Josep Maria Tusell

High-yield isolation of murine Microglia by mild trypsinization

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05–0.12%) in the presence of 0.2–0.5 mM EDTA and 0.5–0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as > 98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony‐stimulating factor and lipopolysaccharide, alone or in the presence of interferon γ, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor‐κB translocation, NO, and tumor necrosis α release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide‐induced NO release and NO synthase‐II expression in a time‐ and concentration‐dependent manner. CGS21680 potentiation of lipopolysaccharide‐induced NO release was suppressed by the A2A receptor antagonist ZM‐241385 and did not occur on mixed glial cultures from A2A receptor‐deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase‐II inhibitor 1400W abolished NO production, and NO synthase‐II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase‐II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.

Upregulation of CCAAT/enhancer binding protein β in activated astrocytes and microglia

The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPβ in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPβ expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPβ in untreated mixed glial cultures. C/EBPβ is upregulated when glial activation is induced by lipopolysaccharide (LPS). The LPS‐induced upregulation of glial C/EBPβ is rapid (2 h at mRNA level, 4 h at protein level). It is elicited by low concentrations of LPS (almost maximal effect at 1 ng/mL) and it is reversed by the protein synthesis inhibitor cycloheximide. C/EBPβ nuclear levels increase both in astrocytes and microglia after LPS treatment, and the response is more marked in microglia. The LPS‐induced increase in microglial C/EBPβ is prevented by coadministration of the MAP kinase inhibitors SB203580 (p38 inhibitor) + SP600125 (JNK inhibitor) or SB203580 + U0126 (ERK inhibitor). Systemic injection of LPS also increases brain nuclear levels of C/EBPβ as shown by Western blot, and this increase is localized in microglial cells as shown by double immunofluorescence, in the first report to our knowledge of C/EBPβ expression in activated glial cells in vivo. These findings support a role for C/EBPβ in the activation of astrocytes and, particularly, microglia. Given the nature of the C/EBPβ‐regulated genes, we hypothesize that this factor participates in neurotoxic effects associated with glial activation.

Astrocytes enhance lipopolysaccharide‐induced nitric oxide production by microglial cells

Several stimuli result in glial activation and induce nitric oxide (NO) production in microglial and astroglial cells. The bacterial endotoxin lipopolysaccharide (LPS) has been widely used to achieve glial activation in vitro, and several studies show that both microglial and, to a lesser extent, astroglial cell cultures produce NO after LPS treatment. However, NO production in endotoxin‐treated astrocyte cultures is controversial. We characterized NO production in microglial, astroglial and mixed glial cell cultures treated with lipopolysaccharide, measured as nitrite accumulation in the culture media. We also identified the NO‐producing cells by immunocytochemistry, using specific markers for the inducible NO synthase (iNOS) isoform, microglial and astroglial cells. Only microglial cells showed iNOS immunoreactivity. Thus, contaminating microglial cells were responsible for NO production in the secondary astrocyte cultures. We then analysed the effect of astrocytes on NO production by microglial cells using microglial–astroglial cocultures, and we observed that this production was clearly enhanced in the presence of astroglial cells. Soluble factors released by astrocytes did not appear to be directly responsible for such an effect, whereas nonsoluble factors present in the cell membrane of LPS‐treated astrocytes could account, at least in part, for this enhancement.

Comparative study of the distribution of calmodulin kinase II and calcineurin in the mouse brain

The Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are Ca2+/calmodulin‐binding proteins that are very abundant in the central nervous system. In the mammalian brain, CaMKII is composed by the association of several similar subunits at different ratios produced by four different genes. CaN is composed of two different subunits produced by two genes. We selected the most abundant subunits of each enzyme in the rodent brain, CaMKII α and CaN A, and compared their pattern of expression in the mouse brain by using in situ hybridization histochemistry and immunohistochemistry. We found that CaMKII and CaN were mainly expressed in cerebral cortex, hippocampus, and striatum and that low levels of expression were observed in midbrain and brainstem. CaN was also expressed in cerebellum. In the cell, the kinase and the phosphatase were detected in the perikarya, the neuronal processes, and the nucleus. The present study shows that all the regions of the mouse brain in which CaMKII is expressed also show CaN expression. This fact is consistent with the presence of common substrates for both enzymes or with a regulatory action of one versus the other. The lack of correspondence in the cerebellum could be explained by the fact that the major subunit of the kinase in this brain region is CaMKII β. J. Neurosci. Res. 57:651–662, 1999.

Effect of different convulsants on calmodulin levels and proto-oncogene c-fos expression in the central nervous system

In the present study, a relationship between convulsant activity and two cellular events, changes in calmodulin (CaM) concentration and proto-oncogene c-fos expression has been considered. c-fos has been found activated after the administration of the organochlorine insecticide lindane, the Ca2+ channel agonist Bay K, and N-methyl-D-aspartate (NMDA). The administration of the voltage-dependent Ca2+ channel antagonist nifedipine was able to block the expression elicited by lindane. The effect of lindane on c-fos expression could not be blocked by prior administration of MK-801, a non-competitive antagonist of the NMDA receptor. These results suggest a possible role for the voltage-dependent Ca2+ channels in the mechanism of action of lindane. By means of in situ hybridization, the different patterns of c-fos expression after the administration of the mentioned compounds have been described. A possible modification of the levels of CaM has also been investigated. Among all the subcellular fractions considered, only levels of nuclear CaM appeared to be affected after the different treatments. The changes observed seemed to follow a similar pattern to that described for c-fos induction. Calcium entry through these voltage-dependent calcium channels would be the link between membrane depolarizing events and expression of c-fos and/or increase in nuclear CaM.

Lindane inhibition of [35S]TBPS binding to the GABAA receptor in rat brain

The inhibition of [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) binding to the GABAA receptor by the insecticide gamma-hexachlorocyclohexane, lindane, was studied in several brain regions and using different membrane preparation methods, both in vitro and after dosing the animals with the chemical. In the latter studies, the amount of lindane remaining in the membrane suspensions used for binding assays was determined. In vitro data showed values of IC50 from 150 to 1675 nM, varying in function of the membrane preparation method used. This may account for the discrepancies in IC50 values found in the literature. IC50 values within the range of 150-250 nM were determined using extensively washed membranes from several brain regions, so no evidence arose for brain regional differences in the affinity of lindane for the TBPS binding site. After different schedules of acute treatment with lindane, we found a manifest relationship between the extent of the observable inhibition of [35S]TBPS binding and the lindane amount remaining in the membrane suspensions used for binding assays. This relationship was in good agreement with the in vitro data, so no support for an in vivo acute regulation of the binding site was obtained.

Relationship between β-AP peptide aggregation and microglial activation

We compared the relationship between the state of aggregation of two peptides (beta-AP 25-35 and beta-AP 1-42) and microglial activation. After 7 days at 37 degrees C beta-AP 25-35 was in an amorphous state and did not activate microglial cells. In the same conditions, aggregated beta-AP 1-42 activated these cells and caused changes in microglial ramification, increasing the proliferation index and inducing tumor necrosis factor alpha (TNF alpha) release. Neither peptide induced a release of nitric oxide (NO). As the toxicity of beta-AP peptides in cell culture is associated with the formation of amyloid fibrils, we also examined the toxicity of both peptides in microglial cell cultures and in PC 12 cell cultures. The results suggest that the two beta-AP fragments studied have similar neurotoxic effects but different pro-inflammatory activities.

Comparative study of the pattern of expression of calmodulin messenger RNAs in the mouse brain

Calmodulin is a major calcium-binding protein in the mammalian brain, playing an important role in neuronal cell function. Its amino acid sequence is highly conserved and the protein is encoded by multiple genes. In the mouse brain, as well as in the rat and the human brain, three different genes have been detected for calmodulin, CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the pattern of expression of the three calmodulin genes and the pattern of calmodulin distribution in the mouse brain by in situ hybridization histochemistry and immunohistochemistry. We found that calmodulin messenger RNAs from the three calmodulin genes were widely expressed in the mouse brain. Nevertheless, there were differences in their patterns of distribution. In general, all calmodulin messenger RNAs were preferentially distributed in hippocampus, cerebral cortex and cerebellar cortex, and CaM II messenger RNA also in caudate-putamen. However, all messenger RNAs showed clearly differentiated patterns of distribution in the hippocampus and the cerebellar cortex. Calmodulin immunoreactivity was present in all cells so far examined. Immunostaining was observed both in the cell nucleus, where it was especially strong, and in the cytoplasm. Our results suggest that the three calmodulin genes are differentially regulated in the mouse brain and also that, although all calmodulin genes have a basal expression, precise regulation of calmodulin levels might be attained through the different contribution of the three calmodulin genes.

The Ca2+/calmodulin system in neuronal hyperexcitability.

Calmodulin (CaM) is a major Ca2+-binding protein in the brain, where it plays an important role in the neuronal response to changes in the intracellular Ca2+ concentration. Calmodulin modulates numerous Ca2+-dependent enzymes and participates in relevant cellular functions. Among the different CaM-binding proteins, the Ca2+/CaM dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. Therefore, the role of the Ca2+/CaM signalling system in different neurotoxicological or neuropathological conditions associated to alterations in the intracellular Ca2+ concentration is a subject of interest. We here report different evidences showing the involvement of CaM and the CaM-binding proteins above mentioned in situations of neuronal hyperexcitability induced by convulsant agents. Signal transduction pathways mediated by specific CaM binding proteins warrant future study as potential targets in the development of new drugs to inhibit convulsant responses or to prevent or attenuate the alterations in neuronal function associated to the deleterious increases in the intracellular Ca2+ levels described in different pathological situations.