Developmental expression patterns of connexin26 and -30 in the rat cochlea

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre- and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and -30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22-week-old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear.     

BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.     

Different regulation of connexin26 and ZO-1 in cochleas of developing rats and of guinea pigs with endolymphatic hydrops.

Using confocal microscopy and morphometry, we analyzed the expression of connexin26 (Cx26) and ZO-1 in rat cochlea during the postnatal period to elucidate spatiotemporal changes in gap junctions and tight junctions during auditory development. We also studied changes in these junctions in experimental endolymphatic hydrops in the guinea pig. In the adult rat cochlear lateral wall, Cx26 was detected in fibrocytes in the spiral ligament and in the basal cell layer of the stria vascularis, whereas ZO-1 was detected in the apical surfaces of marginal cells and in the basal cell layer. During postnatal development, Cx26 expression increased mainly in the spiral ligament, whereas ZO-1 expression increased in the basal cell layer. The morphometry of Cx26 showed a sigmoid time course with a rapid increase on postnatal day (PND) 14, whereas that of ZO-1 showed a marked increase on PND 7. In experimental endolymphatic hydrops, the expression of Cx26 significantly decreased, whereas there were no obvious changes in the expression of ZO-1. These results indicate that gap junctions and tight junctions in the cochlea increase in a different spatiotemporal manner during the development of auditory function and that gap junctions and tight junctions in the cochlea are differentially regulated in experimental endolymphatic hydrops. (J Histochem Cytochem 49:573-586, 2001)     

Roles of gap junctions in glucose transport from glucose transporter 1-positive to -negative cells in the lateral wall of the rat cochlea

Despite the importance of glucose metabolism for auditory function, the mechanisms of glucose transport in the cochlea are not completely understood. We hypothesized that gap junctions mediate intercellular glucose transport in the cochlea in cooperation with facilitative glucose transporter 1 (GLUT1). Immunohistochemistry showed that GLUT1 and the tight junction protein occludin were expressed in blood vessels, and GLUT1, the gap junction proteins connexin26 and connexin30, and occludin were also present in strial basal cells in the lateral wall of the rat cochlea. Gap junctions were found among not only these GLUT1-positive strial basal cells but also GLUT1-negative fibrocytes in the spiral ligaments and strial intermediate cells. Glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the stria vascularis and spiral ligament, whereas EBA was localized only in the vessels. The gap junctional uncouplers heptanol and carbenoxolone inhibited the distribution of 6-NBDG in the spiral ligament without decreasing the fluorescence of EBA in the blood vessels. These findings suggest that gap junctions mediate glucose transport from GLUT1-positive cells (strial basal cells) to GLUT1-negative cells (fibrocytes in the spiral ligament and strial intermediate cells) in the cochlea.     

Expression of gap junction protein connexin43 in the adult rat cochlea: comparison with connexin26.

To elucidate whether the two different gap junction proteins connexin43 (Cx43) and connexin26 (Cx26) are expressed and localized in a similar manner in the adult rat cochlea, we performed three-dimensional confocal microscopy using cryosections and surface preparations. In the cochlear lateral wall, Cx43-positive spots were localized mainly in the stria vascularis and only a few spots were present in the spiral ligament, whereas Cx26-positive spots were detected in both the stria vascularis and the spiral ligament. In the spiral limbus, Cx43 was widely distributed, whereas Cx26 was more concentrated on the side facing the scala vestibuli and in the basal portion. In the organ of Corti, Cx43-positive spots were present between the supporting cells but they were fewer and much smaller than those of Cx26. These data demonstrated distinct differences between Cx43 and Cx26 in expression and localization in the cochlea. In addition, the area of overlap of zonula occludens-1 (ZO-1) immunolabeling with Cx43-positive spots was small, whereas it was fairly large with Cx26-positive spots in the cochlear lateral wall, suggesting that the differences are not associated with the structural difference between carboxyl terminals, i.e., those of Cx43 possess sequences for binding to ZO-1, whereas those of Cx26 lack these binding sequences.     

内耳免疫反应诱导Fas和FasL表达与凋亡的关系

目的研究内耳免疫反应过程中是否存在细胞凋亡,以及细胞凋亡是否与Fas和FasL信号转导有关.方法选用雌性白色豚鼠16只,随机分为实验组和对照组各8只,以钥孔虫戚血蓝蛋白(keyhole limpet hemocyanin,KLH)全身免疫后,实验组以相同抗原进行内耳免疫,对照组内耳注射等量的磷酸盐缓冲生理盐水(phosphate buffered saline,PBS),在内耳免疫5d后处死动物,取内耳免疫侧耳蜗做石蜡切片.通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测内耳凋亡细胞,免疫组化检测内耳Fas和FasL的表达.结果实验组豚鼠内耳Corti器毛细胞,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞,而对照组动物切片仅在支持细胞、血管纹和螺旋神经节细胞中发现极少数TUNEL染色阳性细胞.免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达,FasL蛋白表达阴性.结论内耳免疫反应可诱导细胞凋亡的发生,Fas-FasL途径是参与此过程重要的信号转导途径之一.     

Primary culture of capillary endothelial cells from the spiral ligament and stria vascularis of bovine inner ear

Summary Methods for isolation and culture of microvascular endothelial cells of the inner ear were devised to provide an in-vitro system for studying endothelial functions in this tissue. Capillaries from the stria vascularis and spiral ligament were treated enzymatically to free them from surrounding tissue. Contamination by extraneous tissue was minimized by banding capillary segments in Percoll gradients and culture in plasma-derived serum on a fibronectin-coated substrate. Although only small amounts of inner ear tissue were available, tritiated thymidine autoradiography demonstrated that considerable growth in culture was possible. Addition of heparin and endothelial cell growth supplement to the medium enhanced proliferation. The endothelial origin of the cultured cells was confirmed by immunofluorescent demonstration of the presence of Factor VIII-related antigen and angiotensin-converting enzyme. In addition, tight junctions between cells were observed in both thin sections and platinum replicas obtained by freezefracture techniques. Endothelial cells from neither the stria vascularis nor the spiral ligament allowed passage of horseradish peroxidase across the monolayer during a 5-min period. However, endothelial cells from the stria vascularis exhibited a greater amount of pinocytotic activity than those of the spiral ligament, a difference that is also observed in vivo. Methods for expanding a small population of endothelial cells with retention of specialized properties into one of sufficient size for morphologic and biochemical studies have been demonstrated for the inner ear.     

Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique

We have made several improvements in the method of fixation of the inner ear and the enzyme-histochemical technique for carbonic anhydrase (CA) detection. The results confirmed that CA is localized in the hair cells of the organ of Corti, Deiters' cells or nerve endings, inner pillar cells, Boettcher's cells, stria vascularis, spiral ligament, spiral limbus, and spiral ganglion cells. These results generally agree with previous histochemical observations but showed some differences. Our method preserved tissue morphology and showed more detailed localization of CA activity in the inner ear. In particular, the marginal zone of stria vascularis and the epithelial cells of spiral prominence, facing the endolymph, showed no CA activity, while the suprastrial region of the spiral ligament and the supralimbal region of the spiral limbus, juxtaposed to the perilymph, showed CA activity. In outer hair cells, the cuticular plate, which faces the endolymph showed CA activity, but the lateral membrane, which faces the perilymph showed no CA activity. In contrast, the inner hair cell cytoplasm showed diffuse CA activity. These results will be useful in considering ion exchange between endolymph and its adjacent cells, and between perilymph and its adjacent structures.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to ~1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Expression of different connexin genes in rat uterus during decidualization and at term.

The expression of different connexin genes (cx26, cx32, cx37, cx43) that code for the protein subunits of gap junctions, was investigated in various uterine tissues during the estrous cycle of nonpregnant rats, in pregnant rats at decidualization and at term. Connexin gene expression was studied at the mRNA level by Northern blot hybridization and at the protein level by immunocytochemistry. In gap junctions from uterine epithelium, stroma, or myometrium, connexin 26 and/or connexin 43 are much more abundant than connexins 32 and 37. The expression of connexin 26 and 43 appears to be modulated by maternal steroid hormones. High expression of these connexins is found in developing decidual cells by day 7 to 8 post coitum; furthermore, coexpression of connexins 26 and 43 in myometrium is observed just before delivery on day 21 post coitum. In both the decidua and the myometrium, the connexin 26 protein appears to be distributed in lower abundance than connexin 43. In uterine epithelium only connexin 26 is expressed throughout all of the reproductive phases investigated. The enhanced expression of this gene correlates with higher levels of maternal estrogen both in the proestrus/estrus phase and at term. The distinct spatial and temporal pattern of expression of connexins 26 and 43 in different uterine tissues suggests a physiological role for these proteins during embryo implantation and subsequent contraction of the uterus at birth.     

Inherited connexin mutations associated with hearing loss

One of the most dramatic discoveries in the field of hereditary hearing loss is the association of this sensory defect with connexin mutations. Most significant is the large proportion, 30-50%, of inherited hearing loss that is due to mutations in connexin 26. The proteins these genes encode are expressed in the cochlear duct, in regions containing gap junctions. Together, these findings suggest a crucial role for gap junction proteins in the mammalian inner ear. Mouse models with specific connexin mutations leading to deafness will help resolve the many questions regarding the role of these gap junction proteins in the inner ear.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Cellular characterization of Connexin26 and Connnexin30 expression in the cochlear lateral wall

Gap junctions in the cochlear lateral wall, which consists of the stria vascularis (SV) and spiral ligament (SPL), are important for generating a positive endocochlear potential and high potassium concentration in the endolymph. In this study, the cellular expression of connexin 26 (Cx26) and Cx30 in the cochlear lateral wall of rats and guinea pigs was examined by immunofluorescent staining and confocal microscopy. Co-labeling for Kir4.1 revealed that the stria intermediate cells had extensive labeling for Cx26 and Cx30 with a leaf-like distribution. Cx26 and Cx30 also co-distributed hexagonally around the basal cells. However, no labeling was observed in the marginal cells. In the SPL, punctate Cx26 and Cx30 labeling was distributed along vertical lines orthogonal to the cochlear longitudinal direction. Intense labeling for Cx26 and Cx30 was found in type II fibrocytes in the spiral prominence and central region, but Cx26 labeling was absent in the middle region just beneath the SV, where only Cx30 labeling was observed. Outer sulcus (OS) cells and their root processes also exhibited intense labeling for Cx26 and Cx30. Neither Cx26 nor Cx30 was immunopositive in the hyaline region beneath the OS, in the subcentral region (type IV fibrocytes), or in the tension (type III) fibrocytes beneath the bone. Cx26 and Cx30 labeling was also absent in the lateral wall blood vessels. Thus, Cx26 and Cx30 have distinct cell-specific distributions in the SV and SPL, suggesting that they can form different pathways for transporting ions/nutrients in the cochlear lateral wall.     

Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with “permissive” connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Analysis of connexin phosphorylation sites

Most connexins, the proteins that form gap junction channels, are phosphoproteins. Connexin phosphorylation has been thought to regulate gap junctional protein trafficking, gap junction assembly, channel gating, and turnover. Connexin phosphorylation has been investigated in a variety of ways. Some connexins show mobility shifts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis on phosphorylation. Kinase modulators can change the level of connexin phosphorylation and affect gap junctional communication levels. Metabolic labeling of cultured cells has allowed both phosphoamino acid identification and generation of phosphotryptic peptide maps. However, identification of the location of phosphorylated residues within the connexin sequence has required either targeted peptide synthesis, in vitro phosphorylation of known sites, and two-dimensional comigration studies or liquid chromatographic separation and N-terminal sequencing of peptides. In addition to these conventional methods, we discuss new applications of mass spectrometry to the identification of phosphorylated peptides and the specific residues phosphorylated within the connexin-derived peptide.     

In vitro optimization of antisense oligodeoxynucleotide design: an example using the connexin gene family.

The completion of the human and mouse genomes has identified at least 20 connexin isomers in this family of intercellular channel proteins. However, there are no specific gap junction blockers or channel-blocking mimetic peptides available for the study of specific connexins. We designed antisense oligodeoxynucleotides that functionally reduce targeted connexin protein expression and can be used to reveal the biological function of individual connexins in vivo. Connexin mRNA was firstly exposed in vitro to deoxyribozymes complementing the sense coding sequence. Those that cleaved the target connexin mRNA in defined regions were used as the basis to design oligodeoxynucleotides to the accessible sites, thus taking into account tertiary mRNA configurations rather than relying on computed predictions. Antisense oligodeoxynucleotides designed to bind to accessible mRNA sites selectively reduced connexin26 and -43 mRNA expression in a corneal epithelium ex vivo model. Connexin43 protein levels were reduced correlating with the knockdown in mRNA and the protein's rapid turnover; protein levels of connexin26 did not alter, supporting lower turnover rates reported for that protein. We show, for the first time, an inexpensive and empirical approach to the preparation of specific and functional antisense oligodeoxynucleotides against known gene targets in the post-genomic era.