Induction of superoxide anion and nitric oxide production in cultured microglia.
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Animals↗
Biomedical subjects
C A Colton
Publications and source records attributed to C A Colton.
K+ modulation of microglial superoxide production: involvement of voltage-gated Ca2+ channels.
A variety of cytoactive factors produced during injury and inflammation are known to activate the central nervous system (CNS) macrophage, the microglia. Since extracellular potassium levels are known to rise rapidly at sites of injury in the CNS, we examined the possibility that changes in extracellular potassium could mediate changes in microglial function. The effect of an increase in potassium concentration on microglial superoxide anion production was studied in cultured neonatal rat microglia. Rather than directly inducing superoxide anion production, exposure to media containing 25 and 55 mM potassium enhanced the production of superoxide induced by phorbol 12-myristate 13-acetate. This potentiation was blocked by nifedipine, a voltage-gated calcium channel blocker. Treatment of the microglia with BAY K 8644, an agonist for voltage-gated calcium channels, produced an enhancement of superoxide levels similar to that of potassium. Because these data indicated the presence of a voltage-gated calcium channel, we also examined whole cell current in cultured microglia. A small, voltage-dependent inward calcium current was seen that was increased by exposure of the microglia to BAY K 8644. The presence of a small but finite calcium influx via these channels may be an important factor in the regulation of intracellular microglial events such as activation of the NADPH oxidase and the consequent production of superoxide anion.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Microglial oxyradical production: causes and consequences.
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Amyloid beta-Protein Precursor↗
GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons.
During CNS development, neuroblasts proliferate within germinal zones of the neuroepithelium, and then migrate to their final positions. Although many neurons are thought to migrate along processes of radial glial fibers, increasing evidence suggests environmental factors also influence nerve cell movement. Extracellular matrix molecules are thought to be involved in guiding neuronal migration, and molecules such as NGF and GABA exert trophic effects on immature neurons. The nature of the signals that initiate and direct neuroblast migration, however, is unknown. In vitro, NGF and GABA promote neurite outgrowth from cultured cells, and NGF induces axonal chemotaxis (directed migration along a chemical gradient). At earlier developmental stages, these molecules could influence neuroblast movement. Therefore, we investigated whether these molecules induce embryonic neuronal migration. Using an in vitro microchemotaxis assay, we show that rat embryonic spinal cord neurons migrate toward picomolar NGF and femtomolar GABA beginning at embryonic day 13 (E13). Cells exhibit chemotactic responses to NGF while GABA stimulates chemokinesis (increased random movement). GABA effects are mimicked by muscimol and inhibited by bicuculline and picrotoxin, suggesting GABA motility signals are mediated by GABA receptor proteins. Expression of GABA receptors by embryonic cord cells has been previously reported (Mandler et al., 1990; Walton et al., 1993). We used polymerase chain reaction analysis to demonstrate the presence of NGF and trk mRNA in E13 and E14 cord cells, indicating the cells express message for both NGF and high-affinity NGF receptors. Immunohistochemistry of E13 spinal cord sections indicates that NGF and GABA colocalize in fibers close to the target destinations of migrating neurons, suggesting diffusible gradients of these molecules provide chemoattractant signals to migratory cells. Thus, in vitro, neuroblast migration is induced by specific signaling molecules that are present in the developing spinal cord, and may stimulate migration of embryonic neurons prior to synaptogenesis.
Animals↗
Protease production by cultured microglia: substrate gel analysis and immobilized matrix degradation.
The production of collagen-degrading proteases by cultured neonatal rat microglia was examined using an immobilized fibronectin-gelatin matrix coupled to a fluorescent marker and by substrate gel analysis. When microglia were plated onto the surface of the matrix and incubated under resting (nonstimulated) conditions, a small but visible amount of immobilized matrix was degraded. Treatment with lipopolysaccharide (LPS) or interleukin-1 (IL-1) significantly increased the number of microglia demonstrating substrate degradation. Substrate-SDS polyacrylamide gel electrophoresis of samples of supernatants from untreated cultured microglia indicated the presence of a 72 and a 92 kD metalloproteinase with characteristics corresponding to collagenases. Supernatants from untreated astrocyte cultures were shown to have primarily a 72 kD metalloproteinase. Proteinase activity increased on stimulation of the microglia with LPS and IL-1 in a dose-dependent fashion. These results indicate that cultured microglia release active proteases capable of degrading the extracellular matrix in a localized region. The production of proteases by activated microglia may have important physiological and pathophysiological consequences within the restricted extracellular matrix of the CNS.
Animals↗
Microglia, an in vivo source of reactive oxygen species in the brain.
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Animals↗
Characterization of interleukin-1 production by microglia in culture.
The production of interleukin-1 (IL-1) by cultured neonatal rat microglia was studied using the D10 cell assay. The results show that IL-1 was secreted in response to lipopolysaccharide (LPS) in a dose- and time-dependent fashion. IL-1 production was specific to microglia and was not induced in astrocytes. Indomethacin, which is known to modulate the release of IL-1 from monocytes, had no effect on LPS-stimulated microglia. Aging of the microglia from two weeks to 4 weeks in culture, however, reduced the release of IL-1 in response to LPS. Our data indicate that microglia are a major source of IL-1 and that the release of IL-1 depends on the presence of inflammatory mediators such as LPS and the age of the culture.
Animals↗
Regulation of microglial function by interferons.
Cultured neonatal rat microglia were pretreated with varying doses of either purified interferon (IFN) alpha/beta or recombinant IFN gamma for 24 or 48 h and the following functional parameters examined; superoxide anion production, interleukin-1 secretion and chemotaxis. IFN gamma produced a marked increase in superoxide anion levels when PMA was used to initiate superoxide anion production but had no effect in OPZ-stimulated microglia. Treatment with IFN alpha/beta potentiated superoxide anion production in both PMA and OPZ-stimulated cells. Interleukin-1 activity was decreased by treatment with IFN gamma for 24 h while IFN alpha/beta increased IL-1 activity at 48 h. Removal of serum from the treatment media prevented the action of IFN alpha/beta on IL-1 production. Treatment with IFN alpha/beta or gamma decreased chemotaxis of microglia in response to zymosan activated serum. The data indicate that IFN gamma and alpha/beta can regulate microglial function and that this regulation may differ from that seen for other monocytically derived macrophages.
Animals↗
Oxygen dependency of synaptic transmission at the squid Loligo pealei giant synapse.
1. Synaptic transmission at the squid giant synapse was blocked in 45 min by exposure to 0.02 atm oxygen but was maintained for more than 90 min in air (0.21 atm oxygen) or pure oxygen (1 atm). 2. Excitatory post-synaptic potential amplitude decreased in 0.02 atm oxygen but did not change in either 1 or 0.21 atm oxygen. Fast facilitation was increased in 0.02 atm oxygen only. 3. Post-synaptic resting membrane potential (Vm) and input resistance (Ro) remained unchanged in 1 or 0.21 atm oxygen but Ro was increased in 0.02 atm oxygen. 4. Our results suggest that severe hypoxia decreased the release of transmitter from the pre-synaptic terminal.
Animals↗
Lectin staining of cultured CNS microglia.
Carbohydrate binding proteins, known as lectins, bind to specific sugar groups on most membranes. We used fluorescent and light microscopy to study the interaction of various lectins with the membranes of microglia cultured from neonatal rat or fetal mouse cerebral cortices. Microglia stained intensely with GS-1, RCA, WGA, and ConA and slightly with DBA, UEA, BPA, and SBA. No staining was seen with GS-2, MPA, or PNA. Staining was specific for microglia in the mixed glial cultures and was dose dependent. In addition, microglial lectin binding could be reduced or blocked by competitive inhibition using specific sugars. Treatment of the microglia with agents such as dimethylsulfoxide (DMSO), interleukin-1 (IL-1), interferon (IFN), or lipopolysaccharide (LPS) did not eliminate lectin staining, although the degree of staining was altered. Positive staining of the microglia was also associated with a functional change for at least one lectin, i.e., ConA. Superoxide anion production by microglia was increased in the presence of ConA. Overall, binding of the lectins GS-1, RCA, WGA, and ConA can be used as an identifying tool for microglia in glial cultures, but intensity of staining varies depending on their functional state.
Animals↗
Abnormal production of interleukin-1 by microglia from trisomy 16 mice.
The production of interleukin-1 (IL-1) was examined in cultured CNS microglia obtained from trisomy 16 (Ts16) fetal mouse brain, a model system for studies relevant to Down syndrome (DS). When compared to microglia from their normal littermates, Ts16 microglia produced significantly higher levels of IL-1 activity both before and following stimulation with lipopolysaccharide (LPS). IL-1 release was stimulated by alpha/beta interferon (IFN) in the normal but not Ts16 microglial cultures. The overall level of IL-1 production in normal littermates, however, was still less than that seen in Ts16. Thus, microglia from Ts16 mice may function in an inappropriate manner and, if this abnormality occurs in vivo, may have wide ranging effects on a developing nervous system.
Animals↗
Enhanced production of superoxide anion by microglia from trisomy 16 mice.
Disruption of normal oxygen radical metabolism in the CNS may contribute to the neuropathological changes associated with Down syndrome (trisomy 21) and its mouse counterpart, the trisomy 16 (Ts16) mouse. One potent source of oxyradicals is the CNS-specific macrophage, the microglial cell. We prepared primary glial cultures from the cerebral cortices of Ts16 and normal littermate mice taken at day 15 of gestation. Microglia were isolated from confluent cultures after 14 days in vitro and assayed for superoxide anion production using a cytochrome C reduction assay. Stimulation by either opsonized zymosan (OPZ) or phorbol myristate acetate (PMA), produced significantly higher levels (2.8-20 fold) of superoxide per mg protein in Ts16 microglial cultures. Resting, i.e. unstimulated secretion, was not significantly different from littermate controls. Astrocyte enriched cultures, stimulated by OPZ, exhibited low levels of superoxide production which was higher in Ts16 mice than normal littermates. Microglial enriched cultures from rat neonatal cerebral cortices were exposed for 24 h to medium from the Ts16 glial cultures. Superoxide production in the Ts16 media treated rat microglia was significantly higher than in those treated with littermate conditioned media.
Animals↗
Chemotaxis by a CNS macrophage, the microglia.
Microglia demonstrate many characteristics similar to those seen in monocytes and tissue-specific macrophages, including phagocytosis, production of oxygen radicals, and growth factors and expression of MHC antigens. We have examined the ability of microglia, cultured from the cerebral cortices of neonatal rats, to demonstrate another important functional characteristic of monocytic-derived cells, that is, chemotaxis. Our results show that cultured rat microglia demonstrate chemotaxis to complement dependent chemoattractants such as recombinant C5a, zymosan activated serum, and to rat serum as well as to transforming growth factor-beta, a chemoattractant produced by platelets. Microglia fail to migrate to bacterial dependent chemoattractants such as the N-formyl peptides. The failure to respond is not dependent on maturational state of the microglia. Treatment with DMSO or casein, agents known to induce morphological and functional changes in cultured microglia reminescent of a "resting" and an "activated" macrophage, respectively, do not alter the response to fMet-Leu-Phe. In addition, the chemotactic response to serum in DMSO or casein-treated cells is the same as the response seen in untreated day 10 cultured microglia or untreated age-matched controls. The ability of microglia to migrate in response to inflammatory mediators suggests that these cells can move to sites of injury, thereby enabling them to participate in an inflammatory response.
Animals↗
Effect of helium and heliox on glutamate decarboxylase activity.
The activity of glutamate decarboxylase (GAD) in pure helium at 6.8 MPa was significantly enhanced when compared to GAD activity in air at 0.1 MPa (906 vs. 602 nmol.h-1.mg-1 protein, respectively). No significant difference was found between GAD activities in heliox at 6.8 MPa (0.87% O2 at 6.8 MPa) and in air at 0.1 MPa. On the other hand, the activities in heliox at 0.1 MPa (0.87% O2 at 0.1 MPa) and air at 0.1 MPa were significantly different (655 and 446 nmol.h-1.mg-1 protein, respectively). These data indicate that pressures up to 6.8 MPa do not affect the GAD-catalyzed synthesis of gamma-aminobutyric acid, but that the enzyme, as previously reported, is sensitive to low levels of oxygen.
Animals↗
The action of hydrogen peroxide on paired pulse and long-term potentiation in the hippocampus.
The action of a reactive oxygen intermediate, that is, hydrogen peroxide (H2O2) on modulation of synaptic transmission was examined in the hippocampal brain slice preparation. Microinjection of H2O2 into the apical dendritic region of the CA1 pyramidal cells produced no change in either the pattern or amplitude of paired pulse facilitation compared to saline injection (control). Long term potentiation (LTP), induced by high frequency stimulation of homosynaptic inputs, however, was blocked by microinjection of H2O2 into the dendritic tree. LTP was seen in only 2 out of 10 slices investigated when treated with H2O2 while LTP was seen in 4 out of 5 slices when saline injected. The results suggest that a reactive oxygen intermediate can selectively modify synaptic mechanisms in the hippocampus.
Action Potentials↗
An endogenous source of the superoxide anion in the central nervous system.
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Animals↗
Production of superoxide anions by a CNS macrophage, the microglia.
Microglia have been implicated in both physiological and pathological processes of the brain. Their possible roles have been compared to those of macrophages and granulocytes. Here we demonstrate that specific ability of microglia to secrete the superoxide radical ion in response to a complement activated agent, opsonized zymosan, and to phorbol myristate acetate. As in other organs, this endogenously produced reactive oxygen intermediate could have both beneficial and deleterious effects.
Animals↗
Trigeminal responses to thermal stimulation of the oral cavity in rattlesnakes (Crotalus viridis) before and after bilateral anesthetization of the facial pit organs.
Multiunit neural responses from the superficial maxillary branch of the trigeminal nerve in prairie rattlesnakes were elicited by intraoral thermal stimulation. The responses to oral stimulation were shown to be independent of responses obtained by thermal stimulation of the loreal pits. Histological examination of the dorsal lip, palate, and fang sheath regions revealed dense ramifying neurons in the epidermal layers of the fang sheaths that were morphologically similar to suspected infrared sensitive neurons in the pit membranes.
Anesthesia, Local↗