Christiane Nolte

GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue.

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. GLIA 33:72–86, 2001.

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange

Mitochondrial Ca2+ efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na+-dependent mechanism mediates mitochondrial Ca2+ efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na+/Ca2+ exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca2+ and Na+ fluorescent imaging, we demonstrate that mitochondrial Na+-dependent Ca2+ efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca2+ transport was inhibited, moreover, by CGP-37157 and exhibited Li+ dependence, both hallmarks of mitochondrial Na+-dependent Ca2+ efflux. Finally, NCLX-mediated mitochondrial Ca2+ exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na+/Ca2+ exchanger.

CXCR3-Dependent Microglial Recruitment Is Essential for Dendrite Loss after Brain Lesion

Microglia are the resident macrophage population of the CNS and are considered its major immunocompetent elements. They are activated by any type of brain pathology and can migrate to the lesion site. The chemokine CXCL10 is expressed in neurons in response to brain injury and is a signaling candidate for activating microglia and directing them to the lesion site. We recently identified CXCR3, the corresponding receptor for CXCL10, in microglia and demonstrated that this receptor system controls microglial migration. We have now tested the impact of CXCR3 signaling on cellular responses after entorhinal cortex lesion. In wild-type mice, microglia migrate within the first 3 d after lesion into the zone of axonal degeneration, where 8 d after lesion denervated dendrites of interneurons are subsequently lost. In contrast, the recruitment of microglia was impaired in CXCR3 knock-out mice, and, strikingly, denervated distal dendrites were maintained in zones of axonal degeneration. No differences between wild-type and knock-out mice were observed after facial nerve axotomy, as a lesion model for assessing microglial proliferation. This shows that CXCR3 signaling is crucial in microglia recruitment but not proliferation, and this recruitment is an essential element for neuronal reorganization.

Complement 5a controls motility of murine microglial cells in vitro via activation of an inhibitory G-protein and the rearrangement of the actin cytoskeleton

Microglial cells respond to most pathological events by rapid transformation from a quiescent to an activated phenotype characterized by increased cytotoxicity and motile activity. To investigate the regulation of microglial motility by different inflammatory mediators, we studied cultured murine microglia by time-lapse video microscopy and a computer-based motility assay. Microglial cells exhibited a high resting motility. The acute application of complement 5a (C5a) immediately induced intense ruffling of microglial membranes followed by lamellipodia extension within few seconds, while formyl-Met-Leu-Phe-OH, bacterial endotoxin (lipopolysaccharide) or inflammatory cytokines did not increase motility. This process was accompanied by a rapid rearrangement of the actin cytoskeleton as demonstrated by labelling with fluorescein isothiocyanate-phalloidin and could be inhibited by cytochalasin B. A GTP-binding protein was involved in the signal cascade, since pertussis toxin inhibited motility and actin assembly in response to C5a. Chemotactic migration in a gradient of C5a was also completely blocked by pertussis toxin and cytochalasin B. The C5a-induced motility reaction was accompanied by an increase in intracellular calcium ([Ca2+]i) as measured by a Fluo-3 based imaging system. Ca2+ transients were, however, not a prerequisite for triggering the increase in motility; motility could be repeatedly evoked by C5a in nominally Ca(2+)-free solution, while Ca2+ signals occurred only upon the first stimulation. Moreover, conditions mimicking intracellular Ca2+ transients, like incubation with thapsigargin or Ca2+ ionophore A23187, were not able to induce any motility reaction, suggesting that Ca2+ transients are not necessary for, but are associated with, microglial motility. Motile activity was shown to be restricted to a defined concentration range of [Ca2+]i as revealed by lowering [Ca2+]i with BAPTA-AM or increasing [Ca2+]i with A23187. Since complement factors are released at pathological sites, this signal cascade could serve to increase motility and to direct microglial cells to the lesioned or damaged area by means of a G-protein-dependent pathway and via the rearrangement of the actin cytoskeleton.

Astrocytes of the mouse neocortex express functional N-methyl-D-aspartate receptors.

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 Ca2+ 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 Mg2+; and were accompanied by focal Ca2+ 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.

Phagocytic clearance of apoptotic neurons by Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition.

Abstract: Microglia, the tissue macrophages of the brain, play a crucial role in recognition and phagocytic removal of apoptotic neurons. The microglial receptors for recognition of apoptotic neurons are not yet characterized. Here we established a co‐culture model of primary microglia and cerebellar granule neurons to examine the receptor systems involved in recognition/uptake of apoptotic neurons. Treatment with 100 μM S‐nitrosocysteine induced apoptosis of cerebellar neurons as indicated by nuclear condensation and phosphatidylserine exposure to the exoplasmic leaflet of the plasma membrane. Microglial cells were added to neurons 2 h after apoptosis induction and co‐cultured for 6 h in the presence of ligands that inhibit recognition by binding to respective receptors. Binding/phagocytosis was determined after combined 4′,6‐diamidino‐2‐phenylindole/propidium iodide (for apoptotic/necrotic neurons) and lectin staining (for microglia). Uptake of apoptotic neurons was reduced by N‐acetylglucosamine or galactose, suggesting that recognition involves asialoglycoprotein‐like lectins. Furthermore, the inhibition of microglial binding/uptake of apoptotic neurons by RGDS peptide suggests a role of microglial vitronectin receptor. As microglia selectively bind lipid vesicles enriched in phosphatidylserine and O‐phospho‐L‐serine interfered with the uptake of apoptotic neurons, an involvement of phosphatidylserine receptor is rather likely. Apoptotic neurons do not release soluble signals that serve to attract or activate microglia. Collectively, these results suggest that apoptotic neurons generate a complex surface signal recognized by different receptor systems on microglia.

Secondary Lymphoid Tissue Chemokine (CCL21) Activates CXCR3 to Trigger a Cl− Current and Chemotaxis in Murine Microglia

Microglial cells represent the major immunocompetent element of the CNS and are activated by any type of brain injury or disease. A candidate for signaling neuronal injury to microglial cells is the CC chemokine ligand CCL21, given that damaged neurons express CCL21. Investigating microglia in acute slices and in culture, we demonstrate that a local application of CCL21 for 30 s triggered a Cl− conductance with lasted for tens of minutes. This response was sensitive to the Cl− channel blockers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and 4-acetamide-4′-isothiocyanatostilbene, 2,2′-disulfonic acid. Moreover, CCL21 triggered a chemotaxis response, which was sensitive to Cl− channel blockers. In microglial cells cultured from CCR7 knockout mice, CCL21 produced the same type of Cl− current as well as a chemotaxis response. In contrast, in microglial cells from CXCR3 knockout mice, CCL21 triggered neither a Cl− conductance nor a chemotaxis response after CCL21 application. We conclude that the CCL21-induced Cl− current is a prerequisite for the chemotaxis response mediated by the activation of CXCR3 but not CCR7 receptors, indicating that in brain CCL21 acts via a different receptor system than in lymphoid organs.

Synaptic transmission onto hippocampal glial cells with hGFAP promoter activity

Glial cells increasingly gain importance as part of the brains communication network. Using transgenic mice expressing green fluorescent protein (EGFP) under the control of the human GFAP promoter, we tested for synaptic input to identified glial cells in the hippocampus. Electron microscopic inspection identified synapse-like structures with EGFP-positive postsynaptic compartments. Sub-threshold stimulation to Schaffer collaterals resulted in stimulus-correlated, postsynaptic responses in a subpopulation of EGFP-positive cells studied with the patch-clamp technique in acute slices. This cell population can be recognized by its distinct morphology and has been termed GluR cells in a preceding study. These cells are distinct from the classical astrocytes due to their antigen profile and functional properties, but also lack characteristic features of oligodendrocytes or neurons. GluR cells also received spontaneous synaptic input. Stimulus-correlated and spontaneous responses were quantitatively analysed by ascertaining amplitude distributions, failure rates, kinetics as well as pharmacological properties. The data demonstrate that GABAergic and glutamatergic neurons directly synapse onto GluR cells and suggest a low number of neuronal release sites. These data demonstrate that a distinct type of glial cells is integrated into the synaptic circuit of the hippocampus, extending the finding that synapse-based brain information processing is not a property exclusive to neurons.

Electrophysiological properties of microglial cells in normal and pathologic rat brain slices

Microglial cells serve as pathologic sensors of the brain. They are highly abundant in all regions of the central nervous system (CNS) and are characterized by a ramified morphology within the normal tissue. In the present study, we have developed a procedure to study the membrane properties of identified, in situ microglia in acutely isolated brain slices from rat cortex, striatum and facial nucleus. Unlike the well characterized cultured microglial cells, ramified microglia of the slice are characterized by little, if any, voltage‐gated membrane currents and a very low membrane potential. They are thus distinct from neurons, other glial cells and nonbrain macrophages. To study the consequences of microglial activation on the membrane channel pattern, we compared cells in the normal facial nucleus and at defined times after facial nerve axotomy. Within 12 h of axotomy, microglial cells expressed a prominent inward rectifier current and thus acquired the physiological properties of cultured microglia. Within 24 h of the lesion, the cells expressed an additional outward current, which is typical for lipopolysaccharide (LPS)‐activated microglia in vitro. Seven days after the lesion, at a time of major regenerative processes in the facial nucleus, the physiological properties of microglial cells had reverted to those present prior to the pathological event. In conclusion: (i) ramified microglial cells represent a physiologically unique population of cells in the brain; (ii) are distinct from their cultured counterparts; and (iii), undergo a defined pattern of physiological states in the course of pathologic events.

Microglia express GABAB receptors to modulate interleukin release

gamma-Aminobutyric acid (GABA) can act as a neuroprotective agent besides its well-established role as the main inhibitory neurotransmitter in the CNS. Here we report that microglial cells express GABA(B) receptors indicating that these prominent immunocompetent cells in the brain are a target for GABA. Agonists of GABA(B) receptors triggered the induction of K(+) conductance in microglial cells from acute brain slices and in culture. Both subunits of GABA(B) receptors were identified in cultured microglia by Western blot analysis and immunocytochemistry, and were detected on a subpopulation of microglia in situ by immunohistochemistry. In response to facial nerve axotomy, we observed an increase in GABA(B) receptor expressing microglial cells in the facial nucleus. We activated microglial cells in culture with lipopolysaccharide (LPS) to induce the release of interleukin-6 and interleukin-12p40. This release activity was attenuated by simultaneous activation of the GABA(B) receptors indicating that GABA can modulate the microglial immune response.