Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures

Kinetics for uptake and release of glutamate were measured in normal, i.e., nontransformed, astrocytes in cultures obtained from the dissociated, cortexenriched superficial parts of the brain hemispheres of newborn DBA mice. The uptake kinetics indicated a minor, unsaturable component together with an intense uptake following Michaelis-Menten kinetics. TheK _m (50 M) was reasonably comparable to the corresponding values in brain slices and in other glial preparations. TheV _max (58.8 nmol min^–1 mg^–1 protein) was, however, much higher than that observed in glial cell lines or peripheral satellite cells, and also considerably higher than that generally reported for brain slices. The release of glutamate was much smaller than the uptake, and only little affected by an increase of the external glutamate concentration, suggesting a net accumulation of glutamate rather than a homoexchange. Such an intense accumulation of glutamate into normal astrocytes may play a major role in brain metabolism and may help keep the extracellular glutamate cohcentration below excitatory levels.     

Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures

Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na⁺/K⁺-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate–glutamine cycle, as well as glutamate and d-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.     

Protective Effects of Resveratrol on Hydrogen Peroxide Induced Toxicity in Primary Cortical Astrocyte Cultures

It is well established that the brain is particularly susceptible to oxidative damage due to its high consumption of oxygen and that astrocytes are involved in a variety of important activities for the nervous system, including a protective role against damage induced by reactive oxygen species (ROS). The use of antioxidant compounds, such as polyphenol resveratrol found in red wine, to improve endogenous antioxidant defenses has been proposed for neural protection. The aim of this study is to evaluate the putative protective effect of resveratrol against acute H₂O₂-induced oxidative stress in astrocyte cultures, evaluating ROS production, glutamate uptake activity, glutathione content and S100B secretion. Our results confirm the ability of resveratrol to counteract oxidative damage caused by H₂O₂, not only by its antioxidant properties, but also through the modulation of important glial functions, particularly improving glutamate uptake activity, increasing glutathione content and stimulating S100B secretion, which all contribute to the functional recovery after brain injury.     

Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity

Huntington disease (HD) is characterized by the preferential loss of striatal medium-sized spiny neurons (MSNs) in the brain. Because MSNs receive abundant glutamatergic input, their vulnerability to excitotoxicity may be largely influenced by the capacity of glial cells to remove extracellular glutamate. However, little is known about the role of glia in HD neuropathology. Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters. As a result, mutant huntingtin (htt) reduces glutamate uptake in cultured astrocytes and HD mouse brains. In a neuron-glia coculture system, wild-type glial cells protected neurons against mutant htt-mediated neurotoxicity, whereas glial cells expressing mutant htt increased neuronal vulnerability. Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity. These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.     

A Culture Model of Reactive Astrocytes: Increased Nerve Growth Factor Synthesis and Reexpression of Cytokine Responsiveness

Abstract: Reactive gliosis, which occurs in response to damage to the central nervous system, has been recognized for years but is not yet understood. We describe here a tissue culture model of reactive astrocytes used to characterize their properties. Cultures are prepared 1 week following 6-hydroxydopamine (6-OHDA) lesion of rat substantia nigra and compared with astrocytes cultured from normal adult rats or rats injected with saline only. Astrocytes from the 6-OHDA-lesioned side contained elevated levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA and were intensely immunoreactive for GFAP, vimentin, and two epitopes that in vivo are found only on reactive astrocytes. The basal content of nerve growth factor (NGF) mRNA and NGF in astrocytes from 6-OHDA-lesioned rats was significantly higher relative to control astrocytes. Two inflammatory cytokines, interleukin-1β and interferon-γ, increased synthesis of NGF up to 20-fold in the reactive cells, whereas there was no response in the normal adult astrocytes. Astrocytes from postnatal day 2 rats shared many of the properties of the reactive adult astrocytes. These cultures offer the possibility to characterize the cellular and molecular properties of reactive astrocytes and to determine the factors responsible for activation of astrocytes.     

Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

Regeneration of injured adult CNS axons is inhibited by formation of a glial scar. Immature astrocytes are able to support robust neurite outgrowth and reduce scarring, therefore, we tested whether these cells would have this effect if transplanted into brain injuries. Utilizing an in vitro spot gradient model that recreates the strongly inhibitory proteoglycan environment of the glial scar we found that, alone, immature, but not mature, astrocytes had a limited ability to form bridges across the most inhibitory outer rim. In turn, the astrocyte bridges could promote adult sensory axon re‐growth across the gradient. The use of selective enzyme inhibitors revealed that MMP‐2 enables immature astrocytes to cross the proteoglycan rim. The bridge‐building process and axon regeneration across the immature glial bridges were greatly enhanced by chondroitinase ABC pretreatment of the spots. We used microlesions in the cingulum of the adult rat brains to test the ability of matrix modification and immature astrocytes to form a bridge for axon regeneration in vivo. Injured axons were visualized via p75 immunolabeling and the extent to which these axons regenerated was quantified. Immature astrocytes coinjected with chondroitinase ABC‐induced axonal regeneration beyond the distal edge of the lesion. However, when used alone, neither treatment was capable of promoting axonal regeneration. Our findings indicate that when faced with a minimal lesion, neurons of the basal forebrain can regenerate in the presence of a proper bridge across the lesion and when levels of chondroitin sulfate proteoglycans (CSPGs) in the glial scar are reduced. © 2010 Wiley Periodicals, Inc.Develop Neurobiol 70: 826–841, 2010     

DNase and RNase Responses of Petunia × hybrida During Transition to Flowering as Affected by Light Treatments

The responses of DNase and RNase isoforms and their specific activities following transition to flowering (1 to 6 weeks) were examined in Petunia × hybrida under different light conditions. Petunia × hybrida plants formed flower buds at the 4^th week in the case of high light and at the 6^th week in the far-red light treatment, while no flower bud formation was observed upon red light and control light treatments. The DNase and RNase activities decreased from the 1^st to the 6^th week during transition to flowering. Native-PAGE analysis revealed the appearance of one DNase (D₁) and seven RNase (R₁ - R₇) isoforms in all light treatments. It is assumed that the progress of the flowering could be related to the disappearance or reduction of D₁ DNase band intensity and disappearance of R₁, R₂ and R₇ RNase isoforms. Consequently, these isoforms could be used as potent biochemical markers of flower bud formation under light intensity as well as light quality treatments.     

Prostaglandin E2 stimulates glutamate receptor‐dependent astrocyte neuromodulation in cultured hippocampal cells

Recent Ca²⁺ imaging studies in cell culture and in situ have shown that Ca²⁺ elevations in astrocytes stimulate glutamate release and increase neuronal Ca²⁺ levels, and that this astrocyte‐neuron signaling can be stimulated by prostaglandin E₂ (PGE₂). We investigated the electrophysiological consequences of the PGE₂‐mediated astrocyte‐neuron signaling using whole‐cell recordings on cultured rat hippocampal cells. Focal application of PGE₂ to astrocytes evoked a Ca²⁺ elevation in the stimulated cell by mobilizing internal Ca²⁺ stores, which further propagated as a Ca²⁺ wave to neighboring astrocytes. Whole‐cell recordings from neurons revealed that PGE₂ evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca²⁺ wave and was mediated through both N‐methyl‐D ‐aspartate (NMDA) and non‐NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE₂‐evoked Ca²⁺ elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 221–229, 1999     

Expression of fibronectin and laminin by different types of mouse glial cells cultured in a serum-free medium

The expression of fibronectin and laminin by cultured glial cells was studied. The glial culture from neonatal mouse cerebra maintained in a chemically defined, serum-free medium consisted of type-1 astrocytes, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, oligodendrocytes and type-2 astrocytes. Double-labelling immunofluorescent experiments performed using the mixed glial culture indicated that fibronectin and laminin are expressed in different patterns among the glial subtypes. The staining intensities with anti-fibronectin or anti-laminin antibodies decreased in the order: type-1 astrocytes, O-2A progenitor cells and type-2 astrocytes. Both molecules were deposited in a fibrillar matrix underneath type-1 astrocytes, whereas only intracytoplasmic localization of these molecules was observed with O-2A progenitor cells and type-2 astrocytes. Western blot analysis showed that glial fibronectin has a slightly higher molecular weight than mouse plasma fibronectin (230 kDa) and that glial laminin is a variant with a 220 kDa B chain present and the 400 kDa A chain missing. Using enzyme-linked immunosorbent assays (ELISA), these molecules were detected in the glial extracellular matrix at the concentration of 4 ng/10⁶ cells. A large amount of fibronectin (82 ng/10⁶ cells) was secreted into the culture medium, while secretion of laminin was not detected.     

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate–aspartate-shuttle (MAS) activity to regenerate NAD⁺ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD⁺ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation.     

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate–aspartate-shuttle (MAS) activity to regenerate NAD⁺ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD⁺ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation.     

α-Ketoisocaproate Alters the Production of Both Lactate and Aspartate from [U-13C]Glutamate in Astrocytes: A 13C NMR Study

Abstract: The present study determined the metabolic fate of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mMα-ketoisocaproate (α-KIC). When astrocytes were incubated with 0.2 mM [U-¹³C]glutamate, 64.1% of the ¹³C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [¹³C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [¹³C]glutamate into aspartate in astrocytes incubated in the presence of α-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [¹³C]glutamate into the 1,2,3-¹³C₃-isotopomer of lactate in cells incubated in the presence of α-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of α-KIC. Altogether more of the [¹³C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of α-KIC than in control cells. Overall, the results demonstrate that the presence of α-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.     

PHOSPHOLTPID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELLS FROM BRAINS OF 17-DAY OLD RATS1

Neuronal and glial cells were isolated from the brains of 17-day old rats and incubated for 5 h with either radioactive inorganic phosphate, palmitate, serine, choline or ethanolamine in a tissue culture medium. A comparison of the results suggests that both neuronal and glial cells exhibit effective de novo, phospholipid synthesis and that the observed differences in the uptake are due more to quantitative rather than qualitative differences in phospholipid metabolism of both cell types. Incubations of the combined neuronal and glial fractions with ³²PO₄ and [³H]palmitate result in incorporations up to 100% higher than calculated from incubations of the separate fractions, suggesting that phospholipid metabolism of neuronal and glial cells may exhibit cooperativity.     

Cerebral Aspartate Utilization: Near-Equilibrium Relationships in Aspartate Aminotransferase Reaction

Abstract: The pathways of nitrogen transfer from 50 μM [¹⁵N]aspartate were studied in rat brain synaptosomes and cultured primary rat astrocytes by using gas chromatography-mass spectrometry technique. Aspartate was taken up rapidly by both preparations, but the rates of transport were faster in astrocytes than in synaptosomes. In synaptosomes, ¹⁵N was incorporated predominantly into glutamate, whereas in glial cells, glutamine and other ¹⁵N-amino acids were also produced. In both preparations, the initial rate of N transfer from aspartate to glutamate was within a factor of 2-3 of that in the opposite direction. The rates of transamination were greater in synaptosomes than in astrocytes. Omission of glucose increased the formation of [¹⁵N]-glutamate in synaptosomes, but not in astrocytes. Rotenone substantially decreased the rate of transamination. There was no detectable incorporation of ¹⁵N from labeled aspartate to 6-amino-¹⁵N-labeled adenine nucleotides during 60-min incubation of synaptosomes under a variety of conditions; however, such activity could be demonstrated in glial cells. The formation of ¹⁵N-labeled adenine nucleotides was marginally increased by the presence of 1 mM aminooxyacetate, but was unaffected by pretreatment with 1 mM 5-amino-4-imidazolecarboxamide ribose. It is concluded that (1) aspartate aminotransferase is near equilibrium in both synaptosomes and astrocytes under cellular conditions, but the rates of transamination are faster in the nerve endings; (2) in the absence of glucose, use of amino acids for the purpose of energy production increases in synaptosomes, but may not do so in glial cells because the latter possess larger glycogen stores; and (3) nerve endings have a very limited capacity for salvage of the adenine nucleotides via the purine nucleotide cycle.     

Ginger (Zingiber Officinale)-derived nanoparticles in Schistosoma mansoni infected mice: Hepatoprotective and enhancer of etiological treatment

BackgroundNanotechnology has been manufactured from medicinal plants to develop safe, and effective antischistosmal alternatives to replace today’s therapies. The aim of the study is to evaluate the prophylactic effect of ginger-derived nanoparticles (GNPs), and the therapeutic effect of ginger aqueous extract, and GNPs on Schistosoma mansoni (S. mansoni) infected mice compared to praziquantel (PZQ), and mefloquine (MFQ).Methodology/principal findingsEighty four mice, divided into nine different groups, were sacrificed at 6^th, 8^th, and 10^th week post-infection (PI), with assessment of parasitological, histopathological, and oxidative stress parameters, and scanning the worms by electron microscope. As a prophylactic drug, GNPs showed slight reduction in worm burden, egg density, and granuloma size and number. As a therapeutic drug, GNPs significantly reduced worm burden (59.9%), tissue egg load (64.9%), granuloma size, and number at 10^th week PI, and altered adult worm tegumental architecture, added to antioxidant effect. Interestingly, combination of GNPs with PZQ or MFQ gave almost similar or sometimes better curative effects as obtained with each drug separately. The highest therapeutic effect was obtained when ½ dose GNPs combined with ½ dose MFQ which achieved 100% reduction in both the total worm burden, and ova tissue density as early as the 6^th week PI, with absence of detected eggs or tissue granuloma, and preservation of liver architecture.Conclusions/significanceGNPs have a schistosomicidal, antioxidant, and hepatoprotective role. GNPs have a strong synergistic effect when combined with etiological treatments (PZQ or MFQ), and significantly reduced therapeutic doses by 50%, which may mitigate side effects and resistance to etiological drugs, a hypothesis requiring further research. We recommend extending this study to humans.     

Problems and experience in the light optical and histological presentation of glia cells]

1. 8 histological techniques and 13 modifications derived from those were tested on usefulness for the demonstration of glial cells in the adult rat brain. From these methods the impregnation techniques of Golgi-Kopsch, Valenzuela y Chacón and Rio del Hortega were modified according to a scheme of variance to find out the optimal variants. 2. The impregnation quality depends on the animal species, the animal age, the health of brains, the brain area, the balanced proportion of the treatment stages and the biochemical state of the glial cells. 3. The silver impregnation techniques are not so specific that only one glial type is stained, but one type prevails. The silver carbonate procedure according to Hortega allows to impregnate oligodendrocytes, microglial cells and astrocytes in frozen as well as in paraffin sections. The method of Golgi-Kopsch is more suited for oligodendrocytes and microglial cells than for astrocytes. Following the procedure of Valenzuela y Chacón especially astrocytes, but also microglial cells allow impregnation in both frozen and paraffin sections. 4. The different demonstration qualities of the proved methods call for critical examination of absolute measurements of cell size, length of processes and ramification density. 5. The presence of cell groups of different disposition towards impregnation within a glial type speaks for a biochemical inhomogeneity of the glial types.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

Structural and physiological properties of leech giant glial cells as studied by confocal microscopy

We have studied the architecture of giant neuropile glial cells of the medicinal leech Hirudo medicinalis L. using confocal laser scanning microscopy. We also measured changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by activation of glutamate receptors or voltage-gated Ca²⁺ channels in different glial cell compartments. Glial cells of isolated segmental ganglia were filled iontophoretically with the Ca²⁺ indicator dye Fluo-3. The three-dimensional structure, calculated from serial sections, showed that numerous fine glial branches extend within the whole neuropile, where most of the synapses between neurones are established. Activation of glial glutamate receptors by glutamate or kainate, or depolarizing the cell membrane by elevating the external K⁺ concentration resulted in a transient increase in [Ca²⁺]_i, as measured by Fluo-3 fluorescence. The comparison of [Ca²⁺]_i changes in glial cell branches with changes in the cell body demonstrated that transients in the branches were 2–3 times larger than those in the cell body. The results suggest that glutamate receptors and voltage-gated Ca²⁺ channels are located in the membrane not only of the glial cell body but also of the cellular branches, which may extend close to synaptic domains.     

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.