Cytoplasmic Polyadenylation Element Binding Proteins CPEB1 and CPEB3 Regulate the Translation of FosB and Are Required for Maintaining Addiction-Like Behaviors Induced by Cocaine.

A recurrent and devastating feature of addiction to a drug of abuse is its persistence, which is mediated by maladaptive long-term memories of the highly pleasurable experience initially associated with the consumption of the drug. We have recently found that members of the CPEB family of proteins (Cytoplasmic Polyadenylation Element-Binding Proteins) are involved in the maintenance of spatial memory. However, their possible role in the maintenance of memories that sustain addictive behavior has yet to be explored. Little is known about any of the mechanisms for maintaining memories for addictive behavior. To address the mechanisms whereby addictive behavior is maintained over time, we utilized a conditional transgenic mouse model expressing a dominant-negative version of CPEB1 that abolishes the activity in the forebrain of two of the four CPEB isoforms (CPEB1 and CPEB3). We found that, following cocaine administration, these dominant-negative (DN) CPEB mice showed a significant decrease, when compared to wild type (WT) mice, in both locomotor sensitizations and conditioned place preference (CPP), two indices of addictive behavior. Supporting these behavioral results, we also found a difference between WT and DN-CPEB1-3 mice in the cocaine-induced synaptic depression in the core of the Nucleus Accumbens (NAc). Finally, we found that (1) CPEB is reduced in transgenic mice following cocaine injections and that (2) FosB, known for its contribution to establishing the addictive phenotype, when its expression in the striatum is increased by drug administration, is a novel target of CPEBs molecules. Thus, our study highlights how CPEB1 and CPEB3 act on target mRNAs to build the neuroadaptative implicit memory responses that lead to the development of the cocaine addictive phenotypes in mammals.

Connexin30 and Connexin43 show a time-of-day dependent expression in the mouse suprachiasmatic nucleus and modulate rhythmic locomotor activity in the context of chronodisruption.

BACKGROUND: The astroglial connexins Cx30 and Cx43 contribute to many important CNS functions including cognitive behaviour, motoric capacity and regulation of the sleep-wake cycle. The sleep wake cycle, is controlled by the circadian system. The central circadian rhythm generator resides in the suprachiasmatic nucleus (SCN). SCN neurons are tightly coupled in order to generate a coherent circadian rhythm. The SCN receives excitatory glutamatergic input from the retina which mediates entrainment of the circadian system to the environmental light-dark cycle. Connexins play an important role in electric coupling of SCN neurons and astrocytic-neuronal signalling that regulates rhythmic SCN neuronal activity. However, little is known about the regulation of Cx30 and Cx43 expression in the SCN, and the role of these connexins in light entrainment of the circadian system and in circadian rhythm generation. METHODS: We analysed time-of-day dependent as well as circadian expression of Cx30 and Cx43 mRNA and protein in the mouse SCN by means of qPCR and immunohistochemistry. Moreover, we analysed rhythmic spontaneous locomotor activity in mice with a targeted deletion of Cx30 and astrocyte specific deletion of Cx43 (DKO) in different light regimes by means of on-cage infrared detectors. RESULTS: Fluctuation of Cx30 protein expression is strongly dependent on the light-dark cycle whereas fluctuation of Cx43 protein expression persisted in constant darkness. DKO mice entrained to the light-dark cycle. However, re-entrainment after a phase delay was slightly impaired in DKO mice. Surprisingly, DKO mice were more resilient to chronodisruption. CONCLUSION: Circadian fluctuation of Cx30 and Cx43 protein expression in the SCN is differently regulated. Cx30 and astroglial Cx43 play a role in rhythm stability and re-entrainment under challenging conditions.

Connexin43, but not connexin30, contributes to adult neurogenesis in the dentate gyrus.

The subgranular zone of the dentate gyrus represents a niche in which radial glia (RG)-like cells generate new neurons throughout postnatal life in the mammalian brain. Previous data showed that RG-like cells are coupled through gap junction channels, primarily formed by connexin43 (Cx43) and Cx30, and that the expression of these proteins is required for adult neurogenesis in the hippocampus. However, their individual function and underlying mechanisms remain unclear. Here we demonstrate that Cx43, but not Cx30, is crucial for adult neurogenesis. To assess whether Cx43-dependent intercellular coupling between RG-like cells or rather channel-independent interactions of the protein regulate neurogenesis, mice bearing a Cx43 point mutation (Cx43G138R) in RG-like cells and protoplasmic astrocytes cells were employed, which was expected to cause channel closure without affecting the trafficking of the protein to the membrane. We confirmed the disruption of coupling between RG-like cells and astrocytes in the hippocampus of Cx43G138R mice. Proliferative activity and neurogenesis in the DG were significantly decreased in the mutant mouse line, indicating that functional Cx43 channels are essential for proper adult neurogenesis. The fate of proliferating cells in the DG was not affected by Cx43 mutation as revealed by 5-bromo-2-deoxyuridine (BrdU) incorporation assays. Together, these findings suggest that adult neurogenesis in the hippocampus does not require Cx30 but channel-dependent functions of Cx43.

Connexin43 Containing Gap Junction Channels Facilitate HIV Bystander Toxicity: Implications in NeuroHIV.

Human immunodeficiency virus-1 (HIV-1) infection compromises the central nervous system (CNS) in a significant number of infected individuals, resulting in neurological dysfunction that ranges from minor cognitive deficits to frank dementia. While macrophages/microglia are the predominant CNS cells infected by HIV, our laboratory and others have shown that HIV-infected astrocytes, although present in relatively low numbers with minimal to undetectable viral replication, play key role in NeuroAIDS pathogenesis. Our laboratory has identified that HIV "hijacks" connexin (Cx) containing channels, such as gap junctions (GJs) and hemichannels (HCs), to spread toxicity and apoptosis to uninfected cells even in the absence of active viral replication. In this study, using a murine model with an astrocyte-directed deletion of Cx43 gene (hGFAP-cre Cx43fl/fl) and control Cx43fl/fl mice, we examined whether few HIV-infected human astrocytoma cells (U87-CD4-CCR5), microinjected into the mouse cortex, can spread toxicity and apoptosis through GJ-mediated mechanisms, into the mouse cells, which are resistant to HIV infection. In the control Cx43fl/fl mice, microinjection of HIV-infected U87-CD4-CCR5 cells led to apoptosis in 84.28 ± 6.38% of mouse brain cells around the site of microinjection, whereas hGFAP-cre Cx43fl/fl mice exhibited minimal apoptosis (2.78 ± 1.55%). However, simultaneous injection of GJ blocker, 18α-glycyrrhetinic acid, and Cx43 blocking peptide along with microinjection of HIV-infected cells prevented apoptosis in Cx43fl/fl mice, demonstrating the Cx43 is essential for HIV-induced bystander toxicity. In conclusion, our findings demonstrate that Cx43 expression, and formation of GJs is essential for bystander apoptosis during HIV infection. These findings reveal novel potential therapeutic targets to reduce astrocyte-mediated bystander toxicity in HIV-infected individuals because despite low to undetectable viral replication in the CNS, Cx channels hijacked by HIV amplify viral neuropathogenesis.

Morphological study of a connexin 43-GFP reporter mouse highlights glial heterogeneity, amacrine cells, and olfactory ensheathing cells.

Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Müller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100ß, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP+ astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc.

Altered splicing leads to reduced activation of CPEB3 in high-grade gliomas.

Cytoplasmic polyadenylation element binding proteins (CPEBs) are auxiliary translational factors that associate with consensus sequences present in 3'UTRs of mRNAs, thereby activating or repressing their translation. Knowing that CPEBs are players in cell cycle regulation and cellular senescence prompted us to investigate their contribution to the molecular pathology of gliomas-most frequent of intracranial tumors found in humans. To this end, we performed methylation analyses in the promoter regions of CPEB1-4 and identified the CPEB1 gene to be hypermethylated in tumor samples. Decreased expression of CPEB1 protein in gliomas correlated with the rising grade of tumor malignancy. Abundant expression of CPEBs2-4 was observed in several glioma specimens. Interestingly, expression of CPEB3 positively correlated with tumor progression and malignancy but negatively correlated with protein phosphorylation in the alternatively spliced region. Our data suggest that loss of CPEB3 activity in high-grade gliomas is caused by expression of alternatively spliced variants lacking the B-region that overlaps with the kinase recognition site. We conclude that deregulation of CPEB proteins may be a frequent phenomenon in gliomas and occurs on the level of transcription involving epigenetic mechanism as well as on the level of mRNA splicing, which generates isoforms with compromised biological properties.

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.

Astrocyte uncoupling as a cause of human temporal lobe epilepsy.

Glial cells are now recognized as active communication partners in the central nervous system, and this new perspective has rekindled the question of their role in pathology. In the present study we analysed functional properties of astrocytes in hippocampal specimens from patients with mesial temporal lobe epilepsy without (n = 44) and with sclerosis (n = 75) combining patch clamp recording, K(+) concentration analysis, electroencephalography/video-monitoring, and fate mapping analysis. We found that the hippocampus of patients with mesial temporal lobe epilepsy with sclerosis is completely devoid of bona fide astrocytes and gap junction coupling, whereas coupled astrocytes were abundantly present in non-sclerotic specimens. To decide whether these glial changes represent cause or effect of mesial temporal lobe epilepsy with sclerosis, we developed a mouse model that reproduced key features of human mesial temporal lobe epilepsy with sclerosis. In this model, uncoupling impaired K(+) buffering and temporally preceded apoptotic neuronal death and the generation of spontaneous seizures. Uncoupling was induced through intraperitoneal injection of lipopolysaccharide, prevented in Toll-like receptor4 knockout mice and reproduced in situ through acute cytokine or lipopolysaccharide incubation. Fate mapping confirmed that in the course of mesial temporal lobe epilepsy with sclerosis, astrocytes acquire an atypical functional phenotype and lose coupling. These data suggest that astrocyte dysfunction might be a prime cause of mesial temporal lobe epilepsy with sclerosis and identify novel targets for anti-epileptogenic therapeutic intervention.

Characterization of Panglial Gap Junction Networks in the Thalamus, Neocortex, and Hippocampus Reveals a Unique Population of Glial Cells.

The thalamus plays important roles as a relay station for sensory information in the central nervous system (CNS). Although thalamic glial cells participate in this activity, little is known about their properties. In this study, we characterized the formation of coupled networks between astrocytes and oligodendrocytes in the murine ventrobasal thalamus and compared these properties with those in the hippocampus and cortex. Biocytin filling of individual astrocytes or oligodendrocytes revealed large panglial networks in all 3 gray matter regions. Combined analyses of mice with cell type-specific deletion of connexins (Cxs), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that Cx30 is the dominant astrocytic Cx in the thalamus. Many thalamic astrocytes even lack expression of Cx43, while in the hippocampus astrocytic coupling is dominated by Cx43. Deletion of Cx30 and Cx47 led to complete loss of panglial coupling, which was restored when one allele of either Cxs was present. Immunohistochemistry revealed a unique antigen profile of thalamic glia and identified an intermediate cell type expressing both Olig2 and Cx43. Our findings further the emerging concept of glial heterogeneity across brain regions.

Characterization of cytoplasmic polyadenylation element binding 2 protein expression and its RNA binding activity.

Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with ß-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.

Germ-line recombination activity of the widely used hGFAP-Cre and nestin-Cre transgenes.

Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies.

Connexin36 (Cx36) expression and protein detection in the mouse carotid body and myenteric plexus.

Although connexin36 (Cx36) has been studied in several tissues, it is notable that no data are available on Cx36 expression in the carotid body and the intestine. The present study was undertaken to evaluate using immunohistochemistry, PCR and Western blotting procedures, whether Cx36 was expressed in the mouse carotid body and in the intestine at ileum and colon level. In the carotid body, Cx36 was detected as diffuse punctate immunostaining and as protein by Western blotting and mRNA by RT-PCR. Cx36 punctate immunostaining was also evident in the intestine with localization restricted to the myenteric plexus of both the ileum and the colon, and this detection was also confirmed by Western blotting and RT-PCR. All the data obtained were validated using Cx36 knockout mice. Taken together the present data on localization of Cx36 gap-junctions in two tissues of neural crest-derived neuroendocrine organs may provide an anatomical basis for future functional investigations.

Dual reporter approaches for identification of Cre efficacy and astrocyte heterogeneity.

Gene inactivation reporters are powerful tools to circumvent limitations of the widely used Cre/loxP system of conditional mutagenesis. With new conditional transgenic mouse lines expressing the enhanced cyan fluorescent protein (ECFP) instead of connexin43 (Cx43) after Cre-mediated recombination, we demonstrate dual reporter approaches to simultaneously examine astrocyte subpopulations expressing different connexins, identify compensatory up-regulation within gene families, and quantify Cre-mediated deletion at the allelic level. Analysis of a newly generated Cx43 knock-in ECFP mouse revealed an unexpected heterogeneity of Cx43-expressing astrocytes across brain areas.

Connexin-based intercellular communication and astrocyte heterogeneity.

This review gives an overview of the current knowledge on connexin-mediated communication in astrocytes, covering gap junction and hemichannel functions mediated by connexins. Astroglia is the main brain cell type that expresses the largest amount of connexin and exhibits high level of gap junctional communication compared to neurons and oligodendrocytes. However, in certain developmental and regional situations, astrocytes are also coupled with oligodendrocytes and neurons. This heterotypic coupling is infrequent and minor in terms of extent of the coupling area, which does not mean that it is not important in terms of cell interaction. Here, we present an update on heterogeneity of connexin expression and function at the molecular, subcellular, cellular and networking levels. Interestingly, while astrocytes were initially considered as a homogenous population, there is now increasing evidence for morphological, developmental, molecular and physiological heterogeneity of astrocytes. Consequently, the specificity of gap junction channel- and hemichannel-mediated communication, which tends to synchronize cell populations, is also a parameter to take into account when neuroglial interactions are investigated. This article is part of a Special Issue entitled Electrical Synapses.

Panglial gap junctional communication is essential for maintenance of myelin in the CNS.

In this study, we have investigated the contribution of oligodendrocytic connexin47 (Cx47) and astrocytic Cx30 to panglial gap junctional networks as well as myelin maintenance and function by deletion of both connexin coding DNAs in mice. Biocytin injections revealed complete disruption of oligodendrocyte-to-astrocyte coupling in the white matter of 10- to 15-d-old Cx30/Cx47 double-deficient mice, while oligodendrocyte-to-oligodendrocyte coupling was maintained. There were no quantitative differences regarding cellular networks in acute brain slices obtained from Cx30/Cx47 double-null mice and control littermates, probably caused by the upregulation of oligodendrocytic Cx32 in Cx30/Cx47 double-deficient mice. We observed early onset myelin pathology, and ∼40% of Cx30/Cx47 double-deficient animals died within 42 to 90 d after birth, accompanied by severe motor impairments. Histological and ultrastructural analyses revealed severe vacuolization and myelination defects in all white matter tracts of the CNS. Furthermore, Cx30/Cx47 double-deficient mice exhibited a decreased number of oligodendrocytes, severe astrogliosis, and microglial activation in white matter tracts. Although less affected concerning motor impairment, surviving double-knock-out (KO) mice showed behavioral alterations in the open field and in the rotarod task. Vacuole formation and thinner myelin sheaths were evident also with adult surviving double-KO mice. Since interastrocytic coupling due to Cx43 expression and interoligodendrocytic coupling because of Cx32 expression are still maintained, Cx30/Cx47 double-deficient mice demonstrate the functional role of both connexins for interastrocytic, interoligodendrocytic, and panglial coupling, and show that both connexins are required for maintenance of myelin.

Functional redundancy and compensation among members of gap junction protein families?

Gap junctions are intercellular conduits for small molecules made up by protein subunits called connexins. A large number of connexin genes were found in mouse and man, and most cell types express several connexins, lending support to the view that redundancy and compensation among family members exist. This review gives an overview of the current knowledge on redundancy and functional compensation - or lack thereof. It takes into account the different properties of connexin subunits which comprise gap junctional intercellular channels, but also the compatibility of connexins in gap junctions. Most insight has been gained by the investigation of mice deficient for one or more connexins and transgenic mice with functional replacement of one connexin gene by another. Most single deficient mice show phenotypical alterations limited to critical developmental time points or to specific organs and tissues, while mice doubly deficient for connexins expressed in the same cell type usually show more severe phenotypical alterations. Replacement of a connexin by another connexin in some cases gave rise to rescue of phenotypical alterations of connexin deficiencies, which were restricted to specific tissues. In many tissues, connexin substitution did not restore phenotypical alterations of connexin deficiencies, indicating that connexins are specialized in function. In some cases, fatal consequences arose from the replacement. The current consensus gained from such studies is that redundancy and compensation among connexins exists at least to a limited extent. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex.

Gap junctions mediate intercellular spread of sodium between hippocampal astrocytes in situ.

Activation of glutamatergic synapses results in long-lasting sodium transients in astrocytes mediated mainly by sodium-dependent glutamate uptake. Sodium elevations activate Na(+) /K(+) -ATPase and glucose uptake by astrocytes, representing key signals for coupling glial metabolism to neuronal activity. Here, we analyzed the spread of sodium signals between astrocytes in hippocampal slice preparations. Stimulation of a single astrocyte resulted in an immediate sodium elevation that spread to neighboring astrocytes within a distance of ∼ 100 µm. Amplitude, slope, and propagation speed of sodium elevations in downstream cells decayed monotonically with increasing distance, indicative of a diffusion process. In contrast to sodium, calcium increases elicited by electrical stimulation were restricted to the stimulated cell and a few neighboring astrocytes. Pharmacological inhibition of mGluR1/5 slightly dampened the spread of sodium, whereas inhibition of glutamate uptake or purinergic receptors had no effect. Spread of sodium to neighboring cells was disturbed on pharmacological inhibition of gap junctions, reduced in animals at P4 and virtually omitted in Cx30/Cx43 double-deficient mice. In contrast to results obtained earlier in cultured astrocytes, our data thus indicate that calcium signaling and metabotropic glutamate receptors are supportive of, but not prerequisites for, the spread of sodium between hippocampal astrocytes in situ, whereas expression of Cx30 and Cx43 is essential. Cx30/Cx43-mediated sodium diffusion between astrocytes could represent a signal indicating increased metabolic needs, independent of concomitant calcium signaling. Spread of sodium might also serve a homeostatic function by supporting the re-establishment of steep sodium gradients and by lowering the metabolic burden imposed on single cells.

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.

Pharmacological and genetic approaches to study connexin-mediated channels in glial cells of the central nervous system.

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.