Epigenetic regulation of TLR2-mediated periapical inflammation.

AIM: To determine the methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis. METHODOLOGY: In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n = 17) and symptomatic apical periodontitis (SAP) (n = 17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P < 0.05). RESULTS: TLR2 depicted a hypomethylated DNA profile at the CpG island in SAP when compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P < 0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P < 0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P < 0.05). CONCLUSIONS: TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.

Gene expression in superior temporal cortex of schizophrenia patients.

We investigated gene expression pattern obtained from microarray data of 10 schizophrenia patients and 10 control subjects. Brain tissue samples were obtained postmortem; thus, the different ages of the patients at death also allowed a study of the dynamic behavior of the expression patterns over a time frame of many years. We used statistical tests and dimensionality reduction methods to characterize the subset of genes differentially expressed in the two groups. A set of 10 genes were significantly downregulated, and a larger set of 40 genes were upregulated in the schizophrenia patients. Interestingly, the set of upregulated genes includes a large number of genes associated with gene transcription (zinc finger proteins and histone methylation) and apoptosis. We furthermore identified genes with a significant trend correlating with age in the control (MLL3) or the schizophrenia group (SOX5, CTRL). Assessments of correlations of other genes with the disorder (RRM1) or with the duration of medication could not be resolved, because all patients were medicated. This hypothesis-free approach uncovered a series of genes differentially expressed in schizophrenia that belong to a number of distinct cell functions, such as apoptosis, transcriptional regulation, cell motility, energy metabolism and hypoxia.

Circadian rhythms investigated on the cellular and molecular levels.

Investigations on circadian rhythms have markedly advanced our understanding of health and disease with the advent of high-throughput technologies like microarrays and epigenetic profiling. They elucidated the multi-level behaviour of interactive oscillations from molecules to neuronal networks and eventually to processes of learning and memory in an impressive manner. The small-world topology of synchronized firing through neuron-neuron and neuron-glia gap junctions is discussed as a mathematical approach to these intensively studied issues. It has become evident that, apart from some disorders caused by gene mutations, the majority of disorders originating from disturbances of rhythms arise from environmental influences and epigenetic changes. In this context, it was mandatory to think of and devise experiments on temporary scales, which exponentially increased the volumes of data obtained from time-series and rapidly became prohibitive of manual inspection. Therefore, more and more sophisticated mathematical algorithms have been developed to identify rhythmic expression of genes and to find coexpression by their clustering. It is expected that disturbed rhythmic behaviour in mental disorders is reflected in altered oscillatory behaviour of gene expression.

Epigenetics of schizophrenia.

Investigations on gene variants as milestones in the development of schizophrenia have not fulfilled the enormous, initial expectations. Neither candidate gene approaches trying to associate single genes with the disorder, nor genome-wide association studies (GWAS), that have been welcomed more recently with great enthusiasm, could end the general disappointment associated with these strategies. Owing to very large numbers of samples and most advanced sequencing technologies, some variants have been found but their effects, even in combination are very small. In summary, most of the tentative heritability of schizophrenia remains unexplained. More hope to find mechanisms connecting genes with the disorder lies in analyses of the epigenome with technologies developed during the last 10 or 15 years and undergoing more and more refinement recently. Although investigations on interactions between DNA methylation patterns and histone modifications will probably be the greatest challenge in molecular genetics for the next decades, they appear to be the most promising approaches on complex brain disorders that typically show a high dependence on environmental factors.

Fluoxetine treatment induces EAAT2 expression in rat brain.

Synaptic pathology and disturbed glutamatergic neurotransmission contribute to the neurobiology of depression. Reduced expression of glutamate transporters, most importantly excitatory amino acid transporter (EAAT2), was reported in human studies and animal models. We therefore assessed the effects of antidepressant treatment upon EAAT2 expression. Male Sprague-Dawley rats received daily intraperitoneal injections of the antidepressants desipramine (DES, N = 7), fluoxetine (FLU, N = 7), tranylcypromine (TRAN, N = 5) or a saline control (CON, N = 5) for a period of 14 days. The expression of the major glial glutamate transporter EAAT2 was evaluated by semi-quantitative in situ hybridizations using a (35)S-labeled cRNA probe. Treatment with FLU significantly induced EAAT2 expression in hippocampal and cortical regions in comparison with saline injections, while DES and TRAN-applications did not exert significant effects. It can be postulated that increased expression of EAAT2 may counterbalance the tonus of glutamatergic neurotransmission. Our findings are in concert with human post-mortem findings, valid animal models of depression, antidepressive effects of NMDA-antagonists, and the glutamatergic theory of depression. Further studies should examine the effects of antidepressant treatments upon EAAT2 expression in rodent models of depression to further elucidate the underlying molecular mechanisms.

Affective disorders as complex dynamic diseases--a perspective from systems biology.

Understanding mental disorders and their neurobiological basis encompasses the conceptual management of "complexity" and "dynamics". For example, affective disorders exhibit several fluctuating state variables on psychological and biological levels and data collected of these systems levels suggest quasi-chaotic periodicity leading to use concepts and tools of the mathematics of nonlinear dynamic systems. Regarding this, we demonstrate that the concept of "Dynamic Diseases" could be a fruitful way for theory and empirical research in neuropsychiatry. In a first step, as an example, we focus on the analysis of dynamic cortisol regulation that is important for understanding depressive disorders. In this case, our message is that extremely complex phenomena of a disease may be explained as resulting from perplexingly simple nonlinear interactions of a very small number of variables. Additionally, we propose that and how widely used complex circuit diagrams representing the macroanatomic structures and connectivities of the brain involved in major depression or other mental disorders may be "animated" by quantification, even by using expert-based estimations (dummy variables). This method of modeling allows to develop exploratory computer-based numerical models that encompass the option to explore the system by computer simulations (in-silico experiments). Also inter- and intracellular molecular networks involved in affective disorders could be modeled by this procedure. We want to stimulate future research in this theoretical context.

Differential expression of presynaptic genes in a rat model of postnatal hypoxia: relevance to schizophrenia.

Obstetric complications play a role in the pathophysiology of schizophrenia. However, the biological consequences during neurodevelopment until adulthood are unknown. Microarrays have been used for expression profiling in four brain regions of a rat model of neonatal hypoxia as a common factor of obstetric complications. Animals were repeatedly exposed to chronic hypoxia from postnatal (PD) day 4 through day 8 and killed at the age of 150 days. Additional groups of rats were treated with clozapine from PD 120-150. Self-spotted chips containing 340 cDNAs related to the glutamate system ("glutamate chips") were used. The data show differential (up and down) regulations of numerous genes in frontal (FR), temporal (TE) and parietal cortex (PAR), and in caudate putamen (CPU), but evidently many more genes are upregulated in frontal and temporal cortex, whereas in parietal cortex the majority of genes are downregulated. Because of their primary presynaptic occurrence, five differentially expressed genes (CPX1, NPY, NRXN1, SNAP-25, and STX1A) have been selected for comparisons with clozapine-treated animals by qRT-PCR. Complexin 1 is upregulated in FR and TE cortex but unchanged in PAR by hypoxic treatment. Clozapine downregulates it in FR but upregulates it in PAR cortex. Similarly, syntaxin 1A was upregulated in FR, but downregulated in TE and unchanged in PAR cortex, whereas clozapine downregulated it in FR but upregulated it in PAR cortex. Hence, hypoxia alters gene expression regionally specific, which is in agreement with reports on differentially expressed presynaptic genes in schizophrenia. Chronic clozapine treatment may contribute to normalize synaptic connectivity.

Expression of apolipoprotein E by cultured vascular smooth muscle cells is controlled by growth state.

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism.

Reduced expression of GABA transporter GAT3 in helpless rats, an animal model of depression.

Mood disorders have been linked to glial and synaptic pathology such as disturbed neurotransmission of gamma-aminobutyric acid (GABA). We evaluated the expression of GABAergic marker genes in rats with helpless behaviour, an animal model of depression. Male Sprague-Dawley rats from inbred lines were tested for helpless behaviour and grouped according to failures in terminating foot shock currents. Expression levels of GABAergic marker genes were assessed using semiquantitative in situ-hybridization. Animals with congenital helpless behaviour (cH) were unable to escape current exposure in contrast to cH-animals derived from the same litters with low failure rates and to non-helpless animals (cNH). We found a significant downregulation of the GABA transporter GAT3 in cLH rats. GAT1 showed small changes, glutamic acid decarboxylase (GAD67) and the vesicular GABA transporter were not significantly altered. Reduced GABA transporter expression is well in concert with the behavioural phenotypes of knockout animals and strengthens the hypothesis of impaired glial functions in depression.

Philosophy of neuroscience and options of systems science.

Molecular biology as a research approach in psychiatry has gathered a huge amount of data that can hardly be used for explanation of mental disorders by cellular dysfunctions. In a philosophical sense "explanation" means the application of general laws on specific cases. This is more than description. Most findings of molecular biology only help to describe these processes more in detail. On contrary, systems biology aims to create a computer-based model of the cell. For this project mathematics plays a crucial role. In that respect systems biology also provides tools for data analysis.

Systems biology and addiction.

The onset of addiction is marked with drug induced positive experiences that keep being repeated. During that time, adaptation occurs and addiction is stabilized. Interruption of those processes induces polysymptomatic withdrawal syndromes. Abstinence is accompanied by risks of relapse. These features of addiction suggest adaptive brain dynamics with common pathways in complex neuronal networks. Addiction research has used animal models, where some of those phenomena could be reproduced, to find correlates of addictive behavior. The major thrust of those approaches has been on the involvement of genes and proteins. Recently, an enormous amount of data has been obtained by high throughput technologies in these fields. Therefore, (Computational) "Systems Biology" had to be implemented as a new approach in molecular biology and biochemistry. Conceptually, Systems Biology can be understood as a field of theoretical biology that tries to identify patterns in complex data sets and that reconstructs the cell and cellular networks as complex dynamic, self-organizing systems. This approach is embedded in systems science as an interdisciplinary effort to understand complex dynamical systems and belongs to the field of theoretical neuroscience (Computational Neuroscience). Systems biology, in a similar way as computational neuroscience is based on applied mathematics, computer-based computation and experimental simulation. In terms of addiction research, building up "computational molecular systems biology of the (addicted) neuron" could provide a better molecular biological understanding of addiction on the cellular and network level. Some key issues are addressed in this article.

Interactive molecular networks obtained by computer-aided conversion of microarray data from brains of alcohol-drinking rats.

Lists of differentially expressed genes in a disease have become increasingly more comprehensive with improvements on all technical levels. Despite statistical cutoffs of 99% or 95% confidence intervals, the number of genes can rise to several hundreds or even thousands, which is barely amenable to a researcher's understanding. This report describes some ways of processing those data by mathematical algorithms. Gene lists obtained from 53 microarrays (two brain regions (amygdala and caudate putamen), three rat strains drinking alcohol or being abstinent) have been used. They resulted from analyses on Affymetrix chips and encompassed approximately 6 000 genes that passed our quality filters. They have been subjected to four mathematical ways of processing: (a) basic statistics, (b) principal component analysis, (c) hierarchical clustering, and (d) introduction into Bayesian networks. It turns out, by using the p-values or the log-ratios, that they best subdivide into brain areas, followed by a fairly good discrimination into the rat strains and the least good discrimination into alcohol-drinking vs. abstinent. Nevertheless, despite the fact that the relation to alcohol-drinking was the weakest signal, attempts have been made to integrate the genes related to alcohol-drinking into Bayesian networks to learn more about their inter-relationships. The study shows, that the tools employed here are extremely useful for (a) quality control of datasets, (b) for constructing interactive (molecular) networks, but (c) have limitations in integration of larger numbers into the networks. The study also shows that it is often pivotal to balance out the number of experimental conditions with the number of animals.

A role for apolipoprotein E, apolipoprotein A-I, and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve.

Recent work has demonstrated that apo E secretion and accumulation increase in the regenerating peripheral nerve. The fact that apoE, in conjunction with apoA-I and LDL receptors, participates in a well-established lipid transfer system raised the possibility that apoE is also involved in lipid transport in the injured nerve. In the present study of the crushed rat sciatic nerve, a combination of techniques was used to trace the cellular associations of apoE, apoA-I, and the LDL receptor during nerve repair and to determine the distribution of lipid at each stage. After a crush injury, as axons died and Schwann cells reabsorbed myelin, resident and monocyte-derived macrophages produced large quantities of apoE distal to the injury site. As axons regenerated in the first week, their tips contained a high concentration of LDL receptors. After axon regeneration, apoE and apoA-I began to accumulate distal to the injury site and macrophages became increasingly cholesterol-loaded. As remyelination began in the second and third weeks after injury, Schwann cells exhausted their cholesterol stores, then displayed increased LDL receptors. Depletion of macrophage cholesterol stores followed over the next several weeks. During this stage of regeneration, apoE and apoA-I were present in the extracellular matrix as components of cholesterol-rich lipoproteins. Our results demonstrate that the regenerating peripheral nerve possesses the components of a cholesterol transfer mechanism, and the sequence of events suggests that this mechanism supplies the cholesterol required for rapid membrane biogenesis during axon regeneration and remyelination.

Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages.

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E2 but did not affect the zymosan-induced release of arachidonic acid, PGD2, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A2, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.

Differential expression, activity and regulation of the sodium/myo-inositol cotransporter in astrocyte cultures from different regions of the rat brain.

The high-affinity sodium/myo-inositol cotransporter (SMIT) is involved in osmoregulation in several cells and tissues. In the CNS the activity of SMIT also determines the individual susceptibility of neural cells to the inositol depleting effect of lithium, which is considered to be important in lithium's therapeutic effects in manic-depressive illness. Among neural cells SMIT is particularly active in astrocytes. In the present work we have cloned the cDNA of SMIT of the rat and assessed its activity, expression and regulation in primary astroglia cultures derived from five different rat brain regions: cerebellum, cortex, diencephalon, hippocampus and tegmentum. After an incubation period of 24 h in medium containing 3[H]labeled myo-inositol different steady-state concentrations were detected which were dependent on the brain region from which the astrocytes were cultured. In addition, myo-inositol uptake in astrocytes from different areas was characterized by two different Km values (27 microM for cerebellum and diencephalon, 50 microM for cortex, hippocampus and tegmentum) and by three different v(max) values (approx. 200 pmol/mg protein/min for astrocytes from cerebellum and tegmentum, 298 for hippocampus and 465 for cortex), indicating that the active myo-inositol uptake into astroglial cells is distinct in the various brain regions. The efficacy of uptake as determined by v(max) values of 3[H]myo-inositol uptake correlated with the level of mRNA of SMIT in the astrocyte cultures from the various brain regions as determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Both 3[H]myo-inositol uptake and SMIT mRNA content was upregulated by incubation of astrocytes in medium of increased osmolarity. In astrocytes from cerebellum, cortex, hippocampus and tegmentum 3[H]myo-inositol uptake was downregulated by chronic incubation with 400 microM inositol. This effect was not observed in astrocytes from diencephalon. Furthermore, in astrocytes from cortex and hippocampus but not from cerebellum, diencephalon and tegmentum incubation with corticosterone for three days upregulated 3[H]myo-inositol uptake. It is concluded that SMIT is differentially expressed and regulated in astrocytes from distinct brain regions. These regional differences suggest particular consideration of localized effects in investigations of the role of myo-inositol in the mechanism of action of antibipolar drugs.

Expression of interleukin-3 and tumor necrosis factor-beta mRNAs in cultured microglia.

The function of interleukin-3 (or multi-CSF) in the hemopoietic system has been studied in great detail. Although its growth promoting activity on brain microglial cells has been confirmed both in vitro and in vivo, its presence in the brain and even in cultured brain cells has repeatedly been questioned. We have shown recently that isolated rat microglia express mRNA(IL-3) and synthesize IL-3 polypeptide. It is shown here by use of the PCR method, that mRNA(IL-3) is found also in C6 glioblastoma, in rat aggregate cultures, and in newborn and adult rat brain. Quantitation of amplified cDNA(IL-3) was achieved by non-competitive RT-PCR using an elongated internal standard. IL-3 messenger RNA was almost undetectable in vivo and low in (serum-free) aggregate cultures. In isolated microglia, mRNA(IL-3) was increased upon treatment with LPS, PHA, with the cytokines IL-1 or TNF-alpha, with retinoic acid, dbcAMP or the phorbol ester TPA. Effects of LPS were inhibited by dexamethasone, while the glucocorticoid by itself had no effect on basal IL-3 expression. LPS increased mRNA(IL-3) in a concentration-dependent manner beginning with 10 pg/ml and reaching plateau levels at 10 ng/ml. LPS also increased mRNAs of TNF-alpha and TNF-beta. TNF-alpha mRNA was already detectable in untreated microglia and LPS-increased levels were sustained for a few days. In contrast, TNF-beta mRNA was observed only between 4 and 16 h of LPS incubation. It was absent in LPS-free microglia, and after 24 h of LPS-treatment or later.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C-mediated regulation of inducible nitric oxide synthase expression in cultured microglial cells.

Nitric oxide (NO) has been implicated in a number of important brain functions, such as long-term potentiation (LTP) and long-term depression (LTD), and in events associated with neurodegeneration and neuroprotection. In response to brain injury or disease NO production is increased by an inducible enzyme (iNOS), which is only expressed under these conditions. Activated microglia are a major cellular source of iNOS in brain. Due to the important role of iNOS in brain injury and disease, a detailed understanding of intracellular events triggering the expression of iNOS in microglia would facilitate pharmacotherapeutic approaches. It is shown here, that iNOS mRNA, protein and NO product are induced in cultured microglia by lipopolysaccharide (LPS). This induction is reduced by a number of substances elevating intracellular cyclic AMP levels. It is unabated, however, in the presence of substances inhibiting cyclooxygenase-1 and/or cyclooxygenase-2 (e.g., acetyl salicylic acid, SC 58125, L 745337), but is decreased by approx. 50% with PDTC, a scavenger of reactive oxygen intermediates (ROI) that inhibits nuclear factor kappaB (NF-kappaB) activation. Furthermore, inhibitors of protein kinase C (PKC) strongly inhibit iNOS mRNA and protein induction. PKC, therefore, constitutes a major second messenger component (besides NF-kappaB) in the signaling pathway regulating iNOS expression in microglia.

Interleukin-4-inhibited mRNA expression in mixed rat glial and in isolated microglial cultures.

Interleukin-4 (IL-4) likely is one of the key players in the concert of immunosuppressive factors in brain. Therefore, influences of the cytokine on mRNA expression of endogenous mediators of inflammation, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interferon-inducible protein 10 (IP-10), interleukin-3 receptor-beta (IL-3R-beta), and of another antiinflammatory cytokine, IL-10, have been evaluated in the present study by semi-quantitative RT-PCR. Primary rat mixed glial cultures and isolated microglial cells, the resident immunocytes of the brain, have been used as rich sources of these mRNAs in response to the bacterial cell wall component lipopolysaccharide (LPS). Time-course studies showed peak levels of LPS-increased mRNAs at approximately 4 h. Interestingly, IL-10 mRNA was elevated also upon the LPS-stimulus. IL-4, given 30 min before LPS, inhibited increases of all mRNAs significantly, including IL-10 mRNA. IL-4, however, induced peroxisome proliferator-activated receptor (PPAR)-gamma in cultured microglia. This induction was completely inhibited by simultaneous administration of LPS. The data confirms IL-4 as an important antiinflammatory cytokine and gives some idea of cross-talk between intracellular signaling evoked by pro- and antiinflammatory substances.

Rat microglial interleukin-3.

Interleukin-3 (IL-3, multi-CSF) is a growth factor for a variety of hematopoietic progenitor cells. Recently, microglial cells, the resident macrophages of the central nervous system (CNS) have been shown to proliferate in the presence of IL-3 both in vivo and in culture. Data obtained from cultured astrocytes gave rise to the hypothesis that astrocytes synthesize the microglial growth factor. This is the first report identifying rat microglial cells themselves as a source of IL-3. Culture media conditioned by isolated microglia enhanced microglial proliferation above fresh media controls. IL-3 polypeptide was detected in both conditioned media (CM) and in microglial cells by Western blotting and immunoprecipitation. Furthermore, anti-IL-3 antibodies were able to inhibit microglial proliferation induced by conditioned media. mRNAIL-3 was present in single microglial cells as revealed by in situ hybridization. Total RNA prepared from purified microglia yielded a single PCR amplification product. Identity of the PCR product was confirmed by Southern blot hybridization using a cDNAIL-3 probe and by DNA sequencing. Expression of mRNAIL-3 was observed in both absence and presence of lipopolysaccharide, a bacterial endotoxin, that commonly induces expression of inflammatory cytokines and inhibits microglial proliferation. It is concluded that IL-3 expression in ensuring the recruitment of enhanced numbers of immunocompetent cells at sites of lesion. In the light of weak immune reactions in the brain, it is hypothesized that the expression of a characteristic T cell feature in monocyte-derived microglia may be a partial compensation of T cell functions in brain lesions.

Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation.

The release of ATP was studied in cultures of astrocytes derived from the brain hemispheres of newborn rats. There was a basal efflux of ATP, which was increased up to 19-fold by glutamate (300-1000 microM). N-methyl-D-aspartate (20-500 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 30-100 microM) and kainate (20 microM). The N-methyl-D-aspartate receptor-selective antagonist 2-amino-5-phosphonopentanoate (100 microM) blocked the effect of N-methyl-D-aspartate but not the effects of AMPA, kainate and glutamate. The AMPA receptor-selective antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (30 microM) blocked the effect of AMPA and also of glutamate and N-methyl-D-aspartate, but not the effect of kainate. The kainate receptor-selective antagonist D-glutamyl-amino-methanesulfonate (30 microM) blocked the effect of kainate but not of glutamate. Glutamate (1000 microM) did not increase the release of lactate dehydrogenase from astrocytes. Excitatory amino acids are known to release adenyl compounds in the brain. The present results identify one adenyl compound thus released, namely ATP, and identify astrocytes as one source. The release is brought about by activation of any of the three ionotropic glutamate receptor types-N-methyl-D-aspartate, AMPA and kainate receptors. AMPA receptors seem to mediate at least a part of the effect of glutamate itself, but the involvement of other receptors cannot be ruled out. ATP and its degradation products, such as adenosine, once released, may exert acute as well as trophic effects on neurons and glial cells.