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Microglia-brain macrophages are immune-competent cells of the CNS and respond to pathologic events. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration ([Ca 2+]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of basal [Ca 2+]i along with a suppression of evoked calcium signaling, as indicated by reduced [Ca 2+]i transients during stimulation with UTP and complement factor 5a. Presence of the calcium chelator BAPTA prevented the activation-associated changes in [Ca 2+]i and restored much of the signaling efficacy. We also evaluated downstream consequences of a basal [Ca 2+]i lifting during microglial activation and found BAPTA to strongly attenuate the LPS-induced release of nitric oxide (NO) and certain cytokines and chemokines. Furthermore, microglial treatment with ionomycin, an ionophore elevating basal [Ca 2+]i, mimicked the activation-induced calcium signal suppression but failed to induce release activity on its own. Our findings suggest that chronic elevation of basal [Ca 2+]i attenuates receptor-triggered calcium signaling. Moreover, increased [Ca 2+]i is required, but by itself is not sufficient, for release of NO and certain cytokines and chemokines. Elevation of basal [Ca 2+]i could thus prove a central element in the regulation of executive functions in activated microglia.

We have generated transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. In all regions of the CNS, such as cortex, cerebellum, striatum, corpus callosum, hippocampus, retina, and spinal cord, EGFP-positive cells with morphological properties of astrocytes could be readily visualized by direct fluorescence microscopy in living brain slices or whole mounts. Also in the PNS, nonmyelinating Schwann cells from the sciatic nerve could be identified by their bright green fluorescence. Highest EGFP expression was found in the cerebellum. Already in acutely prepared whole brain, the cerebellum appeared green-yellowish under normal daylight. Colabeling with GFAP antibodies revealed an overlap with EGFP in the majority of cells. Some brain areas, however, such as retina or hypothalamus, showed only low levels of EGFP expression, although the astrocytes were rich in GFAP. In contrast, some areas that were poor in immunoreactive GFAP were conspicuous for their EGFP expression. Applying the patch clamp technique in brain slices, EGFP-positive cells exhibited two types of membrane properties, a passive membrane conductance as described for astrocytes and voltage-gated channels as described for glial precursor cells. Electron microscopical investigation of ultrastructural properties revealed EGFP-positive cells enwrapping synapses by their fine membrane processes. EGFP-positive cells were negative for oligodendrocyte (MAG) and neuronal markers (NeuN). As response to injury, i.e., by cortical stab wounds, enhanced levels of EGFP expression delineated the lesion site and could thus be used as a live marker for pathology.

Pathologic impacts in the brain lead to a widespread activation of microglial cells far beyond the site of injury. Here, we demonstrate that glial Ca2+ waves can trigger responses in microglial cells. We elicited Ca2+ waves in corpus callosum glial cells by electrical stimulation or local adenosine triphosphate (ATP) ejection in acute brain slices. Macroglial cells, but not microglia, were bulk-loaded with Ca2+-sensitive dyes. Using a transgenic animal in which astrocytes were labeled by the enhanced green fluorescence protein (EGFP) allowed us to identify the reacting cell populations: the wave activated a Ca2+ response in both astrocytes and non-astrocytic glial cells and spread over hundreds of micrometers even into the adjacent cortical and ventricular cell layers. Regenerative ATP release and subsequent activation of metabotropic purinergic receptors caused the propagation of the glial Ca2+ wave: the wave was blocked by the purinergic receptor antagonist Reactive Blue 2 and was not affected by the gap junction blocker octanol, but enhanced in Ca2+ free saline. To test whether microglial cells respond to the wave, microglial cells were labeled with a dye-coupled lectin and membrane currents were recorded with the patch-clamp technique. When the wave passed by, a current with the characteristics of a purinergic response was activated. Thus, Ca2+ waves in situ are not restricted to astrocytic cells, but broadly activate different glial cell types.

A complete understanding of the molecular mechanisms involved in the formation and repair of the central nervous system myelin sheath requires an unambiguous identification and isolation of in vivo-differentiated myelin-forming cells. In order to develop a novel tool for the analysis of in vivo-differentiated oligodendrocytes, we generated transgenic mice expressing a red-shifted variant of the green fluorescent protein under the control of the proteolipid protein promoter. We demonstrate here that green fluorescent protein-derived fluorescence in the central nervous system of 9-day- to 7-week-old mice is restricted to mature oligodendrocytes, as determined by its spatiotemporal appearance and by both immunocytochemical and electrophysiological criteria. Green fluorescent protein-positive oligodendrocytes could easily be visualized in live and fixed tissue. Furthermore, we show that this convenient and reliable identification now allows detailed physiological analyses of differentiated oligodendrocytes in situ. In addition, we developed a novel tissue culture system for in vivo-differentiated oligodendrocytes. Initial data using this system indicate that, for oligodendrocytes isolated after differentiation in vivo, as yet unidentified factors secreted by astrocytes are necessary for survival and/or reappearance of a mature phenotype in culture.

In the brain, N-methyl-D-aspartate (NMDA)-type glutamate receptors are important elements for the manifestation of memory as well as mediators of neurotoxicity, and they are thought to be exclusive to neurons. To test for the expression of functional NMDA receptors on astrocytes, we generated transgenic mice in which glial fibrillary acidic protein (GFAP)-positive astrocytes are labeled by a green fluorescent protein and tested their responses to NMDA in acute cortical slices by patch-clamp recording and Ca 2+ imaging. The NMDA-evoked currents reversed at 0 mV; could be blocked by MK-801; persisted in the absence of synaptic transmission; were sensitive to Mg 2+ ; and were accompanied by focal Ca 2+ elevation, indicating the presence of functional NMDA receptors. Furthermore, we detected mRNAs for NMDA receptor subunits in freshly isolated astrocytes purified by fluorescence-activated cell sorting. We conclude that processes of cortical astrocytes enwrapping synaptic regions express high densities of NMDA receptors that could be involved in neurone-glia signaling.Key words: NMDA receptor • astrocyte • glia • GFAP promoter • enhanced green fluorescent protein • patch-clamp recording • calcium imaging • mouse •ÜFRUWH[ strocytes are in intimate contact with neurons in gray matter, particularly at synapses. Several recent studies indicate that there is an intense cross-talk between these two cell types. The astrocytes express the prerequisite to participate in neuronal communication (i.e., receptors) for a variety of neurotransmitters and -hormones (1). Within neurons the information is propagated by electrical activity, but the astrocytic form of intracellular signal propagation is dominated by Ca 2+ signaling (2). These increases in cytosolic calcium can affect neuronal pathways (3). The first indications for this astrocyte-neuronal cross-talk came from cell A cultures (4), whereas recent studies demonstrate that such signal exchanges can also occur in situ. Studies in the isolated retina, for instance, illustrate that astrocytic calcium signals lead to modulation of light-induced excitation of ganglion cells (5). Glutamate receptors seem to play a key role in this neuron-glia interaction, as has been shown for the hippocampus (6).Numerous glutamate-binding molecules, such as glutamate transporters, the non-N-methyl-D-DVSDUWDWH.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (NMDA/AMPA) / kainatetype glutamate receptors, and metabotropic glutamate receptors, have been identified on astrocytes. For neuron-glia signaling via transmitter receptors, the non-NMDA receptors are the prominent communication channels, whereas there is little evidence for the involvement of NMDA receptors in astrocytes. Studies in cultured astrocytes have long demonstrated that these cells are unresponsive to NMDA but are prominently activated by non-NMDA agonists (7). Despite unanimous agreement that cultured astrocytes are devoid of functional NMDA receptors, there is some evidence that NMDA can elicit signals in astrocytes in situ. Be...

Microglial cells have important functions during regenerative processes after brain injury. It is well established that they rapidly respond to damage to the brain tissue. Stages of activation are associated with changes of cellular properties such as proliferation rate or expression of surface antigens. Yet, nothing is known about signal substances leading to the rapid changes of membrane properties, which may be required to initiate the transition from one cell stage into another. From our present study, using the patch-clamp technique, we report that cultured microglial cells obtained from mouse or rat brain respond to extracellularly applied ATP with the activation of a cation conductance. Additionally, in the majority of cells an outwardly directed K+ conductance was activated with some delay. Since ADP, AMP, and adenosine (in descending order) were less potent or ineffective in inducing the cation conductance, the involvement of a P2 purinergic receptor is proposed. The receptor activation is accompanied by an increase of cytosolic Ca2+ as determined by a fura-2-based Ca(2+)-imaging system. This ATP receptor could enable microglial cells to respond to transmitter release from nerve endings with ATP as a transmitter or cotransmitter or to the death of cells with resulting leakage of ATP.

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