Microglia form satellites with different neuronal subtypes in the adult murine central nervous system.

Microglia are the innate immune cells of the central nervous system (CNS). In the adult uncompromised CNS, they have a highly ramified morphology and continuously extend and retract their processes. A subpopulation of microglial cells forms close soma-to-soma contacts with neurons and have been termed satellite microglia, yet the role of such interaction is largely unknown. Here, we analyzed the distribution of satellite microglia in different areas of the CNS of adult male mice applying transgenic- and immunolabeling of neuronal subtypes and microglia followed by three-dimensional imaging analysis. We quantified satellite microglia associated with GABAergic and glutamatergic neurons in the somatosensory cortex, striatum, and thalamus; with dopaminergic and serotonergic neurons in the basal forebrain and raphe nucleus, respectively; and with cerebellar Purkinje cell neurons. Satellite microglia in the retina were assessed qualitatively. Microglia form satellites with all neuronal subtypes studied, whereas a preference for a specific neuron subtype was not found. The occurrence and frequency of satellite microglia is determined by the histo-architectural organization of the brain area and the densities of neuronal somata therein.

Mitochondrial exchanger NCLX plays a major role in the intracellular Ca2+ signaling, gliotransmission, and proliferation of astrocytes.

Mitochondria not only provide cells with energy, but are central to Ca(2+) signaling. Powered by the mitochondrial membrane potential, Ca(2+) enters the mitochondria and is released into the cytosol through a mitochondrial Na(+)/Ca(2+) exchanger. We established that NCLX, a newly discovered mitochondrial Na(+)/Ca(2+) exchanger, is expressed in astrocytes isolated from mice of either sex. Immunoblot analysis of organellar fractions showed that the location of NCLX is confined to mitochondria. Using pericam-based mitochondrial Ca(2+) imaging and NCLX inhibition either by siRNA or by the pharmacological blocker CGP37157, we demonstrated that NCLX is responsible for mitochondrial Ca(2+) extrusion. Suppression of NCLX function altered cytosolic Ca(2+) dynamics in astrocytes and this was mediated by a strong effect of NCLX activity on Ca(2+) influx via store-operated entry. Furthermore, Ca(2+) influx through the store-operated Ca(2+) entry triggered strong, whereas ER Ca(2+) release triggered only modest mitochondrial Ca(2+) transients, indicating that the functional cross talk between the plasma membrane and mitochondrial domains is particularly strong in astrocytes. Finally, silencing of NCLX expression significantly reduced Ca(2+)-dependent processes in astrocytes (i.e., exocytotic glutamate release, in vitro wound closure, and proliferation), whereas Ca(2+) wave propagation was not affected. Therefore, NCLX, by meditating astrocytic mitochondrial Na(+)/Ca(2+) exchange, links between mitochondria and plasma membrane Ca(2+) signaling, thereby modulating cytoplasmic Ca(2+) transients required to control a diverse array of astrocyte functions.

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

Mitochondrial Ca(2+) efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na(+)-dependent mechanism mediates mitochondrial Ca(2+) efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na(+)/Ca(2+) exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca(2+) and Na(+) fluorescent imaging, we demonstrate that mitochondrial Na(+)-dependent Ca(2+) 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 Ca(2+) transport was inhibited, moreover, by CGP-37157 and exhibited Li(+) dependence, both hallmarks of mitochondrial Na(+)-dependent Ca(2+) efflux. Finally, NCLX-mediated mitochondrial Ca(2+) 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(+)/Ca(2+) exchanger.

TRPM3 is expressed in sphingosine-responsive myelinating oligodendrocytes.

Oligodendrocytes are the myelin-forming cells of the CNS and guarantee proper nerve conduction. Sphingosine, one major component of myelin, has recently been identified to activate TRPM3, a member of the melastatin-related subfamily of transient receptor potential (TRP) channels. TRPM3 has been demonstrated to be expressed in brain with unknown cellular distribution. Here, we show for the first time that TRPM3 is expressed in oligodendrocytes in vitro and in vivo. TRPM3 is present during oligodendrocyte differentiation. Immunohistochemistry of adult rat brain slices revealed staining of white matter areas, which co-localized with oligodendrocyte markers. Analysis of the developmental distribution revealed that, prior to myelination, TRPM3 channels are localized on neurons. On oligodendrocytes they are found after the onset of myelination. RT-PCR studies showed that the transcription of TRPM3 splice variants is also developmentally regulated in vitro. Ca(2+) imaging approaches revealed the presence of a sphingosine-induced Ca(2+) entry mechanism in oligodendrocytes - with a pharmacological profile similar to the profile published for heterologously expressed TRPM3. These findings indicate that TRPM3 participates as a Ca(2+)-permeable and sphingosine-activated channel in oligodendrocyte differentiation and CNS myelination.

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ.

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.

Bradykinin-induced microglial migration mediated by B1-bradykinin receptors depends on Ca2+ influx via reverse-mode activity of the Na+/Ca2+ exchanger.

Bradykinin (BK) is produced and acts at the site of injury and inflammation. In the CNS, migration of microglia toward the lesion site plays an important role pathologically. In the present study, we investigated the effect of BK on microglial migration. Increased motility of cultured microglia was mimicked by B1 receptor agonists and markedly inhibited by a B1 antagonist but not by a B2 receptor antagonist. BK induced chemotaxis in microglia isolated from wild-type and B2-knock-out mice but not from B1-knock-out mice. BK-induced motility was not blocked by pertussis toxin but was blocked by chelating intracellular Ca2+ or by low extracellular Ca2+, implying that Ca2+ influx is prerequisite. Blocking the reverse mode of Na+/Ca2+ exchanger (NCX) completely inhibited BK-induced migration. The involvement of NCX was further confirmed by using NCX+/- mice; B1-agonist-induced motility and chemotaxis was decreased compared with that in NCX+/+ mice. Activation of NCX seemed to be dependent on protein kinase C and phosphoinositide 3-kinase, and resultant activation of intermediate-conductance (IK-type) Ca2+-dependent K+ currents (I(K(Ca))) was activated. Despite these effects, BK did not activate microglia, as judged from OX6 staining. Using in vivo lesion models and pharmacological injection to the brain, it was shown that microglial accumulation around the lesion was also dependent on B1 receptors and I(K(Ca)). These observations support the view that BK functions as a chemoattractant by using the distinct signal pathways in the brain and, thus, attracts microglia to the lesion site in vivo.

Oligodendrocytes in the Mouse Corpus Callosum Maintain Axonal Function by Delivery of Glucose.

In the optic nerve, oligodendrocytes maintain axonal function by supplying lactate as an energy substrate. Here, we report that, in acute brain slices of the mouse corpus callosum, exogenous glucose deprivation (EGD) abolished compound action potentials (CAPs), which neither lactate nor pyruvate could prevent. Loading an oligodendrocyte with 20 mM glucose using a patch pipette prevented EGD-mediated CAP reduction in about 70% of experiments. Loading oligodendrocytes with lactate rescued CAPs less efficiently than glucose. In mice lacking connexin 47, oligodendrocyte filling with glucose did not prevent CAP loss, emphasizing the importance of glial networks for axonal energy supply. Compared with the optic nerve, the astrocyte network in the corpus callosum was less dense, and loading astrocytes with glucose did not prevent CAP loss during EGD. We suggest that callosal oligodendrocyte networks provide energy to sustain axonal function predominantly by glucose delivery, and mechanisms of metabolic support vary across different white matter regions.

Actin dynamics shape microglia effector functions.

Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

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.

Requirement of functional ryanodine receptor type 3 for astrocyte migration.

Astrocyte motility plays an important role in the response of the brain to injury and during regeneration. We used two in vitro assays, a wound-healing model and a chemotaxis assay, to study mechanisms that control astrocyte motility. Ryanodine receptors (RyR), intracellular calcium-release channels, modulate intracellular Ca2+ levels, and also motility: 1) blocking RyR with antagonizing concentration of ryanodine (200 microM) strongly attenuated motility and 2) motility of astrocytes cultured from homozygous RyR type 3 knockout mice was impaired strongly compared with wild-type. In contrast, MIP-1a-induced chemotaxis was neither impaired in the presence of ryanodine nor in the cells from the knockout animals. Reverse transcription-polymerase chain reaction (RT-PCR) analysis combined with Western blotting and immunocytochemistry confirmed the expression of RyR type 3, but not type 1 or 2 in cultured and acutely isolated astrocytes. RyR in astrocytes are linked to Ca2+ signaling because the RyR agonist 4-chloro-m-cresol induced a release of Ca2+ from intracellular stores. These results indicate that astrocytes express only RyR type 3 and that this receptor is important for controlling astrocyte motility.

Accumulation of N-CAM 180 at Contact Sites Between Neuroblastoma Cells and Latex Beads Coated with Extracellular Matrix Molecules.

Neuronal cells expressing neural cell adhesion molecule (N-CAM) accumulate the largest N-CAM component (N-CAM 180) at cell - cell contact sites. To test whether this accumulation is induced by interactions at the surface membrane, latex beads coated with several purified adhesion molecules or extracellular matrix (ECM) components were co-cultured with neuroblastoma cells. Beads coated with L1, N-CAM, the L2/HNK-1 carbohydrate epitope-carrying molecules from adult mouse brain or laminin from Engelbreth-Holm-Swarm (EHS) sarcoma did not induce an accumulation of N-CAM 180 or L1 at sites of contact suggesting that these molecules are not directly involved in N-CAM 180 accumulation or that their mobility is required for this process. Beads coated with ECM components of the PF-HR9 cell line induced accumulation of N-CAM 180 at sites of contact with neuroblastoma cells. Accumulation was seen at cell bodies of undifferentiated and differentiated neuroblastoma cells, as well as on neurites and growth cones of differentiated neuroblastoma cells. Accumulation of the neural adhesion molecule L1 was also seen, but less prominently and reproducibly. These observations suggest that molecules of the ECM can directly or indirectly, e.g. via molecules linked to N-CAM 180 on the cell surface, induce accumulation of N-CAM 180.

Glioma-associated microglia and macrophages/monocytes display distinct electrophysiological properties and do not communicate via gap junctions.

Both brain-resident microglia and peripheral macrophages/monocytes infiltrate into glioma and promote glioma growth. In the present study we analyzed coupling and membrane currents in glioma-associated microglia and macrophages/monocytes and compared this to control and stab wound-associated microglia. Using the Cx3cr1(GFP/wt)Ccr2(RFP/wt) knock-in mouse line, we distinguished membrane currents of glioma-associated microglia and macrophages/monocytes in acute brain slices prepared 14-16 days after inoculation of GL261 glioma cells. The current profile of microglia showed inward rectifying currents reminiscent of an intermediate activation state when compared to other disease models or cell culture. Macrophages/monocytes showed a higher specific outward conductance and a significantly lower capacitance indicative of a smaller membrane area than microglia. As controls, we also recorded currents from control microglia and stab wound-associated microglia. Since there are reports of microglial coupling in vitro, we injected biocytin into these cells and analyzed for cell coupling after fixing the slices and processed for biocytin labeling with Cy3-conjugated-Streptavidin. Neither control microglia nor glioma-associated microglia and macrophages/monocytes nor stab wound-associated microglia showed any sign of coupling. Moreover, performing qRT-PCR revealed that no connexin43 was detectable on isolated and sorted glioma-associated microglia and macrophages/monocytes, indicating that these cells are not part of a coupled network.

Neuron-astrocyte interactions in the medial nucleus of the trapezoid body.

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca(2+) responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca(2+) activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca(2+) responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized.

The principal neurons of the medial nucleus of the trapezoid body and NG2(+) glial cells receive coordinated excitatory synaptic input.

Glial cell processes are part of the synaptic structure and sense spillover of transmitter, while some glial cells can even receive direct synaptic input. Here, we report that a defined type of glial cell in the medial nucleus of the trapezoid body (MNTB) receives excitatory glutamatergic synaptic input from the calyx of Held (CoH). This giant glutamatergic terminal forms an axosomatic synapse with a single principal neuron located in the MNTB. The NG2 glia, as postsynaptic principal neurons, establish synapse-like structures with the CoH terminal. In contrast to the principal neurons, which are known to receive excitatory as well as inhibitory inputs, the NG2 glia receive mostly, if not exclusively, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor-mediated evoked and spontaneous synaptic input. Simultaneous recordings from neurons and NG2 glia indicate that they partially receive synchronized spontaneous input. This shows that an NG2(+) glial cell and a postsynaptic neuron share presynaptic terminals.

The ectonucleotidase cd39/ENTPDase1 modulates purinergic-mediated microglial migration.

Microglia is activated by brain injury. They migrate in response to ATP and although adenosine alone has no effect on wild type microglial migration, we show that inhibition of adenosine receptors impedes ATP triggered migration. CD39 is the dominant cellular ectonucleotidase that degrades nucleotides to nucleosides, including adenosine. Importantly, ATP fails to stimulate P2 receptor mediated migration in cd39(-/-) microglia. However, the effects of ATP on migration in cd39(-/-) microglia can be restored by co-stimulation with adenosine or by addition of a soluble ectonucleotidase. We also tested the impact of cd39-deletion in a model of ischemia, in an entorhinal cortex lesion and in the facial nucleus after facial nerve lesion. The accumulation of microglia at the pathological sites was markedly decreased in cd39(-/-) animals. We conclude that the co-stimulation of purinergic and adenosine receptors is a requirement for microglial migration and that the expression of cd39 controls the ATP/adenosine balance.

Neuroprotective role of bradykinin because of the attenuation of pro-inflammatory cytokine release from activated microglia.

Bradykinin (BK) has been reported to be a mediator of brain damage in acute insults. Receptors for BK have been identified on microglia, the pathologic sensors of the brain. Here, we report that BK attenuated lipopolysaccharide (LPS)-induced release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta from microglial cells, thus acting as an anti-inflammatory mediator in the brain. This effect was mimicked by raising intracellular cAMP or stimulating the prostanoid receptors EP2 and EP4, while it was abolished by a cAMP antagonist, a prostanoid receptor antagonist, or by an inhibitor of the inducible cyclooxygenase (cyclooxygenase-2). BK also enhanced formation of prostaglandin E(2) and expression of microsomal prostaglandin E synthase. Expression of BK receptors and EP2/EP4 receptors were also enhanced. Using physiological techniques, we identified functional BK receptors not only in culture, but also in microglia from acute brain slices. BK reduced LPS-induced neuronal death in neuron-microglia co-cultures. This was probably mediated via microglia as it did not affect TNF-alpha-induced neuronal death in pure neuronal cultures. Our data imply that BK has anti-inflammatory and neuroprotective effects in the central nervous system by modulating microglial function.

The potassium channels Kv1.5 and Kv1.3 modulate distinct functions of microglia.

Activation of microglia by LPS leads to an induction of cytokine and NO release, reduced proliferation and increased outward K(+) conductance, the latter involving the activation of Kv1.5 and Kv1.3 channels. We studied the role of these channels for microglial function using two strategies to interfere with channel expression, a Kv1.5 knockout (Kv1.5(-/-)) mouse and an antisense oligonucleotide (AO) approach. The LPS-induced NO release was reduced by AO Kv1.5 and completely absent in the Kv1.5(-/-) animal; the AO Kv1.3 had no effect. In contrast, proliferation was augmented with both, loss of Kv1.3 or Kv1.5 channel expression. After facial nerve lesion, proliferation rate was higher in Kv1.5(-/-) animals as compared to wild type. Patch clamp experiments confirmed the reduction of the LPS-induced outward current amplitude in Kv1.5(-/-) microglia as well as in Kv1.5- or Kv1.3 AO-treated cells. Our study indicates that induction of K(+) channel expression is a prerequisite for the full functional spectrum of microglial activation.

A1 adenosine receptors in microglia control glioblastoma-host interaction.

We report that experimental glioblastoma grow more vigorously in A(1) adenosine receptor (A(1)AR)-deficient mice associated with a strong accumulation of microglial cells at and around the tumors. A(1)ARs were prominently expressed in microglia associated with tumor cells as revealed with immunocytochemistry but low in microglia in the unaffected brain tissue. The A(1)AR could also be detected on microglia from human glioblastoma resections. To study functional interactions between tumor and host cells, we studied glioblastoma growth in organotypical brain slice cultures. A(1)AR agonists suppressed tumor growth. When, however, microglial cells were depleted from the slices, the agonists even stimulated tumor growth. Thus, adenosine attenuates glioblastoma growth acting via A(1)AR in microglia.

Synaptic transmission onto hippocampal glial cells with hGFAP promoter activity.

Glial cells increasingly gain importance as part of the brain's 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.