Calcium Transport by Primary Cultured Neuronal and Glial Cells from Chick Embryo Brain

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM-Ca²⁺ and 5.4 mM-K⁺. The rate of calcium entry into neurons was increased 2.7-fold by elevating medium potassium to 60 MM. The effect of high external potassium was to increase the V_max value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium-dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM-D-600 (a verapamil derivative) or by 1 mM-CoCl₂, but 0.5 μM-tet-rodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight-of-protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization-sensitive, calcium-specific channel. A similar calcium transport activity was not detected in cultured glial cells.     

CALCIUM FLUXES IN CULTURED AND BULK ISOLATED NEURONAL AND GLIAL CELLS

Abstract— The influx and efflux of ⁴⁵Ca has been studied in cultured human glioma and mouse neuroblastoma cells and in isolated fractions enriched in synaptosomes, neuronal and astrocytic perikarya from rabbit brain.
The uptake of ⁴⁵Ca was somewhat more efficient in glioma compared to neuroblastoma cells, whereas there was little difference in the rate of ⁴⁵Ca uptake by isolated glial cells and neuronal perikarya. Isolated synaptosomes showed the highest rate of ⁴⁵Ca accumulation. An increase of K concentration to 50 m m in the medium, with a corresponding lowering of Na, stimulated both glioma and glial as well as synaptosomal ⁴⁵Ca uptake more markedly than the uptake by neuroblastoma cells and neuronal perikarya. Lowering the Na concentration and replacing it by choline had no effect on the cultured cells and astrocytes. Na-free media caused massive stimulation of ⁴⁵Ca influx in all fractions and cells tested.
The efflux of ⁴⁵Ca was studied after preloading of cells. Three phases could be resolved from the desaturation curves. All cells had nearly similar half-lives for ⁴⁵Ca efflux under standard conditions. Pulses of media containing 50 m m -K stimulated ⁴⁵Ca efflux from glioma cells and astrocytes more efficiently than from neuroblastoma cells, neuronal perikarya and synaptosomes. The stimulated release was exclusively seen in Ca-containing media in experiments with the cultured cells and in Ca-free media in experiments with cell perikarya. The effect of transmitter pulses on the release of ⁴⁵Ca was examined in a limited series. Acetylcholine and isoproterenol were found to stimulate ⁴⁵Ca release more actively from glia than from neurons.     

Neuronal inhibition of astroglial cell proliferation is membrane mediated

Previously we have used a microwell tissue culture assay to show that early postnatal mouse cerebellar astroglia have a flattened morphology and proliferate rapidly when they are cultured in the absence of neurons, but develop specific cell-cell contacts and undergo morphological differentiation when they are co-cultured with purified granule neurons (Hatten, M. E., 1985, J. Cell Biol., 100:384-396). In these studies of cell binding between neurons and astroglia, measurement with light and fluorescence microscopy or with [35S]methionine-labeled cells indicated that the kinetics of the binding of the neurons to astroglial cells are rapid, occurring within 10 min of the addition of the neurons to the growing glia. 6 h after neuronal attachment, astroglial DNA synthesis decreases, as shown by a two- to fivefold decrease in [3H]thymidine incorporation, and glial growth ceases. No effects on astroglial cell growth were seen after adding medium conditioned by purified cerebellar neurons cultured in the absence of astroglia, by astroglia cultured in the absence of neurons, or by a mixed population of cerebellar cells. This result was unchanged when any of these media were concentrated up to 50-fold, or when neurons and astroglia were cultured in separate chambers with confluent medium. Two groups of experiments suggest that membrane-membrane interactions between granule neurons and astroglia control astroglial cell growth. First, neurons fixed with dilute amounts of paraformaldehyde (0.5%) bound to the astroglia with the same kinetics as did living cells, inhibited DNA synthesis, and arrested glial growth within hours. Second, a cell membrane preparation of highly purified granule neurons also bound rapidly to the glia, decreased [3H]thymidine incorporation two- to fivefold and inhibited astroglial cell growth. The rate of the decrease in glial growth depended on the concentration of the granule neural membrane preparation added. A similar membrane preparation from purified cerebellar astroglial cells, PC12 cells, 3T3 mouse fibroblasts, or PTK rat epithelial cells did not decrease astroglial cell growth rates. Living neurons were the only preparation that both inhibited glial DNA synthesis and induced the astroglial cells to transform from the flat, epithelial shapes they have when they are cultured without neurons to highly differentiated forms that resemble Bergmann glia or astrocytes seen in vivo. These results suggest that membrane-membrane interactions between neurons and astroglia inhibit astroglial proliferation in vitro, and raise the possibility that membrane elements involved in glial growth regulation include neuron-glial interaction molecules.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

Composition of gangliosides and phospholipids of neuronal and glial cell enriched fractions

Abstract— Fractions enriched in neuronal cell bodies and in glial cells were isolated from rabbit cerebral cortex by discontinuous gradient centrifugation. The ratio of total lipid to protein was approx. 50 per cent higher in the glial fraction than in the neuronal fraction. The fatty acid composition for the major phosphoglycerides was with few exceptions, similar for neurons and glia. The ganglioside concentration was very low for both cell types, but was approx. twice as high in the glial cells as in the neurons. The pattern of individual gangliosides was, however, very similar for the glial and neuronal fractions and did not differ from that of unfractionated cerebral cortex, synaptosomes and mitochondria. The latter results are discussed in relation to the estimated amounts of plasma membrane in the neuronal and glial fractions.     

L-arginine uptake and release by cultured avian retinal cells: differential cellular localization in relation to nitric oxide synthase

The availability of L-arginine is of pivotal importance for the synthesis of nitric oxide, a signaling molecule in the CNS. Here we show the presence of a high-affinity L-arginine uptake system (Km of 4.4 +/- 0.5 microM and a Vmax of 26.0 +/- 0.9 fmol/well/min) in cultured chick retinal cells. Different compounds, such as N(G)-mono-methyl-L-arginine and L-lysine, were able to inhibit the uptake that was also inhibited 60-70% in the absence of sodium and/or calcium ions. No trans stimulation was observed when cells were preloaded with L-lysine. The data indicate that the L-arginine uptake in cultured retinal cells is partially mediated by the y+ system, but has a great contribution of the B(0,+) system. Autoradiographic studies revealed that the uptake is predominant in glial cells and can also be detected in neurons, whereas immunocytochemistry of nitric oxide synthase and L-citrulline showed that the enzyme is present in neurons and photoreceptors, but not in glial cells. L-[3H]Arginine is released from purified glial cultures incubated with high concentrations of potassium in the extracellular medium. Moreover, the amino acid released from preloaded glial cells was taken up by purified neuronal cultures. These results indicate that L-arginine released from glial cells is taken up by neurons and used as substrate for the synthesis of nitric oxide.     

Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells

1. A procedure has been developed for the separation of intact metabolically active neuronal and glial cells in bulk from rat cerebral cortex. Separation depended on dispersion of the tissue in a Ficoll medium followed by centrifugation on a discontinuous Ficoll gradient. Up to 1.5x10(7) neuronal cells could be collected from 12 brains within 3hr. The morphological appearance of these cells seemed good, and the fraction was 8.5-fold purified in terms of dry weight. Average dry weight per neuron was 2300mumug. Maximum glial contamination of the neuronal fraction was 11% as determined by carbonic anhydrase measurements. The glial fraction was free from neurons but contained various subcellular contaminants. 2. Concentrations of nucleic acids, phospholipid, protein and phosphoprotein were determined in the separated fractions. The neuronal fraction was richer than the glial in all except phospholipid. Succinate dehydrogenase was equally distributed between neurons and glia but the neuronal fraction was 1.8-fold enriched in cytochrome oxidase. 3. Measurement of respiration by the cells showed an endogenous uptake of 117mmumoles of oxygen/mg./hr. in neurons, and 173mmumoles of oxygen/mg./hr. in glia. Addition of substrate at 10mm stimulated uptake to similar values in both fractions. With glucose it was 390, with pyruvate 355, and with glutamate 215mmumoles of oxygen/mg./hr. This represented a larger stimulation of neuronal than of glial respiration compared with the basal level. 4. Respiration in cell suspensions was 70-80% of that of slices, whereas fractionated tissue homogenates had respiratory rates of only one-third those of the cell suspensions. Lactate dehydrogenase content of cell suspensions was maintained during gradient centrifugation and washing. 5. The possible uses of isolated cell preparations are discussed.     

Growth of enteric neurones from isolated myenteric ganglia in dissociated cell culture

Summary Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods. The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro. The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro. Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum-supplemented medium without mitotic inhibitors. Glial cell numbers appeared to decline in serum-free medium after the first week in vitro. Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media. This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall.     

K+--induced changes of oxygen uptake by neuronal enriched and glial enriched fractions from mouse brain cortex.

Neuronal and glial enriched fractions were incubated in a medium with 10mM pyruvate, 5mM fumarate and 0.9mM 5'-AMP and the effect of increased external K+ concentrations was studied upon oxygen uptake. A concentration of 65 mM K+ had a different effect on the oxygen consumption of glial and neuronal perikarya. The rate of oxygen uptake by glia was stimulated by 52.81% whilst an insignificant decrease of 15.79% occurred in the neurones. The highest rate of oxygen uptake by incubated cells was estimated in the presence of the substrate system containing pyruvate, fumarate and 5'-AMP. The significance of components in the substrate system for a high rate of oxygen uptake by cells was also tested with 6.2 mM K+ and 65 mM K+.     

Modulation of Mn2+ accumulation in cultured rat neuronal and astroglial cells

The effects of physiological concentrations of K⁺ on Mn²⁺ accumulation were compared in rat glial cells and neurons in culture. Increasing the K⁺ concentration in growth medium increased significantly the Mn²⁺ level of the cultivated cells, with glial cells more affected than neurons. Ethanol markedly increased the Mn²⁺ accumulation within glia but not within neurons while ouabaïn caused inhibition of Mn²⁺ uptake with neurons and glial cells. A modulation of the total protein synthesis by Mn²⁺ and ethanol level in the growth medium was observed with glial cells. These data suggest that the mechanisms involved in Mn²⁺ accumulation in glial cells are different from those present in neurons. Moreover, the results are consistent with the hypothesis that Mn²⁺ plays a regulatory role in glial cell metabolism.     

EXPERIMENTALLY INDUCED CHANGES IN THE BASE COMPOSITION OF THE RIBONUCLEIC ACIDS OF ISOLATED NERVE CELLS AND THEIR OLIGODENDROGLIAL CELLS

The effect of tricyano-amino-propene, a dimer of malononitrile, on the base composition of the RNA in isolated Deiters' nerve cells and their oligodendroglial cells has been studied using a microelectrophoretic method. Tri-a-p in a dose of 20 mg/kg has the effect of increasing the RNA and protein content per nerve cell by 25 per cent and decreasing the glia RNA by 45 per cent. The RNA base composition of the nerve cells from the control animals differs from that of their glial cells. The guanine of the nerve cell is significantly higher than that of the glia, but the content of cytosine is higher in the glia than in the RNA of nerve cell. The cytosine of nerve cells decreased significantly after tri-a-p administration. In the glial cells the cytosine showed a 20 per cent increase, and the guanine a 25 per cent decrease. Tri-a-p sharpened the difference in RNA composition already existing between the control nerve cells and their glial cells by almost 300 per cent for the guanine and by 400 per cent for the cytosine. The chemical and functional relationship between the nerve cell and its oligodendroglial cells is discussed.     

NEURONAL AND GLIAL SYSTEMS FOR γ-AMINOBUTYRIC ACID TRANSPORT

—The uptake of [2,3-³H]γ-aminobutyric acid (GABA) by bulk prepared neuronal perikarya, nerve endings and glial cells has been studied in an in vitro-system. The uptake in the different fractions had a similar dependence for sodium, potassium and magnesium. Calcium stimulated the synaptosomal GABA uptake at concentrations which inhibited the glial uptake. Bicuculline strongly inhibited the uptake in all fractions. Picrotoxin and strychnine had little effect on the neuronal uptake whereas glial uptake was stimulated. l -2,4-di-aminobutyric acid and chlorpromazine inhibited GABA uptake in all fractions. The effect of cyclic AMP was not significant.     

AMINO ACID INCORPORATION IN VITRO INTO PROTEIN OF NEURONAL AND GLIAL CELL-ENRICHED FRACTIONS

Slices of rabbit cerebral cortex were incubated in the presence of labelled amino acids. Following incubation, neuron- and gliaenriched fractions were obtained by density gradient centrifugation and the TCA-insoluble radioactivity determined. The protein-bound radioactivity was five to six times higher in the neuronal-enriched fraction than in the glial-enriched fraction after incubation with tritiated leucine. The neuronal fraction incorporated also a number of other amino acids to a higher extent than the glial fraction (neuron/glia ratio 2·5-6). A definite dependence of incorporation on the rate of oxygenation was demonstrated. The suppression of amino acid incorporation was more marked for the neuronal fraction than for the glial fraction during incubation in relative hypoxia. An increase of potassium concentration in the incubation medium enhanced the amino acid incorporation in both fractions. Low sodium levels decreased the incorporation. Puromycin inhibited incorporation to approximately 30 per cent of control for both fractions. Addition of cycloheximide and dinitrophenol resulted in greater inhibition of incorporation in the neuronal fraction than in the neuroglial fraction. Actinomycin D did not markedly affect the incorporation in any fraction. These results are discussed in relation to in vivo and in in vitro differences for transport and incorporation of amino acids.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

Bidirectional Movement of γ-Aminobutyric Acid in Rat Spinal Cord Slices

Abstract: The bidirectional movement of GABA (γ-aminobutyric acid) was studied in slices of rat spinal cord which were incubated in small volumes of medium. The appearance in the medium of endogenous GABA and the disappearance from the medium of [¹⁴C]GABA were used to calculate the rates of unidirectional uptake and unidirectional release of GABA. Under these conditions, no net uptake of GABA was observed when slices were incubated in media containing concentrations of GABA as high as 25 μm . Elevated potassium (60 mm ) stimulated the unidirectional release of endogenous GABA from spinal cord slices by a calcium-dependent process. Ouabain (0.1 mm ) more than doubled the unidirectional release of endogenous GABA in a calcium-independent manner, while unidirectional uptake was inhibited by 44%. Nipecotic acid (1.0 mm ) stimulated unidirectional release and inhibited unidirectional uptake of GABA.     

Glia–Neuron Interactions in the Sensory-Motor Cortex of Warm-Blooded Animals (Guinea Pigs and Ground Squirrels) with Different Habitat Conditions and the M-Cholinergic Reaction of the Brain

The glia–neuron interactions were analyzed in the sensory-motor cortex of guinea pigs and ground squirrels (Spermophilus undulatus) during the active summer months. The glial cells were more concentrated in close proximity (15–25 μm) to neurons (38% in guinea pigs and 22.4% in ground squirrels). A more concentrated distribution of glial cells might be very necessary for spontaneous inactive nerve cells (37.2% in guinea pigs and 23% in ground squirrels), since these neurons are associated with the highest energy demand during their functioning and are most susceptible to disturbances of ion homeostasis. The network structure of glia and the close contact between glial cells and brain capillaries provide additional energy for neurons and stabilize the ion balance in the extracellular medium. Glial density in the sensory-motor cortex of ground squirrels is 3 times higher than that in the cortex of guinea pigs. The high content of glial cells in the ground-squirrel cortex is the most important protective factor for survival of animals during long-term hibernation, when the diffusion of K⁺ ions from nerve cells drastically increases due to the high temperature sensitivity of the M-cholinergic response.     

Control of peripheral glial cell proliferation: enteric neurons exert an inhibitory influence on Schwann cell and enteric glial cell DNA synthesis in culture

Neuronal membranes from rat dorsal root ganglia provide a mitogenic signal to cultured Schwann cells and it has been suggested this is an important factor in regulating Schwann cell numbers during development. In this study, the influence of enteric neurons on the DNA synthesis of both Schwann cells and enteric glia has been investigated as well as the effect of axonal membrane fractions (axolemma) on enteric glia. The proliferation rate of rat Schwann cells and enteric glia was assessed in culture using [3H]thymidine uptake and autoradiography in combination with immunolabelling to identify cell types. When purified rat Schwann cells were co-cultured with guinea pig enteric neurons, their DNA synthesis rate was reduced compared with control cultures of pure Schwann cells or Schwann cells not close to neurites or neuronal cell bodies. Nevertheless, in accordance with previous findings that sensory neurons stimulate Schwann cell division, these Schwann cells increased their DNA synthesis rate when in contact with neurites from purified guinea pig or adult rat dorsal root ganglion neurons and on exposure to bovine axolemmal fractions. The enteric neurons also suppressed the DNA synthesis of enteric glia in co-cultures of purified enteric neurons and enteric glia, while bovine axolemma stimulated their DNA synthesis. These results indicate that a mitotic inhibitory signal is associated with enteric neurons and can exert its effect on both Schwann cells and enteric glia, and that enteric glia, like Schwann cells, are stimulated to divide by axolemmal fractions. It thus seems possible that during development glial cell numbers in the peripheral nervous system may be controlled by both positive and negative regulators of cell growth.     

Potassium uptake by the dog erythrocyte

The inward transport of potassium by separated dog erythrocytes has been studied at concentrations of potassium in the medium from 2.9 to 25.0 m.eq./liter and at 38.0 and 33.0 degrees C. At the physiological concentration of external potassium (4.06 m.eq./liter medium), the inward potassium flux is 0.11 m.eq./liter cells hour and the glucose consumption is 2.0 mM/liter cells hour. The dependence of potassium influx on extracellular potassium concentration is given by the following equation, K influx (m.eq./liter cells hour) = 0.028 [K](amb.) - 0.003 in which [K](amb.) refers to the potassium concentration in the medium. In a single 93 hour experiment, 94 per cent of the intracellular potassium was exchanged at an apparently uniform rate. The average apparent activation energy for the process is 7,750 calories +/- 2,000 calories/mol and there is some indication that the apparent activation energy of inward K transport decreases with increasing external K concentration.     

Comparison of α1-Adrenergic Receptor-Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures

alpha 1-Adrenergic receptor binding sites and norepinephrine-stimulated 3H-inositol phosphate (3H-InsP) accumulation were measured in primary cultures of neurons and glia from 1-day-old rat brains. The density of alpha 1-adrenergic receptor binding sites was approximately three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H-InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine-stimulated 3H-InsP formation remained constant for [3H]inositol prelabelling periods of 1-14 days in neurons, whereas the response increased with time in glia and was maximal after 7-10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H-InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pretreatment with phenoxybenzamine decreased norepinephrine-stimulated 3H-InsP formation in a dose-dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H-InsPs were accumulated in neurons and glial cells. These results demonstrate that alpha 1-adrenergic receptors exist on both neurons and glial cells and activate 3H-InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H-InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.     

RESPIRATORY ACTIVITY OF GUINEA PIG BRAIN NUCLEI

Abstract— Preparations of guinea pig brain nuclei, obtained by discontinuous gradient centrifugation in sucrose solutions of pH 6.7–6.8, containing 3 mM-MgCl₂ and phosphate exhibited steady and reproducible oxygen uptake. Oxygen uptake was stimulated 60–70 per cent by glucose, pyruvate, oxalacetate or α-ketoglutarate and 267 per cent by succinate. This respiratory activity was unaffected by the relative sodium or potassium ion content of the medium and by variations in the concentration of inorganic phosphate. Agents known to inhibit citric acid cycle oxidation, oxidative phosphorylation and glycolysis diminished oxygen uptake, but antibiotics inhibiting nucleic acid or protein synthesis did not. Treatment of the nuclear preparation with DNase decreased respiratory capacity, which was partially restored by the addition of polyacrylic acid.