1. Voltage-dependent currents of untreated (proliferating) and lipopolysaccharide (LPS)-treated rat microglial cells in culture were recorded using the whole-cell patch-clamp technique. 2. Membrane potentials showed prominent peaks at -35 mV and -70 mV. Membrane potentials of LPS-treated cells alternated between the two values. This may be due to a negative slope region of the I-V relation resulting in two zero current potentials. 3. From a holding potential of -70 mV, hyperpolarizing steps evoked an inwardly rectifying current both in proliferating and in LPS-treated cells, while depolarizing steps below -50 mV evoked an outwardly rectifying current only in LPS-treated microglia. The currents were K+ selective, as indicated by their reversal potential of approximately 0 mV in symmetric K+ concentrations (150 mM both intra- and extracellularly) and the reversal potential of the outward tail currents of approximately -90 mV at a normal extracellular K+ concentration (4.5 mM). 4. The activation of the outward current could be fitted by Hodgkin-Huxley-type n4 kinetics. The time constant of activation depended on voltage. 5. The inactivation of the inward and outward currents could be fitted by a single exponential. The time constant of the inward current inactivation was dependent on voltage, whereas the time constant of the outward current inactivation was virtually independent of voltage, except near the threshold of activation. Recovery of the outward from inactivation was slow and could be fitted by two exponentials. Responses to depolarizing steps were stable at 0.125 Hz, but greatly decreased from the first to the second pulse at 1 Hz. 6. The inactivation of the inward, but not of the outward, current disappeared in a low Na(+)-containing medium (5 mM). The inward current was selectively inhibited by extracellular Cs+ and Ba2+. The outward current was selectively inhibited by Cd2+, 4-aminopyridine and charybdotoxin. Replacement of intracellular K+ by an equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS-treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS)

No abstract

Primary rat astrocyte cultures were used to isolate a macrophage population that does not adhere to the confluent glial cells. The cells multiplied vigorously in coculture with astrocytes during the 14 d culture period, provided that functionally active lipopolysaccharide (LPS) was either absent or present in very low concentrations. Based on morphological, immunocytochemical, and pharmacological data, it was concluded that the isolated cells were microglia, the resident macrophages of the brain. The findings characterized them as a distinct cell population that shares features both of peritoneal macrophages and of astroglial cells. Like peritoneal macrophages, the isolated cells were able to phagocytize as shown by their ingestion of latex beads and uptake of L-leucyl methylester. Furthermore, they were immunocytochemically stainable by a specific monoclonal antibody (ED 1) against a macrophage-specific antigen (Dijkstra et al., 1985). They also synthesized prostaglandin E2 (PGE2) and secreted interleukin 1 (IL-1) upon stimulation with LPS. Upon stimulation with the ionophore A23187, PGD2, the predominant prostaglandin of the brain, was the major PG metabolite released by these cells. In contrast to peritoneal macrophages, microglial cells were able to multiply. Proliferation of microglial cells in coculture with astrocytes was suppressed when 2 ng LPS/ml or higher concentrations were added to astroglial culture media. These astrocyte cultures, which contained approximately 1% microglia, were used to investigate the influence of LPS on prostaglandin and IL-1 secretion in order to compare astroglial and microglial features. Increasing LPS concentrations induced increased PGE2 secretion, whereas PGD2 secretion was essentially unaffected by LPS. The critical influence of LPS contaminations in most of the commercially available animal sera used for astrocyte cultures on cellular composition in general and on metabolism of hormones and growth factors in particular is discussed.

1 We have investigated the eects of the phosphodiesterase (PDE) type IV inhibitor rolipram and of the glucocorticoid methylprednisolone on the induction of tumour necrosis factor alpha (TNF-a) mRNA and protein in brains of rats after peripheral administration of lipopolysaccharide (LPS). 2 After intravenous administration of LPS, a similar time-dependent induction of both TNF-a mRNA and protein was observed in rat brain. Peak mRNA and protein levels were found 7 h after administration of LPS. 3 In situ hybridization experiments with a speci®c antisense TNF-a riboprobe suggested that the cells responsible for TNF-a production in the brain were microglia. 4 Intraperitoneal administration of methylprednisolone inhibited the induction of TNF-a protein in a dose-dependent manner. A maximal inhibition of TNF-a protein production by 42.9+10.2% was observed at a dose regimen consisting of two injections of each 30 mg kg 71 methylprednisolone. 5 Intraperitoneal administration of rolipram also inhibited the induction of TNF-a protein in a dosedependent manner. The maximal inhibition of TNF-a protein production was 96.1+12.2% and was observed at a dose regimen of three separate injections of each 3 mg kg 71 rolipram. 6 In situ hybridization experiments showed that the level of TNF-a mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of methylprednisolone (2615 mg kg 71 ) and of rolipram (363 mg kg 71 ). 7 We suggest that peripheral administration of LPS induces a time-dependent expression of TNF-a in rat brain, presumably in microglial cells, and that methylprednisolone and rolipram inhibit LPS-induced expression of TNF-a in these cells via a decrease of TNF-a mRNA stability and/or TNF-a gene transcription.

Human brain macrophages (microglia) have been isolated from mixed brain cell cultures initiated from explants of neurosurgical adult human tissue in one step according to a method developed for rat microglia. Cells were characterized enzyme-histochemically (NDPase) in mixed and immunocytochemically (anti-CD 14) in mixed and isolated cultures. Purified cells were used to investigate in more detail membrane currents by the patch clamp technique. In 14 cells microdialyzed with a standard, K(+)-containing intracellular solution there was no indication for a hyperpolarization-induced K(+)-inward current characteristic for newborn rat microglia. However, in 12 cells depolarizing pulses initiated a rapidly inactivating inward current which was followed by an outward current (in 4 cells). The outward current appeared to be carried by K+, since it was absent in another 18 cells, recorded by micropipettes containing Cs+ instead of K+ as the main intracellular cation. The depolarization-induced inward current persisted under these conditions. This current was inhibited by tetrodotoxin (5 microM) and by substitution of Na+ by choline in the bath solution. It is suggested that this Na(+)-current is specifically expressed in macrophages derived from adult brain.