Thyroid hormone and conditioned medium effects on astroglial cells from hypothyroid and normal rat brain: Factor secretion,cell differentiation,and proliferation

The effects of triiodothyronine (T₃)on cell morphology were examined in cerebral hemisphere and cerebellar astrocyte cultures obtained from normal and hypothyroid neonatal rats. T₃-treatment induced morphological changes in astrocytes from cerebral hemispheres. This morphological effect was produced earlier if astrocytes were treated with conditioned medium obtained from cerebral hemisphere astrocyte cultures previously exposed to 50 nM T₃. T₃ or conditioned medium-treatment produced faster morphological changes in hypothyroid rat cerebral hemisphere astrocyte monolayers. Cerebellar astrocytes from normal brain did not respond to thyroid hormone with morphological changes, but proliferated after T₃-treatment. However, hypothyroid cerebellar astrocyte cultures exhibited morphological changes, differently than normal cells. We verified that T₃ may induce astrocyte secretion of factor(s) that promotes morphological differentiation in cerebral hemisphere astroglial cultures and stimulates the proliferation of cerebellar astrocytes. Astrocytes obtained from hypothyroid animals were more sensitive to secreted factors than normal cells. These results emphasize the heterogeneity and the importance of glial cells to normal brain development and open new questions about thyroid hormone therapy in hypothyroidism. © 1995 Wiley-Liss, Inc.     

Binding of atrial and brain natriuretic peptides to cultured mouse astrocytes from different brain regions and effect on cyclic GMP production

We prepared primary cultures of mouse astrocytes from the cerebral cortex, hypothalamus, and cerebellum to examine the possibility of regional disparity in binding of human atrial and porcine brain natriuretic peptides (hANP, pBNP) and their effect on cyclic guanosine monophosphate (cGMP) production. ¹²⁵I-hANP and ¹²⁵I-pBNP bound in a specific and saturable manner to all three regions. For both peptides, Scatchard analysis suggested a single population of binding sites on astrocytes from all three regions. No significant differences were observed in the maximal binding capacities (B_max) or binding dissociation constants (K_D) between the two peptides in the astrocyte preparations from different regions. ANP and BNP also evoked cGMP stimulation in a similar, dose-dependent fashion in astrocytes from all three regions, with maximal responses to both peptides reached at a concentration above 1 μM. While BNP elicited a greater maximal cGMP accumulation than ANP, no difference could be demonstrated in the cGMP responses to either peptide between brain regions. Thus we have been unable to demonstrate regional heterogeneity in the responsiveness of astrocytes to ANP and BNP. © 1993 Wiley-Liss, Inc.     

Beta-amyloid peptides decrease soluble guanylyl cyclase expression in astroglial cells

In astroglial cells beta-amyloid peptides (betaA) induce a reactive phenotype and increase expression of NO synthase. Here we show that treatment of rat brain astrocytes with betaA decreases their capacity to accumulate cyclic GMP (cGMP) in response to NO as a result of a decreased expression of soluble guanylyl cyclase (sGC) at the protein and mRNA levels. Potentiation of betaA-induced NO formation by interferon-gamma did not result in a larger decrease in cGMP formation and inhibition of NO synthase failed to reverse down-regulation of sGC, indicating that NO is not involved. The betaA effect was prevented by the protein synthesis inhibitor cycloheximide. Intracerebral betaA injection also decreased sGC beta1 subunit mRNA levels in adult rat hippocampus and cerebellum. A loss of sGC in reactive astrocytes surrounding beta-amyloid plaques could be a mechanism to prevent excess signalling via cGMP at sites of high NO production.     

Regional,developmental, and cell cycle-dependent differences in μ, δ, and κ-opioid receptor expression among cultured mouse astrocytes

The diversity of opioid receptor expression was examined in astrocytes in low-density and non-dividing (confluent) cultures from the cerebral cortex, hippocampus, cerebellum, and striatum of 1-day-old mice. μ, δ, and κ opioid receptor expression was assessed in individual cells immunocytochemically, by using flow cytometry, and functionally by examining agonist-induced changes in intracellular calcium ([Ca²⁺]_i). Significant spatial and temporal differences were evident in the pattern of expression of μ, δ, and κ receptors among astrocytes. In low-density cultures, greater proportions of astrocytes expressed μ-opioid receptor immunoreactivity in the cerebral cortex and hippocampus (26–34%) than in the cerebellum or striatum (7–12%). At confluence, a greater percentage of astrocytes in cerebellar (26%) and striatal (30%) cultures expressed μ-immunoreactivity. Fewer astrocytes possessed δ-immunoreactivity in low-density striatal cultures (8%) compared to other regions (16–22%). The proportion of δ receptor-expressing astrocytes declined in the cerebellum but increased in the hippocampus. κ-opioid receptors were uniformly expressed by 27–34% of astrocytes from all regions, except in cortical cultures, where the proportion of κ expressing cells was 38% at low-density and decreased to 22% at confluence. Selective μ (PLO 17; H-Tyr-Pro-Phe (N-Me) -D-Pro-NH₂, δ ([D-Pen², D-Pen⁵] enkephalin), or κ (U50,488H; trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeneacetamide methanesulfonate) opioid receptor agonists increased [Ca²⁺]_i in subpopulations of astrocytes indicating the presence of functional receptors. Lastly, opioid receptor immunofluorescence varied during the cell division cycle. A greater proportion of astrocytes in the G₂/M phase of the cell cycle were μ or δ receptor immunofluorescent than at G₀/G₁. When astrocytes were reversibly arrested in G₁, significantly fewer cells expressed δ receptor immunofluorescence; however, upon reentry into the cell cycle immunofluorescent cells reappeared. In conclusion, opioid phenotype varies considerably among individual cultured astrocytes, and this diversity was determined by regional and developmental (age and cell cycle dependent) differences in the brain. These in vitro findings suggest astroglia contribute to regional and developmental idiosyncrasies in opioid function within the brain. GLIA 22:249–259, 1998. © 1998 Wiley-Liss, Inc.     

Regulation by calcium of the nitric oxide/cyclic GMP system in cerebellar granule cells and astroglia in culture

Ca²⁺ entry induced by N-methyl-D-aspartate (NMDA) in neurons and by noradrenaline (NA) in astrocytes is known to increase intracellular cyclic GMP (cGMP) levels through stimulation of the Ca²⁺-dependent nitric oxide synthase type I (NOS-I). The possibility that Ca²⁺ entry could also down-regulate intracellular cGMP by activating a Ca²⁺/calmodulin-dependent phosphodiesterase (CaM-PDE) has been investigated here in primary cultures enriched in granule neurons or in astroglia from rat cerebellum. We show that the same agonists that stimulate nitric oxide (NO) formation (NMDA and NA at 100 μM) and the Ca²⁺ ionophore A23187 (10 μM) decrease cGMP generated in response to direct stimulation of soluble guanylyl cyclase (sGC) by NO donors in both cell types. This effect requires extracellular Ca²⁺ and is prevented by the calmodulin inhibitor W7 (100 μM). Membrane depolarization, manipulations of the Na⁺ gradient, and intracellular Ca²⁺ mobilization also decrease NO donor-induced cGMP formation in granule cells. In astroglia Ca²⁺ entry additionally down-regulates cGMP generated by stimulation of the particulate GC by atrial natriuretic peptide (ANF). Decreases in cGMP produced by A23187 were more pronounced in the absence than in the presence of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), indicating that a CaM-PDE was involved. We also show that astroglial cells can accumulate similar amounts of cGMP than neurons in response to NO donors when IBMX is present but much lower levels in its absence. This may result from a lower ratio of sGC to PDE activities in astroglia. J. Neurosci. Res. 49:333–341, 1997. © 1997 Wiley-Liss, Inc.     

NMDA receptor-mediated cGMP synthesis in primary cultures of mouse cerebellar granule cells appears to involve neuron-astrocyte communication with NO operating as the intercellular messenger

The possibility that neuron-astrocyte communication may be responsible for glutamate (Glu)-stimulated cGMP formation even in relatively homogeneous primary cultures of mouse cerebellar granule cells (7 days in vitro) was investigated. Pharmacological analysis using selective excitatory amino acid (EAA) receptor antagonists showed that cGMP production, stimulated in these cultures by Glu and a variety of endogenous EAAs structurally-related to Glu (namely, L-aspartate, L-cysteine sulphinate, L-homocysteate, S-sulpho-L-cysteine), was mediated wholly by N-methyl-D-aspartate (NMDA) receptor activation. Moreover, EAA-induced responses were dependent on the presence of extracellular calcium but unaffected by addition of the L-type voltage-sensitive calcium channel blockers nifedipine (10 μM) or verapamil (5 μM). The mode of calcium entry was also shown to be important since the calcium ionophore, Λ23187 (10 μM), was unable to stimulate cGMP levels above basal. cGMP formation was blocked by the competitive nitric oxide synthase inhibitor, L-N^G-nitroarginine (100 μM), consistent with a role of nitric oxide (NO) in this signalling pathway. In the presence of added haemoglobin (1 μM), acting as a membrane-impermeable NO scavenger, Glu-stimulated cGMP formation was abolished implying that NO must act as an intercellular messenger. When the neuronal population was destroyed following a 24 hr exposure to the excitotoxin, S-sulpho-L-cysteine (200 μM), Glu-stimulated cGMP formation was abolished; whereas responses to the NO donor, sodium nitroprusside (SNP), although markedly reduced were still double that stimulated by Glu in the absence of the excitotoxin, suggesting the presence of non-neuronal cells that can generate cGMP if supplied directly with NO. Consistent with this suggestion, low levels of the glial specific enzyme, glutamine synthetase, were detected in granule cell cultures. Furthermore, omission or delayed addition of the antimitotic agent, cytosine arabinoside (20 μM), to the growth medium caused a significant increase in the level of Glu-stimulated cGMP formation. © 1996 Wiley-Liss, Inc.     

cAMP-induced stellation in primary astrocyte cultures with regional heterogeneity

It is well known that increased cAMP levels in cultured astrocytes can convert flat polygonal shaped astrocytes into process-bearing, stellate astrocytes. In this study, we have examined the possible existence of astrocyte regional heterogeneity in morphological changes in response to cAMP stimulation. Primary astrocyte cultures were prepared from six different regions of neonatal rat brains, including cerebral cortex, hippocampus, brain stem, mid brain, cerebellum, and hypothalamus. After about 2 weeks in culture, the astrocyte culture medium was changed to DMEM containing various concentrations of 8-CPT-cAMP, a membrane permeable cAMP analog, for 2 h. We found that 250 microM 8-CPT-cAMP produced a maximum effect causing >95% stellation in all regional astrocytes except hypothalamic astrocytes (56% stellation). At lower cAMP concentrations, cell stellation most effectively occurred in cerebellar astrocytes. To examine further the regional heterogeneity of astrocyte morphological changes, glutamate was added together with 8-CPT-cAMP to block cAMP-induced astrocyte stellation. Interestingly, glutamate blockage on cAMP-induced astrocyte stellation was brain region-specific in that cerebral and hippocampal astrocytes were effectively blocked by glutamate when compared to other regional astrocytes. Furthermore, glutamate inhibited isoproterenol-induced astrocyte stellation in a region-specific manner similarly as in cAMP-induced stellation. The present study demonstrates that astrocytes derived from different regions of the neonatal rat brain maintain different levels of morphological plasticity in culture.     

AMPA receptor protein expression and function in astrocytes cultured from hippocampus.

Glutamate receptors guide the proliferation, migration, and differentiation of glial cells. Here, we characterize AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA receptor protein expression and function and mRNA expression in hippocampal glial cultures. By immunocytochemistry, GluR2 (the subunit that limits the Ca(2+) permeability of AMPA receptors) exhibited prominent labeling in hippocampal glial cultures. Double-labeling of GluR2 with GFAP and with A2B5 revealed GluR2 subunit expression on type-1 and type-2 astrocyte lineage cells. GluR1 subunit expression was more prominent in type-1 than in type-2 astrocytes. To characterize functional properties of glutamate receptors expressed in cultured hippocampal astrocytes, we performed whole-cell patch clamp recording. Application of L-glutamate, AMPA, and kainate, but not NMDA, to small, rounded cells (morphologically identified as type-2 astrocytes) elicited inward currents which were blocked by the AMPA/kainate antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Cyclothiazide potentiated AMPA- and kainate-elicited currents, indicative of AMPA-preferring receptors. Current voltage analysis indicated that type-2 astrocyte AMPA receptors were electrically linear, indicative of GluR2-containing, Ca(2+)-impermeable AMPA receptors. By Northern blot analysis, GluR1 mRNA was highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes; GluR2 and GluR3 mRNAs were near background levels. Thus, cultured type-2 astrocytes express functional AMPA receptors in a cell-specific and region-specific manner, consistent with their role in neuronal-glial communication.     

Mutant disrupted‐in‐schizophrenia 1 in astrocytes: Focus on glutamate metabolism

Disrupted‐in‐schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase to regulate production of D‐serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, reuptake, or secretion of GLU remains unexplored. Therefore, we studied the effects of dominant‐negative mutant DISC1 on various aspects of GLU metabolism by using primary astrocyte cultures and hippocampal tissue from transgenic mice with astrocyte‐restricted expression of mutant DISC1. Although mutant DISC1 had no significant effects on astrocyte proliferation, GLU reuptake, glutaminase, or glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine‐serine‐cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus, and elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of N‐methyl‐D‐aspartate (NMDA) receptors in the hippocampus, at postnatal day 21. Our findings indicate that decreased D‐serine production by astrocytic mutant DISC1 might lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse. © 2014 Wiley Periodicals, Inc.     

Immunocytochemistry of cGMP in the Cerebellum of the Immature,Adult, and Aged Rat: the Involvement of Nitric Oxide. A Micropharmacological Study

In this study we describe the localization of formaldehyde-fixed cGMP-immunoreactivity (cGMP-IR) in rat cerebellar tissue slices incubated in vitro. In the absence of phosphodiesterase inhibition, cGMP-immunofluorescence was of low intensity in tissue slices prepared from immature cerebella. Addition of isobutylmethylxanthine (IBMX) to the incubation medium resulted in the appearance of cGMP-IR in clusters of astrocytes in the internal granular layer. Addition of N-methyl-d-aspartate (NMDA), kainic acid, atrial natriuretic factor (ANF), or sodium nitroprusside (SNP) gave an intense cGMP-IR in Bergmann fibres, Bergmann cell bodies, and astrocytes in the internal granular layer. Astrocytes in the white matter showed cGMP-IR after incubation of the slice in the presence of ANF or nitroprusside, but not after NMDA or kainic acid. In addition, after SNP stimulation of cGMP production, cGMP-IR was found in fibres which were not positive for glial fibrillary acidic protein (GFAP). In the adult cerebellar slice, intense basal cGMP-immunostaining was observed in Bergmann fibres, Bergmann cell bodies, and astrocytes in the granular layer. No cGMP-IR was observed in Purkinje cells. Stimulation of the cGMP-content in the glial structures by NMDA, ANF, or SNP, was suggested by the immunocytochemical results. However, when measured biochemically, only the effect of SNP was statistically significant, and immunocytochemistry showed that SNP clearly stimulated cGMP synthesis in neuronal cell structures. In the cerebellum of the aged rat a reduced cGMP-IR was found compared to the adult, in the same structures which showed cGMP-IR in the adult. Basal cGMP-immunostaining was reduced in the presence of haemoglobin, methylene blue, by inhibiting nitric oxide synthesis with NG-monomethyl-l-arginine (NGMAr), or by depletion of external Ca2+. Also the stimulatory effect of NMDA and of ANF (partly) on the cGMP-IR was inhibited by these compounds. cGMP-IR after stimulation of guanylate cyclase by SNP was reduced by the concomitant presence of haemoglobin or methylene blue, but not by NGMAr, or by omission of Ca2+. Our results point to an important role for cGMP in the functioning of glial tissue in the cerebellum and also suggest a role for nitric oxide as an intercellular mediator in the functioning of glutamate and ANF in the cerebellum.     

Characteristics of nitric oxide synthase type I of rat cerebellar astrocytes

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyte-enriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [³H]arginine to [³H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same K_m for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location. © 1996 Wiley-Liss, Inc.     

Age-related Changes in the NMDA Receptor/Nitric Oxide/cGMP Pathway in the Hippocampus and Cerebellum of Freely Moving Rats Subjected to Transcerebral Microdialysis

The N-methyl-d -aspartate (NMDA) receptor/nitric oxide synthase/guanylate cyclase pathway was studied during aging by monitoring extracellular cGMP in the rat hippocampus and cerebellum during in vivo microdialysis. In the hippocampus the basal cGMP efflux decreased by 50% from 3 to 12 months of age, whereas it remained constant with age in the cerebellum. Locally perfused NMDA (1 mM) evoked remarkable cGMP responses in 3-month-old rats; in the hippocampus the cGMP production was already dramatically reduced at 12 months, whereas in the cerebellum a similar impairment occurred much later (24 months). The nitric oxide donor S-nitroso-N-penicillamine (1 mM) elicited cGMP responses which slightly decreased from 3 to 12–24 months in the hippocampus, while no significant decrement with age could be seen in the cerebellum. Local perfusion of the phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine (IBMX, 1 mM) produced large increases in hippocampal cGMP levels. The response decreased at 12 and 24 months, apparently in parallel with the fall in the basal level of cGMP. No significant differences across ages were observed following IBMX infusion in the cerebellum. The decreases in basal outflow and in the NMDA-evoked cGMP response seen in the aged hippocampus were not compensated for by supplying l -arginine. Infusion of d -serine (1 mM) enhanced (150–200%) extracellular cGMP in the cerebellum with no age-related differences. The activity in vitro, of hippocampal nitric oxide synthase at 24 months was 33% lower than at 3 months, whereas the cerebellar enzyme did not show any age-related decay. Aging seems therefore to affect differentially the NMDA receptor/nitric oxide synthase/cGMP pathway in the rat hippocampus versus the cerebellum. In the hippocampus the early fall in the NMDA-evoked cGMP response seems to originate from deficits in NMDA receptor function and nitric oxide synthase and guanylate cyclase activities; in the cerebellum, the decreased response to NMDA in the old animals seems essentially to be due to impairment of NMDA receptor function.     

Steroidogenic acute regulatory protein expression and pregnenolone synthesis in rat astrocyte cultures

Neurosteroids are steroids synthesised by brain cells. The molecular mechanism of neurosteroidogenesis from cholesterol has not yet been revealed. We studied the potential role of the steroidogenic acute regulatory (StAR) protein in neurosterodogenesis by using rat brain astrocytes. The novelty of the study is that regulation of StAR is described in primary cultures from embryonic mesencephalon and cerebellum regions of the brain. Dibutyryl cyclic AMP (dbcAMP) treatment increased StAR protein expression in astrocyte cultures. This was observed in immunoblots of mitochondrial fractions and by immunocytochemistry. Dual-labelling showed that the cyclic AMP-induced increase in StAR immunofluorescence was localised to mitochondria. In addition, mitochondrial cytochrome P450-side chain cleavage enzyme was demonstrated with a specific antibody, indicating the potential for pregnenolone production in these cells. Radioimmunoassay on ether-extracted conditioned media of control and dbcAMP treated cells demonstrated pregnenolone production by mesencephalic and cerebellar astrocyte cultures. Furthermore, 24-h pregnenolone levels, in the presence of inhibitors of further pregnenolone metabolism, were significantly increased by dbcAMP exposure. A murine StAR promoter-luciferase fusion plasmid was activated by dbcAMP in transiently transfected mesencephalic and cerebellar astrocytes. These novel results indicate that cyclic AMP signalling can regulate StAR expression and pregnenolone production in brain astrocytes, and provide additional insight into the role of StAR in neurosteroidogenesis.     

Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.     

Nitric oxide contributes to opioid release from glia during hypoxia

Activation of neuronal nitric oxide (NO) synthase contributes to increased CSF concentrations of the opioids methionine enkephalin and leucine enkephalin during hypoxia in the newborn pig. NO and these opioids, in turn, contribute to hypoxic pial artery dilation. However, the cellular site of origin for opioids detected in CSF cannot be determined using this in vivo model. The present study, therefore, was designed to determine if NO contributes to opioid release from piglet glia grown in primary culture. Glial cell cultures produced more methionine enkephalin than leucine enkephalin under basal conditions. Administration of SNP and 8-Br cGMP to glial cells increased release of both opioids (471±58 vs. 1181±148 pg/mg protein methionine enkephalin before and after SNP 10⁻⁶ M). SNP also increased release of cGMP. Exposure of piglet glial cells to lower than normal O₂ increased the release of both opioids (503±61 vs. 1488±186 pg/mg protein methionine enkephalin before and after hypoxia, (P_O2≈15 mmHg). Hypoxia also increased the release of cGMP from glia while the NO synthase inhibitor N-nitro- -arginine blocked that release. These data show that NO/cGMP and hypoxia release opioids from glia. Additionally, hypoxia releases NO/cGMP from glia. These data therefore suggest that NO contributes to opioid release from glia during hypoxia.     

Evidence for cyclooxygenase activation by nitric oxide in astrocytes

We have evaluated the role of nitric oxide (NO) on the cyclooxygenase pathway in mouse glial cells. Exposure of primary cultures of neonatal mouse cortical astrocytes to bacterial lipopolysaccharide (LPS; 1 μg/ml, 18 h) caused an increase in the release of both nitrite (NO⁻₂) and prostaglandin E₂ (PGE₂2), products of NO synthase (NOS) and cyclooxygenase, respectively. Production of both, NO⁻₂ and PGE₂ by astrocytes, was inhibited by the exposure of the NOS inhibitor Nw-nitro-L-arginine methyl ester (L-NAME: 1, 10, and 100 μM) in a dose related manner. Besides, other NOS inhibitors such as Nitro L-arginine (NNA: 10⁻³ M) prevented the increase in PGE₂ release from LPS-stimulated astrocytes. Sodium nitroprusside (SNP; 100–200 μM) used as a NO donor caused a dose-related enhancement in the accumulation of PGE₂ induced by LPS and the presence of hemoglobin blocked the SNP effects. The exposure to SNP counteracted the decrease of PGE₂ production in LPS-treated astrocytes in which NO synthesis was blocked by L-NAME. In addition, SNP also enhanced the synthesis of PGE₂ following exogenous arachidonic acid astrocytes exposure. Interestingly, this effect was blocked by indomethacin. Treatment of astrocytes cultures with dexamethasone (0.1, 1 μM) blocked dose-relatedly the LPS-induced release of both NO⁻₂ and PGE₂. As expected, the presence of indomethacin (1, 10, and 20 μM) prevented in a dose related fashion, PGE₂ production by astrocytes following exposure to LPS. These results strongly indicate that in astroglial cells, NO is able to activate the cyclooxygenase pathway. © 1995 Wiley-Liss, Inc.     

Production of nitrite by primary rat astrocytes in response to pneumococci

Recent studies using a rat model of pneumococcal meningitis have shown that nitric oxide synthase (NOS) inhibitors greatly attenuated microvascular changes and brain edema formation. The site of NO production during bacterial meningitis is unknown. In this study we tested whether primary astrocyte cultures from neonatal rat cortex can be induced to release NO upon stimulation with pneumococci. NO production was assessed by measuring nitrite in the cell culture supernatant using the Griess reaction. Stimulation with heat-killed unencapsulated pneumococci (HKP) increased nitrite concentrations in astrocyte culture supernatants in a dose-dependent fashion. Administration of AT-nitro-L-arginine (L-NA), aminoguanidine, L-canavanine, cycloheximide, and dexamethasone prevented the increase in nitrite concentrations. Addition of L-arginine, but not of o-arginine, partially reversed the inhibitory effect of L-NA. Administration of SOD increased nitrite accumulation. Moreover, at 72 h after stimulation with heat-killed pneumococci (10⁷ cfu/ml) astrocytes showed an inducible NOS-like immunoreactivity. Accumulation of nitrite was also observed when rat cerebellar neurons and microglia were stimulated with HKP, whereas there was only a slight increase of nitrite in media of rat C6 glioma cells, but no increase of nitrite when the human glioblastoma cell line LN-229 was stimulated with HKP. There was a stronger increase in nitrite levels when astrocytes from Lewis rats were used compared to that from Wistar rats. In conclusion, our study indicates that astrocytes, neurons and microglia are inducible for NO production upon stimulation with pneumococci.     

Phosphorylation of glial fibrillary acidic protein is stimulated by glutamate via NMDA receptors in cortical microslices and in mixed neuronal/glial cell cultures prepared from the cerebellum

In previous work we showed that phosphorylation of glial fibrillary acidic protein (GFAP), an astrocyte marker, is increased by glutamate in hippocampal slices from immature rats via a type II metabotropic receptor. In the present work we show that glutamate also stimulates GFAP phosphorylation in microslices prepared from immature cerebellar cortex, but by a different receptor mechanism from that observed in the hippocampus. Thus, in cerebellar microslices, NMDA consistently stimulated GFAP phosphorylation, whereas no effect of metabotropic or non-NMDA ionotropic agonists was observed. Glutamate and NMDA also stimulated GFAP phosphorylation in mixed neuronal/glial cell cultures from the cerebellum, although no effect of these agonists was observed in primary cultures of cerebellar astrocytes. In both models, the effects of glutamate and NMDA were dependent on external Ca(2+), were reversed by the NMDA receptor antagonist AP5 and were not blocked by tetrodotoxin. In the slice study the effect of NMDA was confined to a period starting with the first detectable expression of GFAP at 10 days and finishing at 16 days postnatal, as previously observed with metabotropic agonists in hippocampal slices. This period in the rat corresponds to the start of synaptogenesis when astrocyte hypertrophy is occurring. The results are discussed in the light of information in the literature on the occurrence of functional NMDA receptor subunits in glia.     

Uptake of [3H]serotonin and [3H]glutamate by primary astrocyte cultures. II. Differences in cultures prepared from different brain regions.

Regional astrocyte cultures were derived by dissecting six regions; brain stem, cerebellum, mesencephalon, basal ganglia plus diencephalon, cerebral cortex, and hippocampus, from 3 to 4-day-old neonatal rat brains. Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to confirm the astrocyte composition of the cultures. The percentage of GFAP (+) cells between regions varied from 75% to 100%. Once confluent these cultures were incubated with radiolabeled serotonin or glutamate for uptake and autoradiographic studies. For the different brain regions Na(+)-dependent, [3H] L-glutamate, and fluoxetine-sensitive [3H] 5-HT uptake varied markedly. The relative order of uptake for [3H] 5-HT was MS (mesencephalon) greater than CC (cerebral cortex) greater than BG + DI (basal ganglia + diencephalon) greater than HP (hippocampus) greater than BS (brain stem) greater than CB (cerebellum). For [3H] L-glutamate the order was HP greater than CC greater than BG + DI greater than MS = BS greater than CB. For [3H] 5-HT this essentially corresponds to the reported order of binding in situ of the [3H] 5-HT-specific uptake ligand [3H] citalopram. For [3H] L-glutamate regional variation of the uptake for the different cultures corresponds to the regional uptake reported for different regions of rat brain. Double-label studies with GFAP and radiolabeled neurotransmitters were also used to study uptake into GFAP(+) astrocytes by autoradiography. Flat GFAP cells with or without processes comprised 65-98% of the cultures and represented most of the uptake. The percentage of all GFAP(+) cells that were positive for uptake of ARG varied from 50% to 90% and also showed differences in grain density both intra- and inter-regionally. These differences in transmitter uptake by GFAP(+) astrocytes in primary culture, which are dependent on the region of origin and correspond to regional differences in situ, suggest that such uptake in vitro may reflect uptake by astrocytes in vivo. Implied in this is that uptake by astrocytes represents a significant component of serotonin uptake in vivo.