Intercellular communication in spinal cord astrocytes: fine tuning between gap junctions and P2 nucleotide receptors in calcium wave propagation.

Electrophysiological properties of gap junction channels and mechanisms involved in the propagation of intercellular calcium waves were studied in cultured spinal cord astrocytes from sibling wild-type (WT) and connexin43 (Cx43) knock-out (KO) mice. Comparison of the strength of coupling between pairs of WT and Cx43 KO spinal cord astrocytes indicates that two-thirds of total coupling is attributable to channels formed by Cx43, with other connexins contributing the remaining one-third of junctional conductance. Although such a difference in junctional conductance was expected to result in the reduced diffusion of signaling molecules through the Cx43 KO spinal cord syncytium, intercellular calcium waves were found to propagate with the same velocity and amplitude and to the same number of cells as between WT astrocytes. Measurements of calcium wave propagation in the presence of purinoceptor blockers indicate that calcium waves in Cx43 KO spinal cord astrocytes are mediated primarily by extracellular diffusion of ATP; measurements of responses to purinoceptor agonists revealed that the functional P2Y receptor subtype is shifted in the Cx43 KO astrocytes, with a markedly potentiated response to ATP and UTP. Thus, the reduction in gap junctional communication in Cx43 KO astrocytes leads to an increase in autocrine communication, which is a consequence of a functional switch in the P2Y nucleotide receptor subtype. Intercellular communication via calcium waves therefore is sustained in Cx43 null mice by a finely tuned interaction between gap junction-dependent and independent mechanisms.     

Enhanced connexin 43 immunoreactivity in penumbral areas in the human brain following ischemia

Astrocytes support neurons not only physically but also chemically by secreting neurotrophic factors and energy substrates. Moreover, astrocytes establish a glial network and communicate through gap junctions in the brain. Connexin 43 (Cx43) is one of major component proteins in astrocytic gap junctions. Heterozygote Cx43 KO mice and astrocyte specific Cx43 KO mice exhibited amplified brain damage after ischemic insults, suggesting a neuroprotective role for astrocytic gap junctions. However, some reports mentioned unfavorable effects of gap junctions in neuronal support. Therefore, the role of astrocytic gap junctions under ischemic condition remains controversial. Since these studies have been performed using animal models, we investigated the Cx43 expression in human brain after stroke. Brain slice sections were prepared from pathological samples in our hospital. Embolic stroke brains sectioned because of the stroke were considered as acute ischemic models. Multiple infarction brains sectioned because of pneumonia or cancer were considered as chronic models. We observed the levels of Cx43 in both lesioned and intact areas, and compared them with acute and chronic models. As the results, astrocytes were strongly activated in penumbral lesions both of acute and chronic ischemic models. The Cx43 immunoreactivity was significantly amplified in the penumbra of chronic model compared to that of the acute model. Neurons were well preserved in chronic model compared to acute model. These findings suggested that the brain may generate neuronal protection by increasing the levels of Cx43 and amplifying the astrocytic gap junctional intercellular communication under hypoxic condition.     

Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species‐specific expression,compensation in Cx36 null mice and co‐localization in neurons vs. glia

Electrical synapses formed by connexin36 (Cx36)‐containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter‐based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36‐puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild‐type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36‐puncta co‐localized with Cx45‐puncta, which in turn was associated with the scaffolding protein zonula occludens‐1. In rats, Cx45‐puncta were also co‐localized with Cx36‐puncta and additionally occurred along Bergmann glial processes adjacent to Cx43‐puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.     

Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole‐cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction‐permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin‐Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47‐deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47‐double‐deficient mice. Cx47‐ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43‐deficient animals, oligodendrocyte‐astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30‐double deficient mice, oligodendrocyte‐to‐astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network. © 2010 Wiley‐Liss, Inc.     

CD38 positively regulates postnatal development of astrocytes cell‐autonomously and oligodendrocytes non‐cell‐autonomously

Glial development is critical for the function of the central nervous system. CD38 is a multifunctional molecule with ADP‐ribosyl cyclase activity. While critical roles of CD38 in the adult brain such as oxytocin release and social behavior have been reported, those in the developing brain remain largely unknown. Here we demonstrate that deletion of Cd38 leads to impaired development of astrocytes and oligodendrocytes in mice. CD38 is highly expressed in the developing brains between postnatal day 14 (P14) and day 28 (P28). In situ hybridization and FACS analysis revealed that CD38 is expressed predominantly in astrocytes in these periods. Analyses of the cortex of Cd38 knockout (Cd38^?/? ) mice revealed delayed development of astrocytes and subsequently delayed differentiation of oligodendrocytes (OLs) at postnatal stages. In vitro experiments using primary OL cultures, mixed glial cultures, and astrocytic conditioned medium showed that astrocytic CD38 regulates the development of astrocytes in a cell‐autonomous manner and the differentiation of OLs in a non‐cell‐autonomous manner. Further experiments revealed that connexin43 (Cx43) in astrocytes plays a promotive role for CD38‐mediated OL differentiation. Finally, increased levels of NAD⁺, caused by CD38 deficiency, are likely to be responsible for the suppression of astrocytic Cx43 expression and OL differentiation. Our data indicate that CD38 is a positive regulator of astrocyte and OL development.     

Mice with astrocyte-directed inactivation of connexin43 exhibit increased exploratory behaviour, impaired motor capacities, and changes in brain acetylcholine levels

Gap junctions mediate communication between many cell types in the brain. Gap junction channels are composed of membrane-spanning connexin (Cx) proteins, allowing the cell-to-cell passage of small ions and metabolites. Cx43 is the main constituent of the brain-spanning astrocytic gap junctional network, controlling activity-related changes in ion and glutamate concentrations as well as metabolic processes. In astrocytes, deletion of Cx43-coding DNA led to attenuated gap junctional coupling and impaired propagation of calcium waves, known to influence neuronal activity. Investigation of the role of Cx43 in behaviour has been impossible so far, due to postnatal lethality of its general deletion. Recently, we have shown that deletion of Cx30, which is also expressed by astrocytes, affects exploration, emotionality, and neurochemistry in the mouse. In the present study, we investigated the effects of the astrocyte-directed inactivation of Cx43 on mouse behaviour and brain neurochemistry. Deletion of Cx43 in astrocytes increased exploratory activity without influencing habituation. In the open field, but not in the elevated plus-maze, an anxiolytic-like effect was observed. Rotarod performance was initially impaired, but reached control level after further training. In the water maze, Cx43 deficient mice showed a steeper learning course, although final performance was similar between groups. Cx43 inactivation in astrocytes increased acetylcholine content in the frontal cortex of water maze-trained animals. Results are discussed in terms of altered communication between astrocytes and neurons, possible compensation processes, and differential effects of Cx30- and astrocyte-specific Cx43 deletion.     

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.     

Coupling of astrocyte connexins Cx26, Cx30, Cx43 to oligodendrocyte Cx29, Cx32, Cx47: Implications from normal and connexin32 knockout mice

Oligodendrocytes in vivo form heterologous gap junctions with astrocytes. These oligodendrocyte/astrocyte (A/O) gap junctions contain multiple connexins (Cx), including Cx26, Cx30, and Cx43 on the astrocyte side, and Cx32, Cx29, and Cx47 on the oligodendrocyte side. We investigated connexin associations at A/O gap junctions on oligodendrocytes in normal and Cx32 knockout (KO) mice. Immunoblotting and immunolabeling by several different antibodies indicated the presence of Cx32 in liver and brain of normal mice, but the absence of Cx32 in liver and brain of Cx32 KO mice, confirming the specificity and efficacy of the antibodies, as well as allowing the demonstration of Cx32 expression by oligodendrocytes. Oligodendrocytes throughout brain were decorated with numerous Cx30-positive puncta, which also were immunolabeled for both Cx32 and Cx43. In Cx32 KO mice, astrocytic Cx30 association with oligodendrocyte somata was nearly absent, Cx26 was partially reduced, and Cx43 was present in abundance. In normal and Cx32 KO mice, oligodendrocyte Cx29 was sparsely distributed, whereas Cx47-positive puncta were densely localized on oligodendrocyte somata. These results demonstrate that astrocyte Cx30 and oligodendrocyte Cx47 are widely present at A/O gap junctions. Immunolabeling patterns for these six connexins in Cx32 KO brain have implications for deciphering the organization of heterotypic connexin coupling partners at A/O junctions. The persistence and abundance of Cx43 and Cx47 at these junctions after Cx32 deletion, together with the paucity of Cx29 normally present at these junctions, suggests Cx43/Cx47 coupling at A/O junctions. Reductions in Cx30 and Cx26 after Cx32 deletion suggest that these astrocytic connexins likely form junctions with Cx32 and that their incorporation into A/O gap junctions is dependent on the presence of oligodendrocytic Cx32.     

Selective Cre-mediated gene deletion identifies connexin 43 as the main connexin channel supporting olfactory ensheathing cell networks

Many functions of glial cells depend on the formation of selective glial networks mediated by gap junctions formed by members of the connexin family. Olfactory ensheathing cells (OECs) are specialized glia associated with olfactory sensory neuron axons. Like other glia, they form selective networks, however, the connexins that support OEC connectivity in vivo have not been identified. We used an in vivo mouse model to selectively delete candidate connexin genes with temporal control from OECs and address the physiological consequences. Using this model, we effectively abolished the expression of connexin 43 (Cx43) in OECs in both juvenile and adult mice. Cx43-deleted OECs exhibited features consistent with the loss of gap junctions including reduced membrane conductance, largely reduced sensitivity to the gap junction blocker meclofenamic acid and loss of dye coupling. This indicates that Cx43, a typically astrocytic connexin, is the main connexin forming functional channels in OECs. Despite these changes in functional properties, the deletion of Cx43 deletion did not alter the density of OECs. The strategy used here may prove useful to delete other candidate genes to better understand the functional roles of OECs in vivo.     

Connexin 43 deletion in astrocytes promotes CNS remyelination by modulating local inflammation

As the most abundant gap junction protein in the central nervous system (CNS), astrocytic connexin 43 (Cx43) maintains astrocyte network homeostasis, affects oligodendroglial development and participates in CNS pathologies as well as injury progression. However, its role in remyelination is not yet fully understood. To address this issue, we used astrocyte-specific Cx43 conditional knockout (Cx43 cKO) mice generated through the use of a hGFAP-cre promoter, in combination with mice carrying a floxed Cx43 allele that were subjected to lysolecithin so as to induce demyelination. We found no significant difference in the demyelination of the corpus callosum between Cx43 cKO mice and their non-cre littermate controls, while the remyelination process in Cx43 cKO mice was accelerated. Moreover, an increased number of mature oligodendrocytes and an unaltered number of oligodendroglial lineage cells were found in Cx43 cKO mouse lesions. This indicates that oligodendrocyte precursor cell (OPC) differentiation was facilitated by astroglial Cx43 depletion as remyelination progressed. Underlying the latter, there was a down-regulated glial activation and modulated local inflammation as well as a reduction of myelin debris in Cx43 cKO mice. Importantly, 2 weeks of orally administrating boldine, a natural alkaloid that blocks Cx hemichannel activity in astrocytes without affecting gap junctional communication, obviously modulated local inflammation and promoted remyelination. Together, the data suggest that the astrocytic Cx43 hemichannel is negatively involved in the remyelination process by favoring local inflammation. Consequently, inhibiting Cx43 hemichannel functionality may be a potential therapeutic approach for demyelinating diseases in the CNS.     

Role of astroglial connexin30 in hippocampal gap junction coupling

The impact of connexin30 (Cx30) on interastrocytic gap junction coupling in the normal hippocampus is matter of debate; reporter gene analyses indicated a weak expression of Cx30 in the mouse hippocampus. In contrast, mice lacking connexin43 (Cx43) in astrocytes exhibited only 50% reduction in coupling. Complete uncoupling of hippocampal astrocytes in mice lacking both Cx30 and Cx43 suggested that Cx30 participates in interastrocytic gap junction coupling in the hippocampus. With comparative reporter gene assays, immunodetection, and cre/loxP-based reporter approaches we demonstrate that Cx30 is more abundant than previously thought. The specific role of Cx30 in interastrocytic coupling has never been investigated. Employing tracer coupling analyses in acute slices of Cx30 deficient mice here we show that Cx30 makes a substantial contribution to interastrocytic gap junctional communication in the mouse hippocampus.     

Downregulation of spinal astrocytic connexin43 leads to upregulation of interleukin‐6 and cyclooxygenase‐2 and mechanical hypersensitivity in mice

Connexin43 (Cx43), involved in intercellular signaling, is expressed in spinal dorsal horn astrocytes and crucial in the maintenance of neuropathic pain. Downregulation of spinal astrocytic Cx43 in mice enhances glutamatergic neurotransmission by decreasing glutamate transporter GLT‐1 expression, resulting in cutaneous hypersensitivity. Decreased expression of astrocytic Cx43 could lead to altered expression of other nociceptive molecules. Transfection of Cx43‐targeting siRNA in cultured spinal astrocytes increased expression of the pronociceptive cytokine interleukin‐6 (IL‐6) and the prostaglandin synthesizing enzyme cyclooxygenase‐2 (COX‐2). Increased expression of IL‐6 and COX‐2 was due to decreased Cx43 expression rather than due to diminished Cx43 channel function. In mice, downregulation of spinal Cx43 expression by intrathecal treatment with Cx43‐targeting siRNA increased IL‐6 and COX‐2 expression and induced hind paw mechanical hypersensitivity. Cx43 siRNA‐induced mechanical hypersensitivity was attenuated by intrathecal treatment with anti‐IL‐6 neutralizing antibody and intraperitoneal treatment of selective COX‐2 inhibitor celecoxib, demonstrating that these molecules play a role in nociceptive processing following Cx43 downregulation. Restoring spinal Cx43 by intrathecal injection of an adenovirus vector expressing Cx43 in mice with a partial sciatic nerve ligation reduced spinal IL‐6 and COX‐2 expression. Suppression of glycogen synthase kinase‐3β (GSK‐3β), a serine/threonine protein kinase, prevented upregulation of IL‐6 and COX‐2 expression induced by Cx43 downregulation in both cultured astrocytes and in mouse spinal dorsal horn. Inhibition of spinal GSK‐3β also ameliorated Cx43 siRNA‐induced mechanical hypersensitivity. The current findings indicate that downregulation of spinal astrocytic Cx43 leads to changes in spinal expression of pronociceptive molecules underlying the maintenance of pain following nerve injury.     

Gap junction protein connexin43 is heterogeneously expressed among glial cells in the adult rabbit retina.

The immunohistochemical distribution and ultrastructural immunolocalization of connexin43 (Cx43) in the neural retina of the rabbit was investigated. Cx43 immunolabeling appeared in the form of distinct puncta distributed on different kinds of glial cells and exclusively in the myelinated fiber region of the neural retina. Double-label immunohistochemistry showed that the most obvious Cx43 labeling occurred at processes of glial fibrillary acidic protein-positive astrocytes and on vimentin-positive Müller cells. Cx43-immunoreactive puncta were also evident on cell bodies and processes of 2'-3'-cyclic nucleotide phosphodiesterase-labeled oligodendrocytes. As shown by electron microscopy, immunoreactivity to Cx43 was restricted to gap junctions among the macroglial cell population. The homologous interastrocytic and Müller cell-to-Müller cell, as well as the heterologous astrocyte-to-Müller cell and astrocyte/Müller-to-oligodendrocyte gap junctions were symmetrically labeled. Our results indicate a specific expression of Cx43 at gap junctions between macroglial cells located in the myelinated streak. The extensive Cx43 immunolabeling suggests a substantial amount of gap junctional coupling that establishes a macroglial syncytium.     

Ablation of connexin30 in transgenic mice alters expression patterns of connexin26 and connexin32 in glial cells and leptomeninges

Expression of connexin26 (Cx26), Cx30 and Cx43 in astrocytes and expression of Cx29, Cx32 and Cx47 in oligodendrocytes of adult rodent brain has been well documented, as has the interdependence of connexin expression patterns of macroglial cells in Cx32- and Cx47-knockout mice. To investigate this interdependence further, we examined immunofluorescence labelling of glial connexins in transgenic Cx30 null mice. Ablation of astrocytic Cx30, confirmed by the absence of immunolabelling for this connexin in all brain regions, resulted in the loss of its coupling partner Cx32 on the oligodendrocyte side of astrocyte-oligodendrocyte (A/O) gap junctions, but had no effect on the localization of astrocytic Cx43 and oligodendrocytic Cx47 at these junctions or on the distribution of Cx32 along myelinated fibres. Surprisingly, gene deletion of Cx30 led to the near total elimination of immunofluorescence labelling for Cx26 in all leptomeningeal tissues covering brain surfaces as well as in astrocytes of brain parenchyma. Moreover northern blot analysis revealed downregulation of Cx26 mRNA in Cx30-knockout brains. Our results support earlier observations on the interdependency of Cx30/Cx32 targeting to A/O gap junctions and further suggest that Cx26 mRNA expression is affected by Cx30 gene expression. In addition, Cx30 protein may be required for co-stabilization of gap junctions or for co-trafficking in cells.     

Distinct types of astroglial cells in the hippocampus differ in gap junction coupling

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morphological and functional characteristics, specifically a nonoverlapping expression of either AMPA-type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter-controlled EGFP expression and patch-clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junctional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing beta-Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individual cells. This study supports the notion that the two subpopulations of hGFAP-EGFP-positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes.     

Chronic endothelin exposure inhibits connexin43 expression in cultured cortical astroglia

Severe brain lesions are accompanied by sustained increases in endothelin (ET) levels, which in turn profoundly affect brain microcirculation and neural cell function. A known response of astrocytes to acute increases in ET levels is the rapid and transient closure of gap junctions and the subsequent decrease of gap junction-mediated intercellular communication (GJIC). Because evidence exists that the loss of GJIC alters astrocytic gene expression, we analyzed the effects of chronic ET exposure on astrocytic gap junction coupling. We found that within 24 hr, cultured cortical astrocytes respond to low nanomolar concentrations (2-10 nM) of either ET-1 or ET-3 with a robust inhibition of connexin (Cx)43 expression, the major junctional protein in astrocytes, and a subsequent decline of GIJC. We further observed that in the continuous presence of ETs, Cx43 expression remained inhibited for at least 7 days. In addition, a similar decrease of Cx43 expression occurred in cultured spinal cord astrocytes maintained with ET-1 for 3 days. Applying ETs in combination with the highly selective ETA and ETB receptor antagonists, BQ123 and BQ788, respectively, revealed that the inhibitory influences on astrocytic Cx43 expression depend on activation of ETB receptors. We suggest that the observed ET-dependent inhibition of Cx43 expression and the resulting decline of GJIC might represent a major pathway by which ETs regulate astrocytic gene expression in the injured brain.     

Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy

Following brain injury, and during the process of neurodegeneration, a reactive astrocytic proliferation occurs. This is accompanied by an increase in the synthesis of neuropeptides, cytokines, growth factors and glial fibrillary acidic protein (GFAP), a cell-specific marker for reactive astrocytes. Astrocytes are extensively coupled by gap junctions of the Cx43 connexin subtype. Several studies have shown that in severe trauma, coupling between astrocytes may add to the spread of the damaged area. In this study we ask whether the astrocytosis which is a feature of other neurodegenerative diseases also occurs in mesial temporal lobe epilepsy (MTLE) and whether it is accompanied by an increase in astrocytic communication through an upregulation of Cx43 gap junction channel proteins. In order to examine the astrocytic response and the expression pattern of Cx43 protein, double immunohistochemical labeling studies were undertaken using antibodies against GFAP and Cx43 applied to human hippocampal tissue resected from patients with MTLE, and to normal human control hippocampal tissue. Immunofluorescent labeling of astrocytes and Cx43 was examined using confocal laser scanning microscopy. The images obtained were quantitatively analysed and reconstructed using three-dimensional volume rendering. The results of this study have established that not only is astrocytosis greater in MTLE-affected tissues than previously suggested, but it is accompanied by a highly significant increase in astrocytic Cx43 protein levels. We hypothesize that this surprisingly large upregulation in Cx43 may exacerbate generalized seizures in the progression of MTLE.     

Connexin29 and connexin32 at oligodendrocyte and astrocyte gap junctions and in myelin of the mouse central nervous system

The cellular localization, relation to other glial connexins (Cx30, Cx32, and Cx43), and developmental expression of Cx29 were investigated in the mouse central nervous system (CNS) with an anti-Cx29 antibody. Cx29 was enriched in subcellular fractions of myelin, and immunofluorescence for Cx29 was localized to oligodendrocytes and myelinated fibers throughout the brain and spinal cord. Oligodendrocyte somata displayed minute Cx29-immunopositive puncta around their periphery and intracellularly. In developing brain, Cx29 levels increased during the first few postnatal weeks and were highest in the adult brain. Immunofluorescence labeling for Cx29 in oligodendrocyte somata was intense at young ages and was dramatically shifted in localization primarily to myelinated fibers in mature CNS. Labeling for Cx32 also was localized to oligodendrocyte somata and myelin and absent in Cx32 knockout mice. Cx29 and Cx32 were minimally colocalized on oligodendrocytes somata and partly colocalized along myelinated fibers. At gap junctions on oligodendrocyte somata, Cx43/Cx32 and Cx30/Cx32 were strongly associated, but there was minimal association of Cx29 and Cx43. Cx32 was very sparsely associated with astrocytic connexins along myelinated fibers. With Cx26, Cx30, and Cx43 expressed in astrocytes and Cx29, Cx32, and Cx47 expressed in oligodendrocytes, the number of connexins localized to gap junctions of glial cells is increased to six. The results suggested that Cx29 in mature CNS contributes minimally to gap junctional intercellular communication in oligodendrocyte cell bodies but rather is targeted to myelin, where it, with Cx32, may contribute to connexin-mediated communication between adjacent layers of uncompacted myelin.