even after several weeks in vitro. These results lead us to propose that brain tubulin microheterogeneity is associated with the presence of neurons and not of glia and may, therefore, play a specific role in maintaining neuronal shape and function.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non‐neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non‐neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7‐day‐old cultures a subgroup of small non‐neural cells with processes expressed ChAT activity, and in 14‐day‐old cultures non‐neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non‐neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1‐day‐old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non‐neural cells during the development of Manduca antenna. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

In vivo and in vitro differentiation of neurons and astrocytes in the rat embryo. Immunofluorescence study with neurofilament and glial filament antisera

Antisera raised against neurofilament (NF) peptides and glial fibrillary acidic protein (GFA) (subunit of glial filaments) have been used to identify neurons and astrocytes in order to study their development and differentiation in rat embryo. In vivo observations showed that NF-positive cells first appeared in 12-day-old embryos, whereas GFA-positive cells appeared in brain and spinal cord on the 18th day. In vitro observations showed that NF-positive cells could be obtained only in cultures from 12-day embryos onwards. The further differentiation of neurons involved neurite elongation, aggregation of cell bodies to form islets, and emergence of very brightly staining prominent neurons with large cell bodies and long neurites which took part in complicate pattern formation. GFA-positive cells appeared in vitro on the 16th day and they could be observed even in cultures obtained from 10-day-old embryos. As the culture aged, the GFA staining became highly fibrillary. There was no physical interaction between neuronal and glial processes.     

Metabolic and temporal studies on pancreatic exocrine cells in culture

Summary An approach is described whereby cells with definitive markers are followed from their source through dissociation and fractionation, then during long-term maintenance in vitro. Such sequential studies should enable investigators to define factors regulating proliferation and function of specific cells since ambiguity concerning identity is readily avoided.Pancreatic cells of guinea pigs were isolated by enzymic dissociation, and exocrine cells were enriched by centrifugation with solutions of serum albumin. Resulting populations consisting of up to 95% exocrine cells were then incubated with gyration to produce aggregates, and these were seeded to standard culture plates for further study. Colonial aggregates of exocrine epithelia develop in culture and can be maintained for 20–30 days. The cells exhibit changes with time that are qualitatively similar to those known to occur during serial cultivation of diploid fibroblastlike cells from human and other species. The uptake of tritiated thymidine decreases with maintenance time. Autoradiographic examination indicates that this is due to a reduction in the number of epithelial cells incorporating the isotope. Cell diameters increase from an average of 21 m at day 0 to 44 m by day 26, and a marked increase in heterogeneity of this parameter is also evident. Cellular DNA and protein accumulate during the same interval. Incorporation of tritiated leucine during 24-h exposures increases until about the 10th day in vitro and remains relatively constant for at least 2 weeks thereafter.The data are consistent with the hypothesis that exocrine pancreatic cells like other diploid cells in culture, progress to terminal differentiation under the culture conditions employed. The role of physical, nutritional, and humoral evironmental factors on this process will be the subject of future reports.Supported in part by National Cancer Institute Contracts NO1-CP-43231 and NO1-CP-65751     

Phenotypic changes in satellite glial cells in cultured trigeminal ganglia

Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24?h after culturing, but undergo major phenotypic changes at later times.     

Modifications in energy metabolism during the development of chick glial cells and neurons in culture

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non-neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non-neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7-day-old cultures a subgroup of small non-neural cells with processes expressed ChAT activity, and in 14-day-old cultures non-neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non-neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1-day-old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non-neural cells during the development of Manduca antenna.     

Time of origin of cells in the histiogenesis of the spinal cord]

In order to establish the moment of appearance of neuroblasts and ectoglia of the spinal cord the autoradiographic study with the use of H3-thymidine and C14-thmidine injected to pregnant mice with the intervals between injections 121/2 or 24 hours was undertaken. It was establised that spinal neurons were removed from the nervous tube beginning from the 10th up to 13th days of embroyogenesis. The motoneurons of the anterior horn were the first to appear (10th-12th days), the neurons of the intermideate zone were the next to appear (11th - 12th days) and the last were the neurons of posterior horn (13th day). Beginning from the 13th day of embryogenesis there appeared the ectoglia which migrated following meurblasts two days later. The saturation of the grey matter with glial cells and the saturation of the white matter with Schwann cells was brought about by means of additional multiplication at the site of the glioblasts removed from the nervous tube. The main function of the matrix layer neuroepithelium of the nervous tube as a provider of cells to the spinal cord terminated on the 15th day of embryogenesis.     

Cell differentiation in the hippocampus of the fetal rat in cell culture

Cell cultures from hippocampus of 16 and 17 days old embryonal rats were cultivated up to 4 weeks. After 24 hours in vitro on 18.4 percent of cells and after 5 days in vitro on 70 percent of cells processes could be recognized. These are neuroblasts. The cells reaggregated. Nerve fibers after 4 weeks in vitro are 200 to 300 mum long. Small and big neurons with 12 mum to 26 mum diameters of perikarya, bi- and multipolar neurons after 4 weeks in vitro were observed. In cultures and meningothel-monolayer developed. Maintenance and differentiation of cultures are possible only by sowing in at least 60,000 cells/ml medium. The advantage of cell culture opposite to organ culture exists in experiments with immediate selective influence.     

Immunocytochemical localization of cell type-specific markers in reaggregating cell cultures of mouse cerebellum

Summary Reaggregate cultures were obtained from single-cell suspensions of fetal and early postnatal cerebellum, and fetal telencephalon and mesencephalon from C57BL/6J and NMRI mice and maintained in suspension under constant rotation as described previously (Seeds 1971). The percentage of dead cells in the aggregates as measured by the uptake of the fluorescent dye propidium iodide was always less than 5% of all cells. During the initial phase of reaggregation up to 20 h in vitro (hiv) several immunocytochemically defined cell types had a random distribution within the aggregate. Astrocytes were identified by indirect immunofluorescence by the use of the markers glial fibrillary acidic protein (GFAP), C1 and M1 antigens; neurons by NS-4 antigen and tetanus-toxin receptors; fibroblasts or fibroblast-like cells by fibronectin and laminin; and oligodendrocytes by myelin basic protein (MBP). Choleratoxin receptors and M 2 antigen served to distinguish the more mature from the less mature neurons. In reaggregates of early postnatal cerebellar cells neurons had started to redistribute after 40 hiv, forming an outer region containing more immature neurons and a core with more mature neurons. After 5 days in vitro (div) immature neurons were no longer detectable. From 3–8 div M1-and GFAP-positive astrocytic processes in the outer region showed a tendency for radial orientation. At later stages the processes appeared more randomly distributed and formed a dense glial network. Few oligodendrocytes and fibronectin-positive cells were present in the reaggregates. When reaggregates were prepared from 15 day-old embryonic cerebella, formation of radially oriented astrocytic processes and redistribution of neurons proceeded more slowly, but in a similar pattern as described for early postnatal cerebellum. GFAP was detectable at earlier ages than in situ. In reaggregates of 15 to 17 day old embryonic telencephalic anlage or midbrain, radially oriented astrocytic processes were not detectable. Similar to cerebellar reaggregates, accumulation of neurons in the inner region was observed.     

Non-Neuronal Cells Are Required to Mediate the Effects of Neuroinflammation: Results from a Neuron-Enriched Culture System

Chronic inflammation is associated with activated microglia and reactive astrocytes and plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s. Both in vivo and in vitro studies have demonstrated that inflammatory cytokine responses to immune challenges contribute to neuronal death during neurodegeneration. In order to investigate the role of glial cells in this phenomenon, we developed a modified method to remove the non-neuronal cells in primary cultures of E16.5 mouse cortex. We modified previously reported methods as we found that a brief treatment with the thymidine analog, 5-fluorodeoxyuridine (FdU), is sufficient to substantially deplete dividing non-neuronal cells in primary cultures. Cell cycle and glial markers confirm the loss of ~99% of all microglia, astrocytes and oligodendrocyte precursor cells (OPCs). More importantly, under this milder treatment, the neurons suffered neither cell loss nor any morphological defects up to 2.5 weeks later; both pre- and post-synaptic markers were retained. Further, neurons in FdU-treated cultures remained responsive to excitotoxicity induced by glutamate application. The immunobiology of the FdU culture, however, was significantly changed. Compared with mixed culture, the protein levels of NFκB p65 and the gene expression of several cytokine receptors were altered. Individual cytokines or conditioned medium from β-amyloid-stimulated THP-1 cells that were, potent neurotoxins in normal, mixed cultures, were virtually inactive in the absence of glial cells. The results highlight the importance of our glial-depleted culture system and identifies and offer unexpected insights into the complexity of -brain neuroinflammation.