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Astrocytes are organized as communicating cellular networks where each cell is connected to others via gap junctions. These connections are not pervasive and there is evidence for the existence of subgroups composed by preferentially connected cells. Despite being unclear how these are established, we hypothesized lineage might contribute to the establishment of these subgroups. To characterize the functional coupling of clonally related astrocytes, we performed intracellular dye injections in clones of astrocytes labeled with the StarTrack method. This methodology revealed sibling astrocytes are preferentially connected when compared to other surrounding astrocytes. These results suggest the role of the developmental origin in the organization of astrocytes as intercellular networks., (© 2019 Wiley Periodicals, Inc.)

Gap junctions (GJs) are dynamic structures composed of hexamers of connexins (Cxs), a class of transmembrane proteins enabling channel-mediated direct intercellular communication through cell-cell diffusion of ions and small metabolites. In defined conditions, Cxs also work as hemichannels allowing exchanges between the cytoplasm and the extracellular medium. The most common GJ channel is formed by connexin 43 (Cx43) and plays an important role in physiological and pathological processes in excitable tissues, such as heart and brain. Hence, Cx43 has been largely envisioned as a new therapeutic target in cancer, neurological and psychiatric indications, or cardiovascular diseases. Identifying new pharmacological inhibitors of Cx43 GJs with different mechanisms of action and from diverse chemical classes is thus highly challenging. We present here a high-content screening method, based on the evaluation of fluorescent dye transfer rates between adjacent cells to monitor the function of GJs in U251 glioblastoma cells expressing high levels of Cx43. This assay was validated using well-described pharmacological GJ inhibitors such as mefloquine. The method was adapted to screen a library of 1,280 Food and Drug Administration- and European Medicines Agency-approved drugs that led to the selection of both known and new inhibitors of GJ channel function. We further focused on a specific class of microtubule-targeting agents, confirming that a proper tubulin network is required for functional Cx43 GJ channels.

By 2040 neurodegenerative diseases will become the world's second leading cause of death after cardiovascular disease (WHO). Major efforts are required to elucidate the underlying molecular and cellular mechanisms of neurodegenerative diseases. Connexin and pannexin membrane channel proteins are conduits through which neuronal, glial, and vascular tissues interact. In the normal brain, this interaction underlies homeostasis, metabolic supply and neuroprotection. In models of neuroinflammation these channels present aberrant functioning. Validation of the molecular mechanisms by which these membrane channels influence neurodegeneration particularly in Alzheimer's disease could lead to new and alternative therapeutic strategies targeting these channels., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Intercellular communication through gap junction channels plays a key role in cellular homeostasis and in synchronizing physiological functions, a feature that is modified in number of pathological situations. In the brain, astrocytes are the cell population that expresses the highest amount of gap junction proteins, named connexins. Several techniques have been used to assess the level of gap junctional communication in astrocytes, but so far they remain very difficult to apply in adult brain tissue. Here, using specific loading of astrocytes with sulforhodamine 101, we adapted the gap-FRAP (Fluorescence Recovery After Photobleaching) to acute hippocampal slices from 9 month-old adult mice. We show that gap junctional communication monitored in astrocytes with this technique was inhibited either by pharmacological treatment with a gap junctional blocker or in mice lacking the two main astroglial connexins, while a partial inhibition was measured when only one connexin was knocked-out. We validate this approach using a mathematical model of sulforhodamine 101 diffusion in an elementary astroglial network and a quantitative analysis of the exponential fits to the fluorescence recovery curves. Consequently, we consider that the adaptation of the gap-FRAP technique to acute brain slices from adult mice provides an easy going and valuable approach that allows overpassing this age-dependent obstacle and will facilitate the investigation of gap junctional communication in adult healthy or pathological brain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Antidepressants, prescribed as first line treatment of neuropathic pain, have a limited efficacy and poorly tolerated side effects. Because recent studies pointed out the implication of astroglial connexins (Cx) in both neuropathic pain and antidepressive treatment, we investigated whether their blockade by mefloquine could modulate the action of the tricyclic antidepressant amitriptyline. Using primary cultures, we found that both mefloquine and amitriptyline inhibited Cx43-containing gap junctions, and that the drug combination acted synergically. We then investigated whether mefloquine could enhance amitriptyline efficacy in a preclinical model of neuropathic pain. Sprague-Dawley rats that underwent chronic unilateral constriction injury (CCI) to the sciatic nerve (SN) were treated with either amitriptyline, mefloquine or the combination of both drugs. Whereas acute treatments were ineffective, chronic administration of amitriptyline reduced CCI-SN-induced hyperalgesia-like behavior, and this effect was markedly enhanced by co-administration of mefloquine, which was inactive on its own. No pharmacokinetic interactions between both drugs were observed and CCI-SN-induced neuroinflammatory and glial activation markers remained unaffected by these treatments in dorsal root ganglia and spinal cord. Mechanisms downstream of CCI-SN-induced neuroinflammation and glial activation might therefore be targeted. Connexin inhibition in astroglia could represent a promising approach towards improving neuropathic pain therapy by antidepressants., Competing Interests: This work was supported by the Commissariat à l’Energie Atomique and Theranexus Company. TJ, AD, CP, FM and MC are employees of Theranexus. The other authors declare no potential conflict of interest.

Astrocytes represent a major non-neuronal cell population actively involved in brain functions and pathologies. They express a large amount of gap junction proteins that allow communication between adjacent glial cells and the formation of glial networks. In addition, these membrane proteins can also operate as hemichannels, through which "gliotransmitters" are released, and thus contribute to neuroglial interaction. There are now reports demonstrating that alterations of astroglial gap junction communication and/or hemichannel activity impact neuronal and synaptic activity. Two decades ago we reported that several general anesthetics inhibited gap junctions in primary cultures of astrocytes (Mantz et al., (1993) Anesthesiology 78(5):892-901). As there are increasing studies investigating neuroglial interactions in anesthetized mice, we here updated this previous study by employing acute cortical slices and by characterizing the effects of general anesthetics on both astroglial gap junctions and hemichannels. As hemichannel activity is not detected in cortical astrocytes under basal conditions, we treated acute slices with the endotoxin LPS or proinflammatory cytokines to induce hemichannel activity in astrocytes, which in turn activated neuronal hemichannels. We studied two extensively used anesthetics, propofol and ketamine, and the more recently developed dexmedetomidine. We report that these drugs have differential inhibitory effects on gap junctional communication and hemichannel activity in astrocytes when used in their respective, clinically relevant concentrations, and that dexmedetomidine appears to be the least effective on both channel functions. In addition, the three anesthetics have similar effects on neuronal hemichannels. Altogether, our observations may contribute to optimizing the selection of anesthetics for in vivo animal studies., (© 2015 Wiley Periodicals, Inc.)

Sleep-wake cycle is characterized by changes in neuronal network activity. However, for the last decade there is increasing evidence that neuroglial interaction may play a role in the modulation of sleep homeostasis and that astrocytes have a critical impact in this process. Interestingly, astrocytes are organized into communicating networks based on their high expression of connexins, which are the molecular constituents of gap junction channels. Thus, neuroglial interactions should also be considered as the result of the interplay between neuronal and astroglial networks. Here, we investigate the effect of modafinil, a wakefulness-promoting agent, on astrocyte gap junctional communication. We report that in the cortex modafinil injection increases the expression of mRNA and protein of connexin 30 but not those of connexin 43, the other major astroglial connexin. These increases are correlated with an enhancement of intercellular dye coupling in cortical astrocytes, which is abolished when neuronal activity is silenced by tetrodotoxin. Moreover, gamma-hydroxybutyric acid, which at a millimolar concentration induces sleep, has an opposite effect on astroglial gap junctions in an activity-independent manner. These results support the proposition that astroglia may play an important role in complex physiological brain functions, such as sleep regulation, and that neuroglial networking interaction is modified during sleep-wake cycle. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'., (Copyright © 2013. Published by Elsevier Ltd.)

A hallmark of neurodegenerative diseases is the reactive gliosis characterized by a phenotypic change in astrocytes and microglia. This glial response is associated with modifications in the expression and function of connexins (Cxs), the proteins forming gap junction channels and hemichannels. Increased Cx expression is detected in most reactive astrocytes located at amyloid plaques, the histopathological lesions typically present in the brain of Alzheimer's patients and animal models of the disease. The activity of Cx channels analyzed in vivo as well as in vitro after treatment with the amyloid β peptide is also modified and, in particular, hemichannel activation may contribute to neuronal damage. In this review, we summarize and discuss recent data that suggest glial Cx channels participate in the neurodegenerative process of Alzheimer's disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the brain, glia represents the cell population that expresses the highest level of connexins, the membrane protein constituents of gap junction channels and hemichannels. This statement has initially led to propose the existence of a glial syncytium. Since then, functional studies have established that connexin channel-mediated communication between glial cells was more restricted and plastic that primarily thought. In particular, this is the case for astrocytes that form functional networks of communicating cells. Altogether these findings lead to reconsider the interaction between neurons and glia that should not be solely studied at the single cell level but also at a more integrated level as the interplay between neuronal circuits and glial networks., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Astrocytes are extensively coupled through gap junctions (GJs) that are composed of channels mostly constituted by connexin43 (Cx43). This astroglial gap junctional intercellular communication (GJIC) allows propagation of ions and signaling molecules critical for neuronal activity and survival. It is drastically inhibited by a short-term exposure to endothelin-1 (ET-1) or to sphingosine-1-phosphate (S1P), both compounds being inflammatory mediators acting through activation of GTP-binding protein-coupled receptors (GPCRs). Previously, we have identified the GTPases G(i/o) and Rho as key actors in the process of S1P-induced inhibition. Here, we asked whether similar mechanisms underlied the effects of ET-1 and S1P by investigating changes in the phosphorylation status of Cx43 and in the molecular associations of Cx43 with zonula occludens (ZO) proteins and occludin. We showed that the inhibitory effect of ET-1 on GJIC was entirely dependent on the activation of G(i/o) but not on Rho and Rho-associated kinase. Both ET-1 and S1P induced dephosphorylation of Cx43 located at GJs through a process mediated by G(i/o) and calcineurin. Thanks to co-immunoprecipitation approaches, we found that a population of Cx43 (likely junctional Cx43) was associated to ZO-1-ZO-2-occludin multiprotein complexes and that acute treatments of astrocytes with ET-1 or S1P induced a G(i/o)-dependent increase in the amount of Cx43 linked to these complexes. As a whole, this study identifies a new mechanism of GJIC regulation in which two GPCR agonists dynamically alter interactions of Cx43 with its molecular partners., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Astrocytes express high levels of connexin43, a protein that forms two types of channels: gap junction channels for direct intercellular communication, and hemichannels for exchanges with the extracellular space. Inflammation induces connexin43 hemichannel activation, which has been proposed to be involved in neuroglial interactions. Here, we investigated the contribution of connexin43 to NMDA-induced excitotoxicity in neuron/astrocyte co-cultures, after treatment with a pro-inflammatory cytokine mixture, containing TNF-alpha and IL1-beta (Mix), that stimulated astroglial connexin43 hemichannel activity. Interestingly, NMDA treatment induced a higher amount of neurotoxicity in Mix-treated co-cultures than in untreated ones, whereas this extent of neurotoxicity was absent in enriched neuron cultures or in co-cultures with connexin43 knock-out astrocytes. Furthermore, application of connexin43 hemichannel blockers or a synthetic cannabinoid prevented the Mix-induced potentiated NMDA neurotoxicity. Altogether, these data demonstrate that inflammation-induced astroglial hemichannel activation plays a critical role in neuronal death and suggest a neuroprotective role of connexin43 hemichannel blockade.

This review gives an overview of connexin expression in glial cells of the central nervous system, the different modes of connexin action, including gap junctional channels and hemichannels, as well as the available methodologies to measure their activity. We summarize the strengths and limitations of current pharmacological and genetic approaches to interfere with connexin channel functions. We outline new avenues not only to study specific mechanisms by which connexins exert these functions but also to selectively investigate well-defined coupling compartments among glial networks., (Copyright 2009 Elsevier B.V. All rights reserved.)

Gap junctions in astrocytes play a crucial role in intercellular communication by supporting both biochemical and electrical coupling between adjacent cells. Despite the critical role of electrical coupling in the network organization of these glial cells, the electrophysiological properties of gap junctions have been characterized in cultures while no direct evidence has been sought in situ. In the present study, gap-junctional currents were investigated using simultaneous dual whole-cell patch-clamp recordings between astrocytes from rat hippocampal slices. Bidirectional electrotonic coupling was observed in 82% of the cell pairs with an average coupling coefficient of 5.1%. Double patch-clamp analysis indicated that junctional currents were independent of the transjunctional voltage over a range from -100 to +110 mV. Interestingly, astrocytic electrical coupling displayed weak low-pass filtering properties compared to neuronal electrical synapses. Finally, during uncoupling processes triggered by either the gap-junction inhibitor carbenoxolone or endothelin-1, an increase in the input resistance in the injected cell paralleled the decrease in the coupling coefficient. Altogether, these results demonstrate that hippocampal astrocytes are electrically coupled through gap-junction channels characterized by properties that are distinct from those of electrical synapses between neurons. In addition, gap-junctional communication is efficiently regulated by endogenous compounds. This is taken to represent a mode of communication that may have important implications for the functional role of astrocyte networks in situ.

In normal brain, neurons, astrocytes, and oligodendrocytes, the most abundant and active cells express pannexins and connexins, protein subunits of two families forming membrane channels. Most available evidence indicates that in mammals endogenously expressed pannexins form only hemichannels and connexins form both gap junction channels and hemichannels. Whereas gap junction channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, hemichannels communicate the intra- and extracellular compartments and serve as a diffusional pathway for ions and small molecules. A subthreshold stimulation by acute pathological threatening conditions (e.g., global ischemia subthreshold for cell death) enhances neuronal Cx36 and glial Cx43 hemichannel activity, favoring ATP release and generation of preconditioning. If the stimulus is sufficiently deleterious, microglia become overactivated and release bioactive molecules that increase the activity of hemichannels and reduce gap junctional communication in astroglial networks, depriving neurons of astrocytic protective functions, and further reducing neuronal viability. Continuous glial activation triggered by low levels of anomalous proteins expressed in several neurodegenerative diseases induce glial hemichannel and gap junction channel disorders similar to those of acute inflammatory responses triggered by ischemia or infectious diseases. These changes are likely to occur in diverse cell types of the CNS and contribute to neurodegeneration during inflammatory process.

In previous studies, we showed that endothelin-1 increased astrocyte proliferation and glucose uptake. These effects were similar to those observed with other gap junction inhibitors, such as carbenoxolone (CBX). Because 24-h treatment with endothelin-1 or CBX downregulates the expression of connexin43, the main protein forming astrocytic gap junctions, which can also be involved in proliferation, in this study, we addressed the possible role of connexin43 in the effects of endothelin-1. To do so, connexin43 was silenced in astrocytes by siRNA. The knock down of connexin43 increased the rate of glucose uptake, characterized by the upregulation of GLUT-1 and type I hexokinase. Neither endothelin-1 nor CBX were able to further increase the rate of glucose uptake in connexin43-silenced astrocytes. In agreement, no effects of endothelin-1 and CBX on GLUT-1 and type I hexokinase were observed in connexin-43 silenced astrocytes or in astrocytes from connexin43 knock-out (KO) mice. Our previous studies suggested a close relationship between glucose uptake and astrocyte proliferation. Consistent with this, connexin43-silenced astrocytes exhibited an increase in Ki-67, a marker of proliferation. The effects of ET-1 on retinoblastoma phosphorylation on Ser780 and on the upregulation of cyclins D1 and D3 were affected by the levels of connexin43. In conclusion, our results indicate that connexin43 participates in the effects of endothelin-1 on glucose uptake and proliferation in astrocytes. Interestingly, although the rate of growth in connexin43 KO astrocytes has been reported to be reduced, we observed that an acute reduction in connexin43 by siRNA increased proliferation and glucose uptake.

Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.

The barrel field of the somatosensory cortex constitutes a well documented example of anatomofunctional compartmentalization and activity-dependent interaction between neurons and astrocytes. In astrocytes, intercellular communication through gap junction channels composed by connexin 43 and 30 underlies a network organization. Immunohistochemical and electrophysiological experiments were undertaken to determine the coupling properties of astrocyte networks in layer IV of the developing barrel cortex. The expression of both connexins was found to be enriched within barrels compared with septa and other cortical layers. Combination of dye-coupling experiments performed with biocytin and immunostaining with specific cell markers demonstrated that astrocytic networks do not involve neurons, oligodendrocytes or NG2 cells. The shape of dye coupling was oval in the barrel cortex whereas it was circular in layer IV outside the barrel field. Two-dimensional analysis of these coupling areas indicated that gap junctional communication was restricted from a barrel to its neighbor. Such enrichment of connexin expression and transversal restriction were not observed in a transgenic mouse lacking the barrel organization, whereas they were both observed in a double-transgenic mouse with restored barrels. Direct observation of sulforhodamine B spread indicated that astrocytes located between two barrels were either weakly or not coupled, whereas coupling within a barrel was oriented toward its center. These observations indicated a preferential orientation of coupling inside the barrels resulting from subpopulations of astrocytes with different coupling properties that contribute to shaping astrocytic networks. Such properties confine intercellular communication in astrocytes within a defined barrel as previously reported for excitatory neuronal circuits.

Astrocytes have a role in maintaining normal neuronal functions, some of which depend on connexins, protein subunits of gap junction channels and hemichannels. Under inflammatory conditions, microglia release cytokines, including interleukin-1beta and tumor necrosis factor-alpha, that reduce intercellular communication via gap junctions. Now, we demonstrate that either conditioned medium harvested from activated microglia or a mixture of these two cytokines enhances the cellular exchange with the extracellular milieu via Cx43 hemichannels. These changes in membrane permeability were not detected in astrocytes cultured from Cx43 knock-out mice and were abrogated by connexin hemichannel blockers, including La3+, mimetic peptides, and niflumic acid. Both the reduction in gap junctional communication and the increase in membrane permeability were mediated by a p38 mitogen-activated protein kinase-dependent pathway. However, the increase in membrane permeability, but not the gap junction inhibition, was rapidly reversed by the sulfhydryl reducing agent dithiothreitol, indicating that final regulatory mechanisms are different. Treatment with proinflammatory cytokines reduced the total and cell surface Cx43 levels, suggesting that the increase in membrane permeability was attributable to an increase in hemichannels activity. Indeed, unitary events of approximately 220 pS corresponding to Cx43 hemichannels were much more frequent in astrocytes treated with microglia conditioned medium than under control conditions. Finally, the effect of cytokines enhanced the uptake and reduced the intercellular diffusion of glucose, which might explain changes in the metabolic status of astrocytes under inflammatory conditions. Accordingly, this opposite regulation may affect glucose trafficking and certainly will modify the metabolic status of astrocytes involved in brain inflammation.

Astrocytes play a well-established role in brain metabolism, being a key element in the capture of energetic compounds from the circulation and in their delivery to active neurons. Their metabolic status is affected in many pathological situations, such as gliomas, which are the most common brain tumors. This proliferative dysfunction is associated with changes in gap junctional communication, a property strongly developed in normal astrocytes studied both in vitro and in vivo. Here, we summarize and discuss the findings that have lead to the identification of a link between gap junctions, glucose uptake, and proliferation. Indeed, the inhibition of gap junctional communication is associated with an increase in glucose uptake due to a rapid change in the localization of both GLUT-1 and type I hexokinase. This effect persists due to the up-regulation of GLUT-1 and type I hexokinase and to the induction of GLUT-3 and type II hexokinase. In addition, cyclins D1 and D3 have been found to act as sensors of the inhibition of gap junctions and have been proposed to play the role of mediators in the mitogenic effect observed. Conversely, in C6 glioma cells, characterized by a low level of intercellular communication, an increase in gap junctional communication reduces glucose uptake by releasing type I and type II hexokinases from the mitochondria and decreases the exacerbated rate of proliferation due to the up-regulation of the Cdk inhibitors p21 and p27. Identification of the molecular actors involved in these pathways should allow the determination of potential therapeutic targets that could lead to the testing of alternative strategies to prevent, or at least slow down, the proliferation of glioma cells.

Brain inflammation is characterized by a reactive gliosis involving the activation of astrocytes and microglia. This process, common to many brain injuries and diseases, underlies important phenotypic changes in these two glial cell types. One characteristic feature of astrocytes is their high level of intercellular communication mediated by gap junctions. Previously, we have reported that astrocyte gap junctional communication (AGJC) and the expression of connexin 43 (Cx43), the main constitutive protein of gap junctions, are inhibited in microglia (MG)-astrocyte cocultures. Here, we report that bacterial lipopolysaccharide activation of microglia increases their inhibitory effect on Cx43 expression and AGJC. This inhibition is mimicked by treating astrocyte cultures with conditioned medium harvested from activated microglia. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were identified as being the main factors responsible for this conditioned medium-mediated activity. Interestingly, an inflammatory response characterized by MG activation and reactive astrocytes occurs in Alzheimer's disease, at sites of beta-amyloid (Abeta) deposits. We found that this peptide potentiates the inhibitory effect of a conditioned medium diluted at a concentration that is not effective per se. This potentiation is prevented by treating astrocytes with specific blockers of IL-1beta and TNF-alpha activities. Thus, the suppression of communication between astrocytes, induced by activated MG could contribute to the proposed role of reactive gliosis in this neurodegenerative disease.

Sphingosine-1-phosphate (S1P) is a potent and pleiotropic bioactive lysophospholipid mostly released by activated platelets that acts on its target cells through its own G protein-coupled receptors. We have previously reported that mouse striatal astrocytes expressed mRNAs for S1P1 and S1P3 receptors and proliferate in response to S1P. Here, we investigated the effect of S1P on gap junctions. We show that a short-term exposure of astrocytes to S1P causes a robust inhibition of gap junctional communication, as demonstrated by dye coupling experiments and double voltage-clamp recordings of junctional currents. The inhibitory effect of S1P on dye coupling involves the activation of both Gi and Rho GTPases. Rho-associated kinase (ROCK) also plays a critical role. The capacity of S1P to activate a Rho/ROCK axis in astrocytes is demonstrated by the typical remodeling of actin cytoskeleton. Connexin43, the protein forming gap junction channels, is a target of the Gi- and Rho/ROCK-mediated signaling cascades. Indeed, as shown by Western blots and confocal immunofluorescence, its nonphosphorylated form increases following S1P treatment and this change does not occur when both cascades are disrupted. This novel effect of S1P may have an important physiopathological significance when considering the proposed roles for astrocyte gap junctions on neuronal survival.

A number of studies have contributed to demonstrate that neurons and astrocytes tightly and actively interact. Indeed, the presence of astrocytes in neuronal cultures increases the number of synapses and their efficiency, and thanks to enzymatic and uptake processes, astrocytes play a role in neuroprotection. A typical feature of astrocytes is that they establish cell-cell communication in vitro, as well as in situ, through intercellular channels forming specialized membrane areas defined as gap junctions. These channels are composed of junctional proteins termed connexins (Cxs): in astrocytes connexin 43 (Cx43) and 30 (Cx30) have been shown to prevail. Several recent works indicate that gap junctional communication (GJC) and/or connexin expression in astrocytes are controlled by neurons. Altogether, these observations lead to the concept that neuronal and astrocytic networks interact through mutual setting of their respective mode of communication and that astrocyte gap junctions represent a target in neuroglial interaction.

Cultured astrocytes are highly coupled by gap junction channels mainly constituted by connexin 43. We have previously shown that gap junctional communication (GJC) represents a functional property of astrocytes that is a target for their interaction with other brain cell types, including neurons and brain macrophages. In pathological situations, neurons as well as brain macrophages produce superoxide ions leading to the formation of hydrogen peroxide (H2O2) that can be cytotoxic. We report here that 10-min exposure to 100 microM H2O2 increases GJC in astrocytes. Moreover, 30-min exposure to 100 microM H2O2 induces, 24 h later, an astrocyte cell death by both apoptosis and necrosis. This H2O2-induced astrocyte cell death is not affected when gap junctions are inhibited by several uncoupling agents, including 18alpha-glycyrrhetinic acid, halothane, heptanol, and endothelin-1, indicating that the proportion of cell death is not related to the level of GJC. The effect of H2O2 on gap junction channels does not result from the production of free radicals but is rather linked to modification of the redox equilibrium in astrocytes. Indeed, an oxidative agent reproduces the H2O2-evoked response while reducing agents prevent the effect of H2O2. Finally, when astrocytes are cocultured with brain macrophages, the effects of H2O2 on both GJC and toxicity are not observed, revealing a new protective role of brain macrophages during oxidative stress., (Copyright 2003 Wiley-Liss, Inc.)

A unique workshop was recently held focusing on enhancing collaborations leading to identify and update the development of therapeutic strategies targeting connexin/pannexin large pore channels. Basic scientists exploring the functions of these channels in various pathologies gathered together with leading pharma companies which are targeting gap junction proteins for specific therapeutic applications. This highlights how paths of discovery research can converge with therapeutic strategies in innovative ways to enhance target identification and validation. [ABSTRACT FROM AUTHOR]

Glial cells, and in particular astrocytes, are crucial to maintain neuronal microenvironment by regulating energy metabolism, neurotransmitter uptake, gliotransmission, and synaptic development. Moreover, a typical feature of astrocytes is their high expression level of connexins, a family of membrane proteins that form gap junction channels allowing intercellular exchanges and hemichannels that provide release and uptake pathways for neuroactive molecules. Interestingly, several studies have revealed unexpected changes in astrocytes from depressive patients and rodent models of depressive-like behavior. Moreover, changes in the expression level of the astroglial connexin 43 (Cx43) have been reported in a depressive context. On the other hand, antidepressive drugs have also been shown to impact the expression of this connexin in astrocytes. However, so far there is little information concerning the functional consequence of these changes, i.e., the status of gap junctional communication and hemichannel activity in astrocytes exposed to antidepressants. In the present work we focused our attention on the action of seven antidepressants from four different therapeutic classes and tested their effects on Cx43 expression and on the two connexin-based channels functions studied in cultured astrocytes. We here report that when used at non-toxic and clinically relevant concentrations they have no effects on Cx43 expression but differential effects on Cx43 gap junction channels. Moreover, all tested antidepressants inhibit Cx43 hemichannel with different efficiency depending on their therapeutic classe. By studying the impact of antidepressants on the functional status of astroglial connexin channels, contributing to dynamic neuroglial interactions, our observations should help to better understand the mechanism by which these drugs provide their effect in the brain. [ABSTRACT FROM AUTHOR]

In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2011) 118, 826-840. Abstract Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-β peptide (Aβ) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3 h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aβ-treated microglia (CM-Aβ). The effects of CM-Aβ were mimicked by TNF-α and IL-1β and were abrogated by neutralizing TNF-α with soluble receptor and IL-1β with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aβ made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aβ induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia. [ABSTRACT FROM AUTHOR]

Neurons and brain macrophages (BM), respectively, increase and inhibit gap junctional communication (GJC) and connexin expression in cultured astrocytes. Thus, in brain diseases and injuries, neuronal death associated with the BM activation may decrease GJC in astrocytes and therefore have a physiopathological relevance. [ABSTRACT FROM AUTHOR]