Role of connexin43 in central nervous system injury

Gap junctions are specialized cell-to-cell contacts that provide direct intercellular communication. In the central nervous system (CNS), gap junction coupling occurs between both neurons and glial cells. One of the most abundant gap junction proteins in the CNS is connexin43 (Cx43). The functional syncytium formed by astrocytes via Cx43 gap junction intercellular communication has, for example, been implicated in maintaining the homeostasis of the extracellular milieu of neurons. In particular, astrocytes are involved in the spatial buffering of many ions, signalling molecules and energy sources. In this review, the role of Cx43 following CNS injury is examined by combining evidence surrounding the response of Cx43 to CNS injury and the effects of Cx43 gap junction blockade on neuronal survival in various models of injury. Combined evidence suggests that transient blockade targeting the window of initial Cx43 upregulation observed following injury is potentially therapeutic.     

Connexins in neurons and glia：targets for intervention in disease and injury

Both neurons and glia throughout the central nervous system are organized into networks by gap junctions. Among glia, gap junctions facilitate metabolic homeostasis and intercellular communication. Amo...     

Role of gap junctions in chronic pain

Gap junctions are specialized transmembrane channels that allow rapid electrical signalling and direct intercellular communication for maintenance and coordination of normal cellular activities and homeostasis. Although gap junction channels in the nervous system mediate intercellular coupling between glial cells and between neurons, they also contribute to the spread of secondary damage and inflammation under pathological conditions. There is now evidence of the involvement of gap junctions in chronic pain caused by nervous system damage or tissue inflammation. In this Mini-Review, we highlight recent studies demonstrating the dynamic plasticity of gap junctions in response to nervous system injury and the effects of gap junction blockade on neuronal survival and modulation of pain in animal models of neuropathic and inflammatory pain. The involvement of dorsal root ganglia and spinal cord gap junctions in mediating chronic pain and the potential for targeting connexins as a novel modality for the treatment of intractable pain syndromes arising from nervous system injury and disorders are discussed.     

Gap junction-mediated bidirectional signaling between human fetal hippocampal neurons and astrocytes

Gap junctions are clusters of intercellular channels that connect the interiors of coupled cells. In the brain, gap junctions function as electrotonic synapses between neurons and as pathways for the exchange of metabolites and second-messenger molecules between glial cells. Astrocytes, the most abundant glial cell type coupled by gap junctions, are intimately involved in the active control of neuronal activity including synaptic transmission and plasticity. Previous studies have suggested that astrocytic-neuronal signaling may involve gap junction-mediated intercellular connections; this issue remains unresolved. In this study, we demonstrate that second-trimester human fetal hippocampal neurons and astrocytes in culture are coupled by gap junctions bidirectionally; we show that human fetal neurons and astrocytes express both the same and different connexin subtypes. The formation of functional homotypic and heterotypic gap junction channels between neurons and astrocytes may add versatility to the signaling between these cell types during human hippocampal ontogeny; disruption of such signaling may contribute to CNS dysfunction during pregnancy.     

Role of gap junctional coupling in astrocytic networks in the determination of global ischaemia-induced oxidative stress and hippocampal damage

While there is evidence that gap junctions play important roles in the determination of cell injuries, there is not much known about mechanisms by which gap junctional communication may exert these functions. Using a global model of transient ischaemia in rats, we found that pretreatment with the gap junctional blockers carbenoxolone, 18alpha-glycyrrhetinic acid and endothelin, applied via cannulae implanted into the hippocampus in one hemisphere, resulted in decreased numbers of TUNEL-positive neurons, as compared with the contralateral hippocampus that received saline injection. Post-treatment with carbenoxolone for up to 30 min after the stroke injury still resulted in decreased cell death, but post-treatment at 90 min after the ischaemic insult did not result in differences in cell death. However, quinine, an inhibitor of Cx36-mediated gap junctional coupling, did not result in appreciable neuroprotection. Searching for a possible mechanism for the observed protective effects, possible actions of the gap junctional blockers in the electrical activity of the hippocampus during the ischaemic insult were assessed using intracerebral recordings, with no differences observed between the saline-injected and the contralateral drug-injected hippocampus. However, a significant reduction in lipid peroxides, a measure of free radical formation, in the hippocampus treated with carbenoxolone, revealed that the actions of gap junctional coupling during injuries may be causally related to oxidative stress. These observations suggest that coupling in glial networks may be functionally important in determining neuronal vulnerability to oxidative injuries.     

Fibroblast growth factors-5 and -9 distinctly regulate expression and function of the gap junction protein connexin43 in cultured astroglial cells from different brain regions

Astroglial cells contribute to neuronal maintenance and function in the normal and diseased brain. Gap junctions formed predominantly by connexin43 (cx43) provide important pathways to coordinate astroglial responses. We have previously shown that fibroblast growth factor (FGF)-2, which occurs ubiquitously in the CNS, downregulates gap junction communication in cortical and striatal, but not in mesencephalic astroglial cells in vitro (Reuss et al. Glia 22:19-30, 1998). Other members of the FGF family expressed in the CNS include FGF-5 and FGF-9. We show that both FGF-5 and FGF-9, like FGF-2, downregulate astroglial gap junctions and functional coupling. However, their effects are strikingly different from different brain regions, with regard to astroglial cells. FGF-5 specifically affects mesencephalic astroglial cells without changing coupling of cortical and striatal astroglia, while FGF-9 reduces gap junctional coupling in astroglia from all three brain regions. Both cx43 mRNA and protein levels as well as functional coupling assessed by dye spreading are affected. To clarify whether brain region-specific effects of FGFs on astroglial coupling are due to differential expression of FGF receptors (FGFR), we monitored expression of the four known FGFR mRNAs in astroglial cultures by RT-PCR. Irrespective of their regional origin, astroglial cells express mRNAs for FGFR-2 and FGFR-3. In summary, our results provide evidence for an important role of FGF-2, -5, and -9 in a distinct, CNS region-specific regulation mechanism of astroglial gap junction communication. The molecular basis underlying the regionally distinct responsiveness of astrocytes to different FGFs may be sought beyond distinct FGFR expression.     

Mathematics and the gap junctions: in-phase synchronization of identical neurons

A close consideration of some mathematical results with regards to neuronal synchronization mechanisms are examined. It is well known that intercellular coupling via gap junctions normally occurs between identical neurons, such as the coupling between inhibitory interneurons belonging to the same class. It is unknown why this should happen. The theory of coupled oscillators offers some explanations to answer the questions of the functional role of electrical interactions mediated by gap junctions and the necessity to couple identical neurons. The inference presented here from the mathematical results is that only if the cells are identical will their firing synchronize in-phase. Thus, we propose the concept that the functional role of gap junctional electrical coupling is to synchronize neurons in-phase and therefore this type of coupling will be found between neurons belonging to the same class.     

Hypoxia in high glucose followed by reoxygenation in normal glucose reduces the viability of cortical astrocytes through increased permeability of connexin 43 hemichannels

Brain ischemia causes more extensive injury in hyperglycemic than normoglycemic subjects, and the increased damage is to astroglia as well as neurons. In the present work, we found that in cortical astrocytes from rat or mouse, reoxygenation after hypoxia in a medium mimicking interstitial fluid during ischemia increases hemichannel activity and decreases cell–cell communication via gap junctions as indicated by dye uptake and dye coupling, respectively. These effects were potentiated by high glucose during the hypoxia in a concentration‐dependent manner (and by zero glucose) and were not observed in connexin 43^−/− astrocytes. The responses were transient and persistent after short and long periods of hypoxia, respectively. The persistent responses were associated with a progressive reduction in cell viability that was prevented by La³⁺ or peptides that block connexin 43 (Cx43) hemichannels or by inhibition of p38 MAP kinase prior to hypoxia–reoxygenation but not by treatments that block pannexin hemichannels. Block of Cx43 hemichannels did not affect the reduction in gap junction mediated dye coupling observed during reoxygenation. Cx43 hemichannels may be a novel therapeutic target to reduce cell death following stroke, particularly in hyperglycemic conditions. © 2009 Wiley‐Liss, Inc.     

Altered olivocerebellar activity patterns in the connexin36 knockout mouse

The inferior olive (IO) has among the highest densities of neuronal gap junctions in the nervous system. These gap junctions are proposed to be the underlying mechanism for generating synchronous Purkinje cell complex spike (CS) activity. Gap junctions between neurons are formed mostly by connexin 36 proteins. Thus, the connexin 36 knockout (Cx36KO) mouse provides an opportunity to test whether gap junction coupling between IO neurons is the basis of CS synchrony. Multiple electrode recordings of crus 2 CSs were obtained from wildtype (Wt) and Cx36KO mice. Wts showed statistically significant levels of CS synchrony, with the same spatial distribution as has been reported for other species: high CS synchrony levels occurred mostly among Purkinje cells within the same parasagittally-oriented cortical strip. In contrast, in Cx36KOs, synchrony was at chance levels and had no preferential spatial orientation, supporting the gap junction hypothesis. CS firing rates for Cx36KOs were significantly lower than for Wts, suggesting that electrical coupling is an important determinant of IO excitability. Rhythmic CS activity was present in both Wt and Cx36KOs, suggesting that individual IO cells can act as intrinsic oscillators. In addition, the climbing fiber reflex was absent in the Cx36KOs, validating its use as a tool for assessing electrical coupling of IO neurons. Zebrin II staining and anterograde tracing showed that cerebellar cortical organization and the topography of the olivocerebellar projection are normal in the Cx36KO. Thus, the differences in CS activity between Wts and Cx36KOs likely reflect the loss of electrical coupling of IO cells.     

Human and mouse microglia express connexin36, and functional gap junctions are formed between rodent microglia and neurons

Microglia, the tissue macrophages of the central nervous system (CNS), intimately interact with neurons physically and through soluble factors that can affect microglial activation state and neuronal survival and physiology. We report here a new mechanism of interaction between these cells, provided by the formation of gap junctions composed of connexin (Cx) 36. Among eight Cxs tested, expression of Cx36 mRNA and protein was found in microglial cultures prepared from human and mouse, and Cx45 mRNA was found in mouse microglial cultures. Electrophysiological measurements found coupling between one-third of human or mouse microglial pairs that averaged below 30 pico-Siemens and displayed electrical properties consistent with Cx36 gap junctions. Importantly, similar frequency of low-strength electrical coupling was also obtained between microglia and neurons in cocultures prepared from neocortical or hippocampal rodent tissue. Lucifer yellow dye coupling between neurons and microglia was observed in 4% of pairs tested, consistent with the low strength and incidence of electrical coupling. Cx36 expression level and/or the degree of coupling between microglia did not significantly change in the presence of activating agents, including lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha, except for some reduction of Cx36 protein when exposed to the latter two agents. Our findings that intercellular coupling occurs between neuronal and microglial populations through Cx36 gap junctions have potentially important implications for normal neural physiology and microglial responses in neuronopathology in the mammalian CNS.     

The effects of bone morphogenetic protein 2 and 4 (BMP2 and BMP4) on gap junctions during neurodevelopment

Nervous system deficits account for the third largest group of fatal birth defects (after heart and respiratory problems) in North America. Although considerable advance has been made in neuroscience research, the early events involved in neurogenesis remain to be elucidated. More specifically, the effects of signaling molecules on intercellular communication during neurodevelopment have not yet been studied. The development of the central nervous system is regulated, at least in part, by signaling molecules such as bone morphogenetic proteins (BMPs). In this study, we have used the embryonal mouse P19 cell line to examine the effects of BMP2 and BMP4 on gap junctional communication as well as neuronal and astrocytic differentiation. The undifferentiated P19 cells show high levels of the gap junction protein, connexin43 (Cx43), and functional intercellular coupling. However, Cx43 expression and dye coupling decrease as these cells differentiate into neurons and astrocytes. In contrast, cells treated with BMP2 or BMP4 lose their capacity to differentiate into neurons but not astrocytes, while they maintain extensive gap junctional communication. The very few neurons that remain in the BMP-treated cultures are coupled (a characteristic not seen in the control neurons). Together, our data suggest that BMPs may play a critical role in morphogenesis of P19 cells while they affect gap junctions.     

Brain macrophages inhibit gap junctional communication and downregulate connexin 43 expression in cultured astrocytes

Astrocytes are typically interconnected by gap junction channels that allow, in vitro as well as in vivo, a high degree of intercellular communication between these glial cells. Using cocultures of astrocytes and neurons, we have demonstrated that gap junctional communication (GJC) and connexin 43 (Cx43) expression, the major junctional protein in astrocytes, are controlled by neuronal activity. Moreover, neuronal death downregulates these two parameters. Because in several brain pathologies neuronal loss is associated with an increase in brain macrophage (BM) density, we have now investigated whether coculture with BM affects astrocyte gap junctions. We report here that addition of BM for 24 h decreases the expression of GJC and Cx43 in astrocytes in a density-dependent manner. In contrast, Cx43 is not detected in BM and no heterotypic coupling is observed between the two cell types. A soluble factor does not seem to be involved in these inhibitions because they are not observed either in the presence of BM conditioned media or in the absence of direct contact between the two cell types by using inserts. These observations could have pathophysiological relevance as neuronal death, microglial proliferation and astrocytic reactions occur in brain injuries and pathologies. Because astrocyte interactions with BM and dying neurons both result in the downregulation of Cx43 expression and in the inhibition of GJC, a critical consequence on astrocytic phenotype in those situations could be the inhibition of gap junctions.     

Brief Note: MATHEMATICS AND THE GAP JUNCTIONS: IN-PHASE SYNCHRONIZATION OF IDENTICAL NEURONS

A close consideration of some mathematical results with regards to neuronal synchronization mechanisms are examined. It is well known that intercellular coupling via gap junctions normally occurs between identical neurons, such as the coupling between inhibitory interneurons belonging to the same class. It is unknown why this should happen. The theory of coupled oscillators offers some explanations to answer the questions of the functional role of electrical interactions mediated by gap junctions and the necessity to couple identical neurons. The inference presented here from the mathematical results is that only if the cells are identical will their firing synchronize in-phase. Thus, we propose the concept that the functional role of gap junctional electrical coupling is to synchronize neurons in-phase and therefore this type of coupling will be found between neurons belonging to the same class.­­     

Immunohistochemical detection of connexin36 in sympathetic preganglionic and somatic motoneurons in the adult rat

Gap junctional communication in the adult CNS plays an important role in the synchronization of neuronal activities. In vitro studies have shown evidence of electrotonic coupling through gap junctions between sympathetic preganglionic motoneurons and between somatic motoneurons in the neonatal and adult rat spinal cord. Electrotonic transmission of membrane oscillations might be an important mechanism for recruitment of neurons and result in the generation of rhythmic sympathetic and somato-motor activity at the population level. Gap junctions in the adult spinal cord are constituted principally by connexin36 (Cx36). However, the distribution of Cx36 in specific neuronal populations of the spinal cord is unknown. Here, we identify Cx36-like immunoreactivity in sympathetic preganglionic and somatic motoneurons in thoracic spinal cord segments of the adult rat. For this purpose, double immunostaining against Cx36 and choline acetyltransferase (ChAT) was performed on transverse sections (20 microm) taken from spinal segments T6-T8. Cx36 punctate immunostaining was detected in the majority of ChAT-immunoreactive (-ir) neurons from lamina VII [intermediolateral cell column (IML) and intercalated cell group (IC)], lamina X [central autonomic nucleus (CA)] and in ventral horn neurons from laminae VIII and IX. Cx36 puncta were distributed in the neuronal somata and along dendritic processes. The presence of Cx36 in ChAT-ir neurons is consistent with electrical coupling between sympathetic preganglionic motoneurons and between somatic motoneurons through gap junctions in the adult spinal cord.     

Neurodegeneration in Excitotoxicity, Global Cerebral Ischemia, and Target Deprivation: A Perspective on the Contributions of Apoptosis and Necrosis

In the human brain and spinal cord, neurons degenerate after acute insults (e.g., stroke, cardiac arrest, trauma) and during progressive, adult-onset diseases [e.g., amyotrophic lateral sclerosis, Alzheimer’s disease]. Glutamate receptor-mediated excitotoxicity has been implicated in all of these neurological conditions. Nevertheless, effective approaches to prevent or limit neuronal damage in these disorders remain elusive, primarily because of an incomplete understanding of the mechanisms of neuronal death in in vivo settings. Therefore, animal models of neurodegeneration are crucial for improving our understanding of the mechanisms of neuronal death. In this review, we evaluate experimental data on the general characteristics of cell death and, in particular, neuronal death in the central nervous system (CNS) following injury. We focus on the ongoing controversy of the contributions of apoptosis and necrosis in neurodegeneration and summarize new data from this laboratory on the classification of neuronal death using a variety of animal models of neurodegeneration in the immature or adult brain following excitotoxic injury, global cerebral ischemia, and axotomy/target deprivation. In these different models of brain injury, we determined whether the process of neuronal death has uniformly similar morphological characteristics or whether the features of neurodegeneration induced by different insults are distinct. We classified neurodegeneration in each of these models with respect to whether it resembles apoptosis, necrosis, or an intermediate form of cell death falling along an apoptosis-necrosis continuum. We found that N-methyl-d-aspartate (NMDA) receptor- and non-NMDA receptor-mediated excitotoxic injury results in neurodegeneration along an apoptosis-necrosis continuum, in which neuronal death (appearing as apoptotic, necrotic, or intermediate between the two extremes) is influenced by the degree of brain maturity and the subtype of glutamate receptor that is stimulated. Global cerebral ischemia produces neuronal death that has commonalities with excitotoxicity and target deprivation. Degeneration of selectively vulnerable populations of neurons after ischemia is morphologically nonapoptotic and is indistinguishable from NMDA receptor-mediated excitotoxic death of mature neurons. However, prominent apoptotic cell death occurs following global ischemia in neuronal groups that are interconnected with selectively vulnerable populations of neurons and also in nonneuronal cells. This apoptotic neuronal death is similar to some forms of retrograde neuronal apoptosis that occur following target deprivation. We conclude that cell death in the CNS following injury can coexist as apoptosis, necrosis, and hybrid forms along an apoptosis–necrosis continuum. These different forms of cell death have varying contributions to the neuropathology resulting from excitotoxicity, cerebral ischemia, and target deprivation/axotomy. Degeneration of different populations of cells (neurons and nonneuronal cells) may be mediated by distinct or common causal mechanisms that can temporally overlap and perhaps differ mechanistically in the rate of progression of cell death.     

Neuronal and glial gap junctions in the goldfish preoptic area, a thin section and freeze-fracture study

In freeze-fracture, both large macular gap junctions and long thin gap junctions surrounded by a strand of tight junction were found on neurosecretory cells. Preoptic neurons show large areas of soma-to-soma apposition, but thin section showed no evidence for gap junctions between neuronal somata. Neurosecretory cell neurites formed parallel bundles in neuropil lateral to the nucleus, and gap junctions were found between the neurites. These junctions apparently correspond to macular junctions seen on neurosecretory elements in freeze-fracture. Some large macular gap junctions found in freeze-fracture presumably correspond to junctions seen between glial cells in thin section. However, glial membranes lacked characteristics distinguishing them from neuronal membranes. In one instance, a large apparent glial sheet process formed both macular and long thin gap junctions on different surfaces. The long thin gap junctions that were surrounded by a strand of tight junction were formed with a large neurosecretory cell soma. Extensive pinocytosis was observed at some membranes forming gap junctions.     

Gap junctions: multifaceted regulators of embryonic cortical development

The morphological development of the cerebral cortex from a primitive neuroepithelium into a complex laminar structure underlying higher cognition must rely on a network of intercellular signaling. Gap junctions are widely expressed during embryonic development and provide a means of cell-cell contact and communication. We review the roles of gap junctions in regulating the proliferation of neural progenitors as well as the migration and differentiation of young neurons in the embryonic cerebral cortex. There is substantial evidence that although gap junctions act in the classical manner coupling neural progenitors, they also act as hemichannels mediating the spread of calcium waves across progenitor cell populations and as adhesive molecules aiding neuronal migration. Gap junctions are thus emerging as multifaceted regulators of cortical development playing diverse roles in intercellular communication.     

Grid-mapped freeze-fracture analysis of gap junctions in gray and white matter of adult rat central nervous system,with evidence for a “panglial syncytium” that is not coupled to neurons

In white matter regions of the brain and spinal cord of adult mammals, gap junctions previously were observed linking astrocytes to astrocytes, as well as to oligodendrocytes and ependymacytes. The resulting “functional syncytium” was proposed to modulate the ion fluxes that occur during electrical activity of the associated axons. Gap junctions also have been reported linking neurons with glia, and functional neuronal-glial coupling has been postulated. To investigate the glial syncytium and the neuron-to-glia coupling hypotheses, we used “grid-mapped freeze fracture,” conventional thin-section electron microscopy, and light microscope immunocytochemistry to examine and characterize neurons and glia in gray and white matter of adult rat brain and spinal cord. We have obtained quantitative evidence for the abundance and widespread distribution of gap junctions interlinking the three primary types of macroglia throughout both gray and white matter of the mammalian central nervous system (CNS), thereby extending the concept to that of a functional panglial syncytium. In contrast to previous reports, we show that of more than 400 gap junctions in which both participating cells were identified, none were between neurons and glia. Thus, neuronal coupling and glial coupling involved separate and distinct pathways. Finally, putative water channels (i.e., “square arrays”) were confirmed to be abundant and in close association with gap junctions in astrocytes and ependymacytes. Because the astrocyte “intermediaries” extend cytoplasmic conduits throughout gray and white matter of brain and spinal cord, from the ependymal layer to the pia-glial limitans, and from oligodendrocytes surrounding axons to astrocyte endfeet surrounding capillaries, the proposed panglial syncytium, with its abundance of water channels and intercellular ion channels, is optimally positioned and equipped to modulate water and ion fluxes across broad regions of the CNS. J. Comp. Neurol. 388:265–292, 1997. © 1997 Wiley-Liss, Inc.