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1-O-Acylceramides (1-OACs) have a fatty acid esterified to the 1-hydroxyl of the sphingosine head group of the ceramide, and recently we identified these lipids as natural components of human and mouse epidermis. Here we show epidermal 1-OACs arise shortly before birth during the establishment of the water permeability barrier in mice. Fractionation of human epidermis indicates 1-OACs concentrate in the stratum corneum. During in vitro maturation into reconstructed human epidermis, human keratinocytes dramatically increase 1-OAC levels indicating they are one source of epidermal 1-OACs. In search of potential enzymes responsible for 1-OAC synthesis in vivo, we analyzed mutant mice with deficiencies of ceramide synthases (Cers2, Cers3, or Cers4), diacylglycerol acyltransferases (Dgat1 or Dgat2), elongase of very long fatty acids 3 (Elovl3), lecithin cholesterol acyltransferase (Lcat), stearoyl-CoA desaturase 1 (Scd1), or acidic ceramidase (Asah1). Overall levels of 1-OACs did not decrease in any mouse model. In Cers3 and Dgat2-deficient epidermis they even increased in correlation with deficient skin barrier function. Dagt2 deficiency reshapes 1-OAC synthesis with an increase in 1-OACs with N-linked non-hydroxylated fatty acids and a 60% decrease compared to control in levels of 1-OACs with N-linked hydroxylated palmitate. As none of the single enzyme deficiencies we examined resulted in a lack of 1-OACs, we conclude that either there is functional redundancy in forming 1-OAC and more than one enzyme is involved, and/or an unknown acyltransferase of the epidermis performs the final step of 1-OAC synthesis, the implications of which are discussed.

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life.

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.

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells.

Complex sphingolipids are strongly expressed in neuronal tissue and contain ceramides in their backbone. Ceramides are synthesized by six ceramide synthases (CerS1-6). Although it is known that each tissue has a unique profile of ceramide synthase expression and ceramide synthases are implicated in several neurodegenerative disorders, the expression of ceramide synthase isoforms has not been investigated in the retina. Here we demonstrate CerS1, CerS2 and CerS4 expression in mouse retina and cornea, with CerS4 ubiquitously expressed in all retinal neurons and Müller cells. To test whether ceramide synthase deficiency affects retinal function, we compared electroretinograms and retina morphology between wild-type and CerS1-, CerS2- and CerS4-deficient mice. Electroretinograms were strongly reduced in amplitude in ceramide synthase-deficient mice, suggesting that signalling in the outer retina is affected. However, the number of photoreceptors and cone outer segment length were unaltered and no changes in retinal layer thickness or synaptic structures were found. Mass spectrometric analyses of ceramides, hexosyl-ceramides and sphingomyelins showed that C20 to C24 acyl-containing species were decreased whereas C16-containing species were increased in the retina of ceramide synthase-deficient mice. Similar but smaller changes were also found in the cornea. Thus, we hypothesize that the replacement of very long-chain fatty acyl residues by shorter C16 residues may affect the electrical properties of retina and cornea, and alter receptor-mediated signal transduction, vesicle-mediated synaptic transmission or corneal light transmission. Future studies need to identify the molecular targets of ceramides or derived sphingolipids in light signal transduction and transmission in the eye.

Correction to:NPG Asia Materials (2018) https://doi.org/10.1038/s41427-018-0014-9 published online on 16 April 2018

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse.SIGNIFICANCE STATEMENTHorizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light responses of transient OFF-α retinal ganglion cells in a newly generated mouse line. In this mouse line, horizontal cell signals were no longer modulated by light. With light response recordings, we show that horizontal cells increase the dynamic range of retinal ganglion cells for contrast and temporal changes and contribute to the center/surround organization of their receptive fields.

Background— Sudden infant death syndrome (SIDS) describes the sudden, unexplained death of a baby during its first year of age and is the third leading cause of infant mortality. It is assumed that ≤20% of all SIDS cases are because of cardiac arrhythmias resulting from mutations in ion channel proteins. Besides ion channels also cardiac gap junction channels are important for proper conduction of cardiac electric activation. In the mammalian heart Connexin43 (Cx43) is the major gap junction protein expressed in ventricular cardiomyocytes. Recently, a novel Connexin43 loss-of-function mutation (Cx43E42K) was identified in a 2-month-old SIDS victim. Methods and Results— We have generated Cx43E42K-expressing mice as a model for SIDS. Heterozygous cardiac-restricted Cx43E42K-mutated mice die neonatally without major cardiac morphological defects. Electrocardiographic recordings of embryonic Cx43+/E42K mice reveal severely disturbed ventricular activation, whereas immunohistochemical analyses show normal localization and expression patterns of gap junctional Connexin43 protein in the Cx43E42K-mutated newborn mouse heart. Conclusions— Because we did not find heterogeneous gap junction loss in Cx43E42K mouse hearts, we conclude that the Cx43E42K gap junction channel creates an arrhythmogenic substrate leading to lethal ventricular arrhythmias. The strong cardiac phenotype of Cx43E42K expressing mice supports the association between the human Cx43E42K mutation and SIDS and indicates that Connexin43 mutations should be considered in future studies when SIDS cases are to be molecularly explained.

Ceramides (Cer) are mediators of inflammatory processes. In a chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we observed a significant elevation of C16-Cer and its synthesizing enzyme, ceramide synthase(CerS)6, in the lumbar spinal cord. In the present study, we have confirmed that C16-Cer and CerS6 are also upregulated in the lumbar spinal cord in a spontaneous relapse-remitting EAE model, using SJL mice overexpressing a transgenic T cell receptor (TCR1640). CerS6 was found to be expressed in macrophages, T cells and B cells in EAE lesions. In macrophages, we demonstrated that interferon gamma (IFN-γ)-induced CerS6 upregulation was amplified by 17ß-estradiol, an action that was further accompanied by increased upregulation of tumor necrosis factor alpha (TNF-α). Accordingly, CerS6 and TNF-α expression was upregulated predominantly in the spinal cord in female TCR1640 mice, which usually develop the relapse-remitting form of EAE, while male TCR1640 mice showed an attenuated regulation of CerS6 and TNF-α and exhibit mostly chronic disease progression. Furthermore, expression of TNFR2, one of two receptors of TNF-α, which is linked to neuroprotection and remyelination, was also upregulated to a greater extent during EAE in female TCR1640 mice in comparison to male TCR1640 mice. Taken together, our results confirm the upregulation of CerS6 and C16-Cer in an adjuvant-independent, physiological EAE model and further suggest an anti-inflammatory role of CerS6 in the regulation of the disease course in female TCR1640 mice via TNF-α/TNFR2.

The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1–6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.

Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured cells.

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.

During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca2+ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd+) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd+ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y1R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca2+ too, which are required for potentiation of contraction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Ceramide synthase 2 (CerS2) catalyzes the synthesis of dihydroceramides from dihydrosphingosine and very long fatty acyl (C22-C24)-CoAs. CerS2-deficient (gene trap) mice were reported to exhibit myelin and behavioral abnormalities, associated with the expression of CerS2 in oligodendrocytes and neurons based on expression of lacZ reporter cDNA instead of the cers2 gene in these mice. In order to clarify the cell-type-specific expression of CerS2 protein, we have raised antibodies that specifically recognize the glycosylated and non-glycosylated CerS2 protein in wild-type but not in CerS2-deficient mouse tissues. In early postnatal, juvenile and adult mouse brain, the new antibodies detect CerS2 protein only in oligodendrocytes but not in neurons, suggesting that the gene trap vector in CerS2-deficient mice led to ectopic expression of the lacZ reporter gene in neurons. In liver, the CerS2 protein is expressed in hepatocytes but not in Ito cells or Kupffer cells. We conclude that the behavioral abnormalities observed in CerS2-deficient mice originate primarily in oligodendrocytes and not in neurons. The identification of specific cell types in which CerS2 protein is expressed is prerequisite to further mechanistic characterization of phenotypic abnormalities exhibited by CerS2-deficient mice. The amount of CerS2 protein detected in different tissues by immunoblot analyses does not strictly correspond to the activity of the CerS2 enzyme. Disproportional results are likely due to post-translational regulation of the CerS2 protein.

Panglial networks are essential for normal physiology in the CNS, and the function of distinct connexins participating in these networks is not well understood. We generated Connexin32 (Cx32)-deficient mice with additional deletion of astrocytic Cx43 to explore the role of both connexins in panglial networks. Cx43/Cx32 double knock-out (dKO) mice revealed strong microglial activation in corpus callosum and cingulum along with severe astrogliosis and scar formation. In addition, most of the fine myelinated fibers projecting from the corpus callosum into the cortex were lost. Myelin loss was caused by a strong decrease of oligodendrocytes in the cingulum of Cx43/Cx32dKO mice. Immunoblot analyses using newly generated specific Cx47 antibodies revealed that oligodendrocytic Cx47 is phosphorylatedin vivodepending on astrocytic Cx43 expression. In Cx43-deficient mice, Cx47 protein levels were strongly decreased, whereas Cx47 mRNA levels were not altered. Using Cx43G138R/Cx30KO mice, we show that Cx47 expression depends on the presence of astrocytic Cx43 protein and that its gap junctional channel function is not necessary for Cx47 stabilization. In consequence, Cx43/Cx32dKO mice additionally lack Cx47 expression and therefore cannot form oligodendrocytic gap junctions, which explains the phenotypic similarities to Cx32/Cx47dKO mice. Our findings provide strong evidence that phosphorylation and stability of oligodendrocytic Cx47 proteins is dependent on astrocytic Cx43 expression. These results further unravel the complexity of panglial networks and show that results of previous studies using astrocytic Cx43-deficient mice have to be reconsidered.

Rationale: The gap junctional protein connexin (Cx) 45 is strongly expressed in the early embryonic myocardium. In the adult hearts of mice and humans, the expression mainly is restricted to the cardiac conduction system. Cx45 plays an essential role for development and function of the embryonic heart because general and cardiomyocyte-directed deficiencies of Cx45 in mice lead to embryonic lethality attributable to morphological and functional cardiovascular defects. The function of Cx45 in the adult mouse has not yet been cleared. Objective: To clarify the function of Cx45 in the adult mouse heart. Methods and Results: To circumvent the embryonic lethality resulting from Cx45 deficiency, mice were generated in which deletion of Cx45 specifically was induced in cardiomyocytes of adult mice. These Cx45-deficient mice were viable but showed a decrease in atrioventricular nodal conductivity. In addition, the Cx30.2 protein that is coexpressed with Cx45 in the cardiac conduction system was posttranscriptionally reduced by 70% in mutant hearts. Furthermore, deletion of both Cx45 and Cx30.2 resulted in viable mice that, however, showed stronger impairment of atrioventricular nodal conduction than the single Cx45-deficient mice. Conclusions: Cx45 is required for optimal impulse propagation in the atrioventricular node and stabilizes the level of the coexpressed Cx30.2 protein in the adult mouse heart. In contrast to the embryo, Cx45 is not essential for the viability of adult mice.

The gap junction channel protein connexin40 (Cx40) is crucial in vascular and renal physiology, because Cx40-deficient mice exhibit impaired conduction of endothelium-dependent dilations and pronounced hypertension. The latter precludes mechanistic insights into the role of endothelial Cx40, because long-lasting hypertension itself may affect conduction and Cx expression. We aimed to identify endothelial Cx40 functions, their dependency on the conductive capability, and to separate these from hypertension-related alterations. We assessed conduction and Cx expression in mice with cell type–specific deletion of Cx40 and in mice expressing a defective Cx40 (Cx40A96S) identified in humans, which forms nonconducting gap junction channels. Confined arteriolar stimulation with acetylcholine or bradykinin elicited local dilations that conducted upstream without attenuation of the amplitude for distances up to 1.2-mm in controls with a floxed Cx40 gene (Cx40 fl/fl ). Conducted responses in hypertensive animals devoid of Cx40 in renin-producing cells were unaltered but remote dilations were reduced in normotensive animals deficient for Cx40 in endothelial cells (Cx40 fl/fl :Tie2-Cre). Surprisingly, Cx37 expression was undetectable by immunostaining in arteriolar endothelium only in Cx40 fl/fl :Tie2-Cre; however, transcriptional activity of Cx37 in the cremaster was comparable with Cx40 fl/fl controls. Cx40A96S mice were hypertensive with preserved expression of Cx40 and Cx37. Nevertheless, conducted responses were blunted. We conclude that endothelial Cx40 is necessary to support conducted dilations initiated by endothelial agonists and to locate Cx37 into the plasma membrane. These functions are unaltered by long-lasting hypertension. In the presence of a nonconducting Cx40, Cx37 is present but cannot support the conduction highlighting the importance of endothelial Cx40.

Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelin-associated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior.

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart–brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.—Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36–containing gap junctions.

Well-defined gradients of the lipid mediator sphingosine-1-phosphate (S1P) direct chemotactic egress of mature thymocytes from the thymus into the circulation. Although it is known that these gradients result from low S1P levels in the thymic parenchyma and high S1P concentrations at the exit sites and in the plasma, the biochemical mechanisms that regulate these differential S1P levels remain unclear. Several studies demonstrated that ceramide synthase 2 (Cers2) regulates the levels of the S1P precursor sphingosine. We, therefore, investigated whether Cers2 is involved in the regulation of S1P gradients and S1P-dependent egress into the circulation. By analyzing Cers2-deficient mice, we demonstrate that Cers2 limits the levels of S1P in thymus and blood to maintain functional S1P gradients that mediate thymocyte emigration into the circulation. This function is specific for Cers2, as we also show that Cers4 is not involved in the regulation of thymic egress. Our study identified Cers2 as an important regulator of S1P-dependent thymic egress, and thus contributes to the understanding of how S1P gradients are maintained in vivo.

Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I–III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I–III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.

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.

Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To investigate this issue further, we examined immunofluorescence labelling of glial connexins in wild-type vs. transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal and non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.

Deletion of the gap-junction-forming protein connexin40 leads to renin-dependent hypertension in mice, but whether observed human variants in connexin40, such as A96S, promote hypertension is unknown. Here, we generated mice with the A96S variant in the mouse connexin40 gene. Although mice homozygous for the A96S mutations had normal expression patterns of connexin40 in the kidney, they were hypertensive, had sixfold higher plasma renin concentrations, and had 40% higher levels of renin mRNA than controls. Renin-expressing cells were aberrantly located outside the media layer of afferent arterioles, and increased renal perfusion pressure did not inhibit renin secretion from kidneys isolated from homozygous A96S mice. Treatment with a low-salt diet in combination with an ACE inhibitor increased renin mRNA levels, plasma renin concentrations, and the number of aberrantly localized renin-producing cells. Taken together, these findings suggest that the A96S mutation in connexin40 leads to renin-dependent hypertension in mice. Modulation of renin secretion by BP critically depends on functional connexin40; with the A96S mutation, the aberrant extravascular localization of renin-secreting cells in the kidney likely impairs the pressure-mediated inhibition of renin secretion.

The role of gap junctions in the neural control of fertility remains poorly understood. Using acute brain slices from adult GnRH-green fluorescent protein transgenic mice, individual GnRH neurons were filled with a mixture of fluorescent dextran and neurobiotin. No dye transfer was found between any GnRH neurons, although approximately 30% of GnRH neurons exchanged neurobiotin with closely apposed cells. Dual electrophysiological recordings from pairs of GnRH neurons revealed an absence of electrical coupling. Using adult connexin 36 (Cx36)-cyan fluorescent protein transgenic mice, Cx36 was identified in cells within several hypothalamic brain regions, including 64% of preoptic area kisspeptin neurons but not in GnRH neurons. To assess the potential role of Cx36 in non-GnRH neurons within the GnRH neuronal network (i.e. neurons providing afferent inputs to GnRH neurons), a calmodulin kinase IIα-Cre (CKC)-LoxP strategy was used to generate mice with a neuron-specific deletion of Cx36 beginning in the first postnatal week. Mutant female mice exhibited normal puberty onset but disordered estrous cyclicity, although their fecundity was normal as was their estrogen-negative and -positive feedback mechanisms. The effects of adult deletion of Cx36 from neurons were assessed using a tamoxifen-dependent inducible CKC-Cx36 transgenic strategy. Mutant mice exhibited the same reproductive phenotype as the CKC-Cx36 animals. Together these observations demonstrate that there is no gap junctional coupling between GnRH neurons. However, it is apparent that other neurons within the GnRH neuronal network, potentially the preoptic kisspeptin neurons, are dependent on Cx36 gap junctions and that this is critical for normal estrous cyclicity.

Gap junctional coupling of renin-producing cells is of major functional importance for the control of renin synthesis and release. This study was designed to determine the relevance of the vascular gap junction protein connexin 45 (Cx45) for the control of renin expression and secretion. By crossbreeding mice which drive Cre recombinase under the control of the endogenous renin promoter with mice harboring floxed Cx45 gene alleles, we generated viable mice with a deletion of Cx45 in the renin cell lineage. These mice were normotensive, and renin cells in their kidneys were normal with regard to localization and number. Sodium deficiency induced typical recruitment of renin-producing cells along afferent arterioles, whereas sodium overload resulted in a decrease in the number of cells expressing renin. Regulation of renin secretion by perfusion pressure, catecholamines, and angiotensin II from isolated kidneys of mice with renin cell-specific deletion of Cx45 was normal. Analyzing Cx45 promoter activity in cells of the preglomerular arteriolar tree by using mice driving the reporter gene LacZ under the control of the Cx45 promoter revealed strong staining in smooth muscle cells of the media, whereas renin-expressing cells were almost devoid of LacZ staining. Conversely, renin-producing cells, but not vascular smooth muscle cells expressed the gap junction protein Cx40. These findings suggest that Cx45 plays no major functional role in renin-producing cells, probably because the expression of Cx45 is downregulated in these cells. Since renin-producing cells in the adult kidney can reversibly transform into vascular smooth muscle cells and vice versa, our findings on connexin expression indicate that these phenotype switches are paralleled by characteristic reciprocal changes in the transcriptional activity of Cx40 and Cx45 genes.

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons. J. Comp. Neurol. 519:433–450, 2011. © 2010 Wiley-Liss, Inc.

The gap junction protein connexin-45 (Cx45) is expressed in the conduction system of the heart and in certain neurons of the retina and brain. General and cardiomyocyte-directed deficiencies of Cx45 in mice lead to lethality on embryonic day 10.5 as a result of cardiovascular defects. Neuron-directed deletion of Cx45 leads to defects in transmission of visual signals. Connexin-36 (Cx36) is co-expressed with Cx45 in certain types of retinal interneurons. To determine whether these two connexins have similar functions and whether Cx36 can compensate for Cx45, we generated knock-in mice in which DNA encoding Cx45 was replaced with that encoding Cx36. Neuron-directed replacement of Cx45 with Cx36 resulted in viable animals. Electroretinographic and neurotransmitter coupling analyses demonstrated functional compensation in the retina. By contrast, general and cardiomyocyte-directed gene replacement led to lethality on embryonic day 11.5. Mutant embryos displayed defects in cardiac morphogenesis and conduction. Thus, functional compensation of Cx45 by Cx36 did not occur during embryonic heart development. These data suggest that Cx45 and Cx36 have similar functions in the retina, whereas Cx45 fulfills special functions in the developing heart that cannot be compensated by Cx36.

Arterial PCO2, a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of PCO2 in the blood and brain, yet the mechanisms by which the brain senses CO(2) remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO(2)-dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO(2)-dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of PCO2. ATP release is independent of extracellular Ca(2+) and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO(2)-dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO(2)-dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO(2)-dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in PCO2 is an important mechanism contributing to central respiratory chemosensitivity.

Mutations in the GJB2 gene coding for connexin26 (Cx26) can cause a variety of deafness and hereditary hyperproliferative skin disorders in humans. In this study, we investigated the Cx26S17F mutation in mice, which had been identified to cause the keratitis-ichthyosis-deafness (KID) syndrome in humans. The KID syndrome is characterized by keratitis and chronic progressive corneal neovascularization, skin hyperplasia, sensorineural hearing loss and increased carcinogenic potential. We have generated a conditional mouse mutant, in which the floxed wild-type Cx26-coding DNA can be deleted and the Cx26S17F mutation is expressed under control of the endogenous Cx26 promoter. Homozygous mutants are not viable, whereas the surviving heterozygous mice show hyperplasia of tail and foot epidermis, wounded tails and annular tail restrictions, and are smaller than their wild-type littermates. Analyses of auditory brainstem responses (ABRs) indicate an ∼35 dB increased hearing threshold in these mice, which is likely due to the reduction of the endocochlear potential by 20-40%. Our results indicate that the Cx26S17F protein, which does not form functional gap junction channels or hemichannels, alters epidermal proliferation and differentiation in the heterozygous state. In the inner ear, reduced intercellular coupling by heteromeric channels composed of Cx26S17F and Cx30 could contribute to hearing impairment in heterozygous mice, while remaining wild-type Cx26 may be sufficient to stabilize Cx30 and partially maintain cochlear homeostasis. The phenotype of heterozygous mice resembles many of the symptoms of the human KID syndrome. Thus, these mice represent an appropriate model to further investigate the disease mechanism.

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30(T5M/T5M) mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca(2+) signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.

Neuronal gap junctions, allowing fast intercellular electrotonic signal transfer, have been implicated in mechanisms governing learning and memory processes. We have examined conditional neuron-directed (Cx45fl/fl:Nestin-Cre) connexin45 deficient mice in terms of behavioral and electrophysiological correlates of learning and memory. Behavioral habituation to a novel environment and motor learning were not changed in these mice. Novel object recognition after delays of up to 60min was impaired in neuronal Cx45 deficient mice. However, object-place recognition was not significantly different from controls. Analysis of enhanced green fluorescent reporter protein expression controlled by the endogenous mouse Cx45 promoter in the brain of neuronal Cx45 deficient mice suggested that Cx45 is expressed in the perirhinal cortex and the CA3 subregion of the hippocampus. The neuronal Cx45 deficient mice were also examined for aberrations in the generation and synchronization of network oscillations in the hippocampus. General excitability, synaptic short time plasticity, and spontaneous high-frequency oscillations (sharp-wave ripples) in the hippocampus were not different from controls. However, bath stimulation of hippocampal slices with kainate induced significantly lower gamma-oscillation amplitudes in the CA3, but not in the CA1 subfield of the neuronal Cx45 deficient mice. Additionally, they exhibited a significantly larger full width half maximum of the frequency distribution in the CA1 subfield as compared to the controls. In conclusion, the neuron-directed deletion of Cx45 impaired one-trial novel object recognition and altered kainate-induced gamma-oscillations possibly via the disruption of inter-neuronal gap junctional communication in the hippocampus or perirhinal cortex.

The study shows the dynamic expression of connexin47 (Cx47) in oligodendrocytes and myelin of mice, either in myelinogenesis occurring in early development or in an experimental model of new-myelinogenesis of adult mice. Cx47 first appeared in the embryonic mouse brain at E10.5 successively the expression increased, principally in regions populated by developing oligodendrocytes. The expression declined postnatally toward adulthood and immunoreactivity was restricted to a few specific areas, such as the corpus callosum, the striatum, the cerebellum, and the spinal cord. Since the expression of Cx47 in developing oligodendrocytes preceded those of Cx32 and Cx29, a role of Cx47 in myelinogenesis was postulated. This hypothesis was tested in a model of re-myelination, which principally involved the corpus callosum, occurring in adult mice by treatment with cuprizone. Cx47 was upregulated during demyelination and recovered during the remyelination phase. During demyelination, Cx47 was first over-expressed in the corpus callosum and later, when the myelin virtually disappeared in the injured areas, Cx47 was expressed in astrocytes located inside and closely around the demyelinated areas. The remyelination of injured areas occurred after stopping the administration of cuprizone and continued to complete recovery. In this period the expression of Cx47 shifted from astrocytes to newly-formed myelin. Thus, Cx47 exhibits in this model a transient and de novo expression in astrocytes with a topographic segregation in the injured areas, only when oligodendrocytes and the myelin were most severely affected. Taken as a whole the evidence suggests that Cx47 play a key role in myelination.

Connexin43 plays an important role in neuroprotection in experimental stroke models; reducing the expression of this gap junction protein in astrocytes enhances injury upon middle cerebral artery occlusion (MCAO). Because the C-terminal region of connexin43 isimportant for channel activity, we carried out MCAO stroke experiments in mice expressing a truncated form of connexin43 (Cx43DeltaCT mice). Brain sections were analyzed for infarct volume, astrogliosis, and inflammatory cell invasion 4 days after MCAO. Adult cortices and astrocyte cultures were examined for connexin43 (Cx43) expression by immunohistochemistry and Western blot. Cultured astrocytes were also examined for dye coupling, channel conductance, hemichannel activity, and Ca wave propagation. The Cx43DeltaCT mice exhibit enhanced cerebral injury after stroke. Astrogliosis was reduced and inflammatory cell invasion was increased inthe peri-infarct region in these mice compared with controls; Cx43 expression was also altered. Lastly, cultured astrocytes from Cx43DeltaCT mice were less coupled and displayed alterations in channel gating, hemichannel activity, and Ca wave properties. These results suggest that astrocytic Cx43 contributed to the regulation of cell death after stroke and support the view that the Cx43 C-terminal region is important in protection in cerebral ischemia.

In the renal tubule, ATP is an important regulator of salt and water reabsorption, but the mechanism of ATP release is unknown. Several connexin (Cx) isoforms form mechanosensitive, ATP-permeable hemichannels. We localized Cx30 to the nonjunctional apical membrane of cells in the distal nephron and tested whether Cx30 participates in physiologically important release of ATP. We dissected, partially split open, and microperfused cortical collecting ducts from wild-type and Cx30-deficient mice in vitro. We used PC12 cells as ATP biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the apical surface of either intercalated or principal cells. ATP biosensor responses, triggered by increased tubular flow or by bath hypotonicity, were approximately three-fold greater when positioned next to intercalated cells than next to principal cells. In addition, these responses did not occur in preparations from Cx30-deficient mice or with purinergic receptor blockade. After inducing step increases in mean arterial pressure by ligating the distal aorta followed by the mesenteric and celiac arteries, urine output increased 4.2-fold in wild-type mice compared with 2.6-fold in Cx30-deficient mice, and urinary Na(+) excretion increased 5.2-fold in wild-type mice compared with 2.8-fold in Cx30-deficient mice. Furthermore, Cx30-deficient mice developed endothelial sodium channel-dependent, salt-sensitive elevations in mean arterial pressure. Taken together, we suggest that mechanosensitive Cx30 hemichannels have an integral role in pressure natriuresis by releasing ATP into the tubular fluid, which inhibits salt and water reabsorption.

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to ∼400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is ∼90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pHi from 7.2 to 7.8 increased gap junctional conductance (gj) of ∼100-fold with pKa= 7.41. We show that this gj increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca2+]o=∼0.3 mm or below, demonstrating that a moderate reduction of [Ca2+]o can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pHi with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell–cell coupling in horizontal cell in the retina.

In the human heart connexin(Cx)40, Cx43 and Cx45-containing gap junctional channels electrically couple cardiomyocytes, forming a functional syncytium. In the mouse heart, additionally, Cx30.2-containing gap junctions have been detected in the atrioventricular node where they are implicated, together with Cx45, in impulse delay. However, whether the human ortholog of Cx30.2, Cx31.9, is expressed in the human heart has not previously been investigated. We therefore generated Cx31.9 specific antibodies to test for the expression of Cx31.9 in the human heart. These antibodies recognized the Cx31.9 protein in HeLaCx31.9 transfectants by immunofluorescence and immunoblot analyses. However, we did not find punctate Cx31.9 specific immunofluorescence signals in the working myocardium or in the impulse generation and conduction system of adult or fetal human heart. Complementary immunoblot analyses did not reveal Cx31.9 protein in the adult atrial or ventricular myocardium. We conclude that the Cx31.9 protein, unlike its counterpart in the mouse, is not expressed in detectable quantities and is thus unlikely to contribute to the impulse generation and conduction system or the working myocardium of the human heart.

Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flatmounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57-deficient mice. Significantly more connexin57-positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57-immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions. J. Comp. Neurol. 513:363–374

(Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22-C24). In order to study the biological role of CERS2, we have inactivated its coding region in transgenic mice using gene-trapped embryonic stem cells that express lacZ reporter DNA under control of the cers2 promoter. The resulting mice lack ceramide synthase activity toward C24:1 in the brain as well as the liver and show only very low activity toward C18:0-C22:0 in liver and reduced activity toward C22:0 residues in the brain. In addition, these mice exhibit strongly reduced levels of ceramide species with very long fatty acid residues (or=C22) in the liver, kidney, and brain. From early adulthood on, myelin stainability is progressively lost, biochemically accompanied by about 50% loss of compacted myelin and 80% loss of myelin basic protein. Starting around 9 months, both the medullary tree and the internal granular layer of the cerebellum show significant signs of degeneration associated with the formation of microcysts. Predominantly in the peripheral nervous system, we observed vesiculation and multifocal detachment of the inner myelin lamellae in about 20% of the axons. Beyond 7 months, the CERS2-deficient mice developed hepatocarcinomas with local destruction of tissue architecture and discrete gaps in renal parenchyma. Our results indicate that CERS2 activity supports different biological functions: maintenance of myelin, stabilization of the cerebellar as well as renal histological architecture, and protection against hepatocarcinomas.

In the adult dentate gyrus, radial glia-like cells represent putative stem cells generating neurons and glial cells. Here, we combined patch-clamp recordings, biocytin filling, immunohistochemistry, single-cell transcript analysis, and mouse transgenics to test for connexin expression and gap junctional coupling of radial glia-like cells and its impact on neurogenesis. Radial glia-like cells were identified in mice expressing EGFP under control of the nestin and gfap promoters. We show that a majority of Radial glia-like cells are coupled and express Cx43. Neuronal precursors were not coupled. Mice lacking Cx30 and Cx43 in GFAP-positive cells displayed almost complete inhibition of proliferation and a significant decline in numbers of radial glia-like cells and granule neurons. Inducible virus-mediated ablation of connexins in the adult hippocampus also reduced neurogenesis. These findings strongly suggest the requirement of connexin expression by radial glia-like cells for intact neurogenesis in the adult brain and point to possible communication pathways of these cells.