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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.

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.

IntroductionRecent work on glial cell physiology has disclosed that these cells are much more actively involved in brain information processing than hitherto thought. This new insight stimulates a new view according to which the active brain has to be regarded as an integrated circuit of interactive neurons and glial cells. Astrocytes in particular are now regarded as direct communication partners of neurons, by dynamically interacting with synapses through the uptake and release of neurotransmitters and receptor-mediated intracellular Ca 2+ signalling (for reviews, see Haydon, 2001;Newman, 2003;Volterra and Steinhäuser, 2004;Schipke and Kettenmann, 2004). Intriguingly, a distinct subset of glial cells in the hippocampus was reported to receive direct synaptic input from glutamatergic and GABAergic neurons. These glial cells expressed the proteoglycan, NG2, and on this basis were regarded as oligodendrocyte precursor cells (OPCs) (Bergles et al., 2000;Lin and Bergles, 2003). However, the identity of these cells needs further consideration because the specificity of NG2 as an OPC marker becomes increasingly questionable. Current work suggests that NG2 cells comprise a distinct, heterogeneous type of neuroglial cells (Nishiyama et al., 2002;Stallcup, 2002;Greenwood and Butt, 2003;Aguirre et al., 2004;Peters, 2004).Using transgenic mice expressing green fluorescent protein under control of the human GFAP promoter (hGFAP/EGFP mice), we have recently reported a co-existence of two types of glial cells in the hippocampus, distinguishable from each other by mutually exclusive expression of glutamate transporters (GluT type) and ionotropic glutamate receptors (GluR cells). GluT type cells were extensively coupled via gap junctions and contacted blood vessels, thus matching properties of classical astrocytes. By contrast, GluR cells lacked junctional coupling and did not enwrap capillaries (Matthias et al., 2003;Wallraff et al., 2004). Moreover, GluR cells co-expressed S100␤, a common astrocyte marker, NG2, as well as neuronal genes, and hence escaped classification into neurons, astrocytes, or oligodendrocytes.Here we used the hGFAP/EGFP transgenic animal to identify distinct types of glial cells in live slices. We combined ultrastructural analysis and post-recording immunocytochemistry to test whether the two populations of hGFAP/EGFP-positive glial cells in the hippocampus receive synaptic input. Electron microscopic inspection identified synapse-like structures with EGFP-positive postsynaptic compartments. Patch clamp recordings revealed stimulus- 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 synaps...

(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.

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Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by CAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA-and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 pM cAMP and 3.1 pM catalytic subunit of PKA, which is equivalent to 6.2 pM CAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 pM cGMP and 7.3 pM PKG monomer, equivalent to 14.6 pM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 pM.Treatment of washed human platelets with 10 pM (or 10 nM) prostaglandin El (PGE1) elevated the intracellular cAMP concentration to 27 pM (10 pM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein).Treatment of washed human platelets with 100 pM (or 1 pM) sodium nitroprusside elevated the platelet cGMP level to 4 pM (0.9 pM with 1 pM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 pM sodium nitroprusside). PGEl and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increases in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cCMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA-and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation. In particular, the high concentration of the converter enzymes PKA and PKG suggest that rapidity in reaching steady-state levels of phosphorylation in response to CAMP-or cGMP-elevating agents is of considerable importance for the regulation of human platelets.Regulation of protein phosphorylation/dephosphorylation by second messengers and second-messenger-regulated protein kinases is an essential component in the signal-transduction mechanism of those hormones, growth factors,