Carsten Ohlemeyer

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.

Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices

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.

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.

Elevation of Basal Intracellular Calcium as a Central Element in the Activation of Brain Macrophages (Microglia): Suppression of Receptor-Evoked Calcium Signaling and Control of Release Function

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.

Nmda-activated currents in Bergmann glial cells

NMDA receptors play a crucial role in synaptic plasticity of the central nervous system and were thought to be exclusive to neurones. In this study we provide evidence that Bergmann glial cells from mouse cerebellar slices show intrinsic responses to NMDA. As in neurones, NMDA increased membrane conductance and the responses were blocked by the NMDA antagonist ketamine, but not by the non-NMDA glutamate receptor antagonist CNQX. In contrast to responses in neurones, the current voltage relation of the glial NMDA-induced current was linear, reversed at -40 mV, currents were not blocked by Mg2+ or enhanced by glycine and NMDA did not induce an increase in cytosolic Ca2+ as recorded with a fura-2 imaging system. These data imply the presence of distinct NMDA receptors on Bergmann glial cells; these glial receptors could be the substitute for complex neurone-glia interactions in the cerebellum.

AMPA/kainate receptor activation in murine oligodendrocyte precursor cells leads to activation of a cation conductance, calcium influx and blockade of delayed rectifying K+ channels

Studies during the last few years have shown that glial cells can express a large repertoire of neurotransmitter receptors. In this study, we have characterized the properties of a glutamate receptor in oligodendrocytes and their precursor cells from cultures of mouse brain, using the patch-clamp technique to measure ligand-activated currents and a fura-2 imaging system to determine changes in free cytosolic Ca2+ concentration ([Ca2+]i). The precursor cells were identified by their characteristic morphology and their voltage-gated currents as described previously [Sontheimer H. et al. (1989) Neuron 2, 1135-1145]. The ligands kainate, domoate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), as well as L-glutamate but not trans-1-amino-1,3-cyclopentanedicarboxylate elicited inward currents at a holding potential of -70 mV and the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the glutamate- and kainate-induced response reversibly, indicating the expression of an AMPA/kainate-type glutamate receptor. The response is due to the activation of a cationic conductance as revealed by analysing the reversal potential of the kainate-activated current. Receptor activation is accompanied by two additional responses: (i) an increase in [Ca2+]i mediated by depolarization and a subsequent activation of voltage-gated Ca2+ channels and (ii) a transient blockade of a delayed rectifying K+ current, but not of the A-type K+ current. The blockade of the K+ current was not due to the increase in [Ca2+]i since it was also observed in Ca(2+)-free bathing solution when no increase in [Ca2+]i was detectable after exposure to kainate. In contrast to precursor cells, oligodendrocytes responded weakly or not at all to glutamate or related ligands. We conclude that glutamate activates a complex pattern of physiological events in the glial precursor cells, which may play a role during the differentiation process of these cells.

Distinct Physiologic Properties of Microglia and Blood-Borne Cells in Rat Brain Slices After Permanent Middle Cerebral Artery Occlusion

The authors investigated the time course of leukocyte infiltration compared with microglial activation in adult rat brain slices after permanent middle cerebral artery occlusion (MCAO). To distinguish peripheral leukocytes from microglia, the blood cells were prelabeled in vivo with Rhodamine 6G (Rhod6G) IV before induction of ischemia. At specific times after infarct, invading leukocytes, microglia, and endothelial cells were labeled in situ with isolectin (IL)B4-FITC (ILB4). Six hours after MCAO only a few of the ILB4+ cells were colabeled by Rhod6G. These cells expressed the voltage-gated inwardly and outwardly rectifying K+ currents characteristic of macrophages. The majority of the ILB4+ cells were Rhod6G− and expressed a lack of voltage-gated channels, recently described for ramified microglial cells in brain slices, or exhibited only an inward rectifier current, a unique marker for cultured (but unstimulated) microglia. Forty-eight hours after MCAO, all blood-borne and the majority of Rhod6G− cells expressed outward and inward currents indicating that the intrinsic microglial population exhibited physiologic features of stimulated, cultured microglia. The ILB4+/Rhod6G− intrinsic microglial population was more abundant in the border zone of the infarct and their morphology changed from radial to ameboid. Within this zone, the authors observed rapidly migrating cells and recorded this movement by time-lapse microscopy. The current findings indicate that microglial cells acquire physiologic features of leukocytes at a later time point after MCAO.

Bradykinin receptors in cultured astrocytes from neonatal rat brain are linked to physiological responses

Specific binding sites for bradykinin (BK) have recently been demonstrated on astrocytes of primary cultures from neonatal rat brain. In this study we demonstrate that BK induces membrane currents in concert with an elevation of [Ca2+]i. In 67% of astrocytes, BK induced an inward current as determined with the perforated patch-clamp technique in the whole-cell recording configuration. In a small population of astrocytes (20%), a BK-activated outward current was observed, while in the remainder of the cells (13%) no apparent current responses were detected. As recorded by fura-2 microfluorimetry, the peptide induced a transient rise of [Ca2+/bdi even when the extracellular calcium was removed. In the majority of astrocytes, the selective B1-agonist des-Arg9-BK elicited physiological responses with a much lower potency, indicating that the BK receptors are predominantly of the B2 subtype. A minor population of astrocytes was present which only responded to des-Arg9-BK.

GABAA receptor-expressing astrocytes in the supraoptic nucleus lack glutamate uptake and receptor currents

An important function of astrocytes is the clearance of excess extracellular glutamate via specific carriers whose expression has become an astrocytic marker. In the present study, we found that a large population of astrocytes in the supraoptic nucleus (SON) of the rat hypothalamus lacks glutamate uptake currents and receptor responses but expresses GABAA receptors. Patch clamp recordings in acute hypothalamic slices that included the SON showed typical astrocytic membrane currents and demonstrated that GABA, via GABAA receptor activation, triggered a conductance increase with the reversal potential close to the Cl− equilibrium potential and a decrease in resting K+ conductance. Intracellular labeling with Lucifer Yellow revealed that these cells had a radial glia‐like morphology, with cell bodies lined up along the base of the brain and long processes traversing the nucleus; they were not dye‐coupled. Parallel immunocytochemical labelings showed that they expressed strong GABAA receptor and glial fibrillary acidic protein (GFAP) immunoreactivities. In addition, our electrophysiological and morphological analyses revealed another population of astrocytes in this nucleus, located next to the subarachnoid space. They were less numerous than the radial type, had a round morphology and few processes, and were dye‐coupled. Unlike the radial astrocytes, they showed little immunoreactivity for GABAA receptor or GFAP. Moreover, they did not respond to GABA but to glutamate, a response that was partially mimicked by aspartate, indicating glutamate transporter expression. Taken together, our observations add to growing evidence illustrating heterogeneity of astrocytes in the adult brain, a heterogeneity that reflects striking differences in form and function of astrocytic populations in regions as discrete as the SON of the hypothalamus.

Analysis of motile oligodendrocyte precursor cells in vitro and in brain slices

Oligodendrocyte precursor cells are purported to migrate over long distances into the various brain regions where they differentiate into oligodendrocytes and fulfill their appropriate tasks, i.e., myelination of axons. Here we characterize motile oligodendrocyte precursor cells in detail. Video–time lapse analysis was performed on isolated precursor cells in single cell cultures, in co‐culture with cerebellar microexplants, and in living brain slices. Motility analysis of individual cells was combined with electrophysiological, immunological, and morphological characterizations. Translocation of the cell bodies was not continuous but occurred in waves. All motile cells exhibited a simple morphology and most, but not all, of them expressed the A2B5 epitope in vitro. Patch clamp analysis of the motile cells confirmed that they belong to the O‐2A lineage. The percentage of motile cells, as well as their velocities, were enhanced on substrate‐coated laminin in comparison to poly‐L‐lysine. Motility was not influenced by the presence of cerebellar microexplants. O‐2A progenitor cells did not migrate strictly along neurite fascicles which were projected from the microexplants. Glial progenitor cells in situ also did not strictly migrate along the main direction of the axonal fibers of the corpus callosum but rather traversed the fibers with an overall direction toward the cortex. After Lucifer Yellow filling of the motile progenitor cells in situ, we could demonstrate that they were dye‐coupled to yet unidentified cells of the corpus callosum. GLIA 20:284–298, 1997.