Human Molecular Genetics

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Human Molecular Genetics, 2003, Vol. 12, No. 1 13-21
© 2003 Oxford University Press

Connexin30 (Gjb6)-deficiency causes severe hearing impairment and lack of endocochlear potential

Barbara Teubner^1,, Vincent Michel², Jörg Pesch³, Jürgen Lautermann⁴, Martine Cohen-Salmon², Goran Söhl¹, Klaus Jahnke⁴, Elke Winterhager⁵, Claus Herberhold³, Jean-Pierre Hardelin², Christine Petit² and Klaus Willecke^1,*

¹Institut für Genetik, Abteilung Molekulargenetik, Universität Bonn, Bonn, Germany, ²Unité de Génétique des Déficits Sensoriels, Institut Pasteur, Paris, France, ³Klinik und Poliklinik für Hals-Nasen-Ohrenkranke, Universität Bonn, Bonn, Germany, ⁴Klinik und Poliklinik für Hals-Nasen-Ohrenheilkunde, Universität Essen, Essen, Germany and ⁵Institut für Anatomie, Universität Essen, Essen, Germany

Received July 19, 2002; Accepted October 30, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
The gap junction protein connexin30 (Cx30) is expressed in a variety of tissues that include epithelial and mesenchymal structures of the inner ear. We generated Cx30 (Gjb6) deficient mice by deletion of the Cx30 coding region. Homozygous mutants (Cx30^(-/-)) were born at the expected Mendelian frequency, developed normally and were fertile. However, they exhibit a severe constitutive hearing impairment. From the age of hearing onset, these mice lack the electrical potential difference between the endolymphatic and perilymphatic compartments of the cochlea, i.e. the endocochlear potential, which plays a key role in the high sensitivity of the mammalian auditory organ. In addition, after postnatal day 18, the cochlear sensory epithelium starts to degenerate by cell apoptosis. This degeneration process is likely to account for the concomitant decrease of the endolymphatic potassium concentration and the aggravation of the hearing loss in adult Cx30^(-/-) mice. The Cx30 ^(-/-) phenotype thus reveals the critical role of Cx30 both in generating the endocochlear potential and for survival of the auditory hair cells after the onset of hearing. The Cx30 deficient mice may represent a valuable model to study the mechanism of the hearing loss in human patients carrying a homozygous deletion of the CX30 gene (del Castillo et al., 2002, New Engl. J. Med., 346, 243–249).

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
The mammalian auditory organ (cochlea) consists of a scala media which is filled with endolymph as well as a scala vestibuli and a scala tympani which are filled with perilymph. The perilymph has the usual ion content of extracellular fluids, whereas the ion content of the endolymph resembles the intracellular fluid with a high K⁺ and low Na⁺ concentration. The endolymphatic K⁺ is secreted by the stria vascularis (1), i.e. a highly vascularized multilayered epithelium adjacent to the spiral ligament in the lateral wall of the cochlear duct (see Fig. 1). The stria vascularis is also responsible for the generation of the difference in electric potential between the endolymphatic and perilymphatic compartments of the cochlea (about +80 mV), called the endocochlear potential (EP) (2). This transepithelial potential, in conjunction with the membrane potential of the auditory hair cells (-70 mV), drives the mechanoelectrical transduction inward current which is primarily carried by K⁺ ions (3). Therefore, normal EP is essential for the high sensitivity of the mammalian auditory organ.

View larger version (92K): [in this window] [in a new window]   Figure 1. Schematic representation of a cross section through the cochlear duct. The membranous labyrinth of the cochlea (cochlear duct) divides the bony labyrinth in three canals, the scala vestibuli and the scala tympani, both filled with perilymph, and the scala media, filled with endolymph. The organ of Corti, which is the auditory transduction apparatus, protrudes in the scala media. This organ is a sensory epithelium made up of an array of sensory cells (in yellow), i.e. the single row of inner hair cells (ihc) and the triple row of outer hair cells (ohc), and different types of supporting cells, namely pillar cells (p), cells of Deiters (d), and cells of Hensen (h). It is covered by an acellular gel, the tectorial membrane. The organ of Corti is flanked by the epithelial cells of the inner sulcus (is) on the medial side and by the cells of Claudius (c) on the lateral side. The stria vascularis, in the lateral wall of the cochlear duct, is responsible for the secretion of K+ into the endolymph and for the production of the endocochlear potential. Different types of fibrocytes surround the cochlear epithelium. Other abbreviations: (i) interdental cells, (sp) spiral prominence. (Adapted from a figure drawn by P. Küssel-Andermann.)

 
Gap junction channels, formed by hexameric hemichannels which are composed of connexin subunits, mediate the diffusion of ions, metabolites and second messenger molecules between adjacent cells (4). In the mouse auditory organ, gap junction channels contribute to two independent intercellular networks (5–8). The epithelial gap junction system forms around embryonic day 16 (E16) and connects all supporting cells of the cochlear neurosensory epithelium as well as adjacent epithelial cells. No gap junctional communication has been found between the hair cells and the neighbouring supporting cells. The gap junction system between connective tissue cells starts to develop around birth; this gap junction system is composed of fibrocytes in the spiral ligament and spiral limbus, and also includes basal, intermediate and marginal cells of the stria vascularis. To date, expression of at least four different connexins (Cx) has been reported in the inner ear, i.e. Cx26 (Gjb2), -30 (Gjb6), -31 (Gjb3) and -43 (Gja1). Cx26 and Cx30 are constituents of both epithelial and connective tissue gap junction systems and appear to have very similar patterns of distribution (5,6,9). Cx43 has also been found in both gap junction systems (9). Cx31 expression in the mature auditory organ is still a matter of debate (7,10). The functional importance of these gap junction networks in man is shown by the fact that CX26 (GJB2), CX30 (GJB6), CX31 (GJB3) and CX43 (GJA1) mutations can cause hearing loss (11–17). However, their roles in the cochlea are poorly understood. By analysing the auditory function of Cx30-deficient mice, we provide genetic evidence that this gap junction protein is required both for producing the EP and for the survival of various epithelial cell types, including hair cells, in the mature cochlea.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation of Cx30-deficient mice
The coding region of the mouse Cx30 (Gjb6) gene was replaced by the E. coli ß-galactosidase reporter gene equipped with a nuclear localization sequence (18) (Fig. 2A). Crossings of chimeric mice that were born after blastocyst injection with recombined HM1-ES cells gave rise to heterozygous, and after backcrossing with C57BL/6 mice homozygous, Cx30-deficient offspring, as verified by Southern blot analysis (Fig. 2B). Cx30^(-/-) mice were born at the expected Mendelian frequency. They developed without gross abnormalities and were fertile. In northern blot and immunoblot analyses, the Cx30 mRNA and protein were reduced to about half in heterozygous animals and were absent in homozygous Cx30-deficient animals (Fig. 2C and D). Immunohistofluorescence analysis consistently showed, in contrast to wild-type mice (Fig. 3A), no Cx30 immunoreactivity in homozygous mutant mice (Fig. 3B), whereas Cx26 was still expressed in the same parts of the cochlea (Fig. 3C and D). The activity of the lacZ reporter gene mirrored the Cx30 expression pattern, with the advantage of a cell autonomous signal detection in the nucleus compared with the connexin membrane-associated immunoreactivity in Cx30 expressing cells, often in branched cell ramifications. We found ß-galactosidase activity in the following structures or tissues: external auditory meatus, inner ear, leptomeninges, ependymal cells, astrocytes in the grey matter of hindbrain and spinal cord, skin and epithelium of oesophagus, kidney and uterus (data not shown). The Cx30 protein had been previously detected in some of these tissues by the use of specific antibodies (9,19,20). No obvious histological abnormalities were detected in brain, skin, lung, kidney or uterus of Cx30-deficient mice, but these organs are currently being analysed in further detail, especially since CX30 missense mutations have been reported to cause a skin disease in man, namely hidrotic ectodermal dysplasia (Clouston syndrome) (21,22).

View larger version (30K): [in this window] [in a new window]   Figure 2. Generation of Cx30-deficient mice. (A) Targeting scheme of replacement of the Cx30 gene by the reporter gene lacZ and a neo resistance cassette. Upper part, targeting vector; middle part, wild-type locus; bottom, recombined allele. Thin line, vector backbone; filled bar, flanking regions of the Cx30 coding region; open bar, lacZ reporter gene with a nuclear localization signal (NLS) and resistance cassette (neo); thick line, wild-type DNA; Cx30, coding region; N, NotI; H, HindIII; S, SacI; x, corresponding DNA regions for homologous recombination. (B) Southern blot analysis of Cx30 wild-type (+/+), heterozygous (+/-) and Cx30-deficient (-/-) mice. Genomic DNA of tail biopsies was digested by HindIII and hybridized to the probe shown in (A). The size of the hybridizing fragments is given in kbp at the left. (C) Northern blot analysis of total brain RNA from wild-type (+/+), heterozygous (+/-) and Cx30-deficient (-/-) adult mice. The probe corresponds to the coding region of Cx30 and detects transcripts of 2.0 and 2.3 kb (34). (D) Western blot analysis of Cx30 in the brain. Antibodies to Cx30 reveal a 30 kDa band in wild-type (+/+) and heterozygous (+/-) mice only.

 

View larger version (38K): [in this window] [in a new window]   Figure 3. Immunohistofluorescence analysis of Cx30 and Cx26 in the Cx30(-/-) cochlea. Cochlear sections of wild-type (A, C) and Cx30(-/-) (B, D) adult mice, immunostained for Cx30 (A), (B) or Cx26 (C), (D). Subregions are indicated in (C): L, spiral limbus; OC, organ of Corti; SL, spiral ligament; SV, stria vascularis. The Cx30 immunoreactivity is absent in the Cx30(-/-) cochlea (B). The Cx26 immunoreactivity in Cx30(-/-) cochlea (D) is similar to that of wild-type mice (C). Scale bar: 50 µm.

 
Severe hearing loss in Cx30^(-/-) mice
It had been reported that a CX30 missense mutation in a human family leads to a middle/high-frequency hearing loss (13). Large deletions in the GJB6 gene or in regulatory elements of GJB2 were also detected which cause recessive non-syndromic deafness in the Mediterranean or Ashkenazi Jewish population (14,16,17). Moreover, the GJB6 mutations G11R and A88V are involved in hidrotic ectodermal dysplasia (21) and the V37E mutation of GJB6 underlies Clouston syndrome (22). We therefore tested the Cx30-deficient mice for auditory brainstem responses (ABR). Auditory thresholds of adult (4 months old) heterozygous mice were not different from those of their wild-type littermates. This further argues in favour of a dominant or trans-dominant negative effect of the CX30 mutation (Thr5Met) that has been found in the human family (13). In contrast, adult homozygous mutant mice (Cx30^(-/-)) had no detectable response up to 100 dB, the loudest stimulus tested. Consistently, Cx30^(-/-) adults had no Preyer reflex (see Materials and Methods). Young Cx30^(-/-) animals at P17–P18, however, still had detectable, though increased, auditory thresholds: 84±20 dB for a click stimulus (n=14 ears), vs 31±2 dB (n=8) in Cx30^(+/+) animals (P<0.001). No dysfunction of the vestibular system, which controls balance, could be detected in Cx30^(+/-) and Cx30^(-/-) mice.

The auditory epithelium degenerates in Cx30^(-/-) mice
In Cx30^(-/-) mice the cochlea and vestibular end organs were normally shaped. Therefore, even though Cx30 is expressed during ear development (6), Cx30-containing channels do not seem to play a crucial role in the morphogenesis of the inner ear.

Histological examination showed that, up to P17, the Cx30^(-/-) cochleae were indistinguishable from those of heterozygous and wild-type mice. However, from P18 onwards, we observed a loss of cells in the sensory epithelium of the Cx30^(-/-) cochleae (Fig. 4A and B). The deterioration profile varied from one mutant animal to the other and concerned all cell types (see Fig. 1), but mainly affected the outer and inner hair cells. This degeneration process occurred gradually with age, since in 3-week-old animals, we found both intact and degenerated hair cells, whereas in 4-month-old animals, hair cell loss was more extensive. No displacement of the Reissner's membrane (see Fig. 1), which would indicate abnormal volume of the endolymphatic fluid compartment, was found in homozygous mutant mice. No gross anomaly of the other cochlear structures, including the stria vascularis and the spiral ligament (Fig. 5), was noted either.

View larger version (57K): [in this window] [in a new window]   Figure 4. Degeneration of the cochlear sensory epithelium in Cx30-deficient mice. (A, B) Transversal sections through the basal turns of cochleae from adult (4 months old) wild-type (A) and Cx30(-/-) (B) mice. Note that the outer hair cells (arrows) are missing in the Cx30-deficient mice. In (C), a section of a Cx30(-/-) cochlea (basal turn) at P18 has been processed for TUNEL assay. The three outer hair cells are labelled (arrow). Scale bar: 10 µm in (A) and (B), 5 µm in (C).

 

View larger version (50K): [in this window] [in a new window]   Figure 5. Structural analysis of the spiral ligament and stria vascularis in Cx30-deficient mice. (A) Semi-thin section reveals no obvious degeneration or alteration in the structure of the spiral ligament (Spl) and stria vascularis (Stv), including intrastrial blood vessels; Rm, Reissner's membrane. (B) Transmission electron microscopy analysis of the stria vascularis shows a continuous sheet of marginal cells (Mc) that lines the cochlear duct. Intermediate cells (Ic) and basal cells (Bc) are present without obvious morphological alterations. Blood vessels (Bv) are lined by intact endothelial cells. Scale bar: 20 µm in (A); 2.5 µm in (B).

 
We investigated the process of cell death in the Cx30^(-/-) sensory epithelium by the TUNEL method. In agreement with the histological findings, different cell types were labelled, but the apoptotic process mainly concerned the outer and inner hair cells (Fig. 4C).

The Cx30^(-/-) phenotype thus reveals the crucial role of the Cx30-containing gap junctions in the survival of the various cell types (including the hair cells) of the mature sensory epithelium. This role remains to be clarified, but might be related to the proposed role of the epithelial gap junction network in the spatial buffering of the K⁺ ions that are released at the basolateral pole of the hair cells after depolarization (3,23). In the absence of Cx30, the deficiency of the gap junctions between supporting cells may lead to a local but significant extracellular accumulation of K⁺ ions around the hair cells, which in turn may trigger cell apoptosis because of chronic depolarization of the cell membrane, or by other hypothetical mechanisms (24). Similar involvement of Cx26 in cell survival was recently revealed by the analysis of mice carrying a Cx26 null mutation targeted to the epithelial gap junctional network of the inner ear (24). However, there are some differences in the time course and extent of the apoptotic process between the Cx26 and the Cx30 mouse mutants, which could be explained by the different properties of the corresponding connexin channels (25), and/or by the presence of an EP in the targeted Cx26-deficient mice only (24) (see below).

Cx30^(-/-) mice lack the endocochlear potential
In the mouse, the endocochlear potential (EP) normally appears at P5 and progressively increases to reach adult values (about +80 mV) by P17–P18 (26). We first explored the EP and the endolymphatic K⁺ concentration in Cx30^(-/-) adult mice (see Materials and Methods), and found that the EP was virtually undetectable (3±3 mV; n=16 ears, vs 83±13 mV; n=20 in controls, P<0.001), and the endolymphatic K⁺ concentration (44±19 mM) was significantly decreased (P<0.001) compared with wild-type mice (148±15 mM). It is well known that the integrity of the epithelium lining the endolymphatic compartment, which prevents unspecific ion exchanges between endolymph and perilymph, is essential for the maintenance of both the EP and the high endolymphatic K⁺ concentration. We therefore measured these two parameters also in young mutant mice at P13–P14, i.e. several days before the first cells of the sensory epithelium undergo apoptosis. At that age, the endolymphatic K⁺ concentration (100±39 mM; n=18 ears) was not different from that of control mice (102±24 mM; n=18 ears), whereas the EP was already absent (0±4 mV, n=26 ears, vs 74±9 mV, n=8 in controls; P<0.01).

As recently shown in Cx26-deficient mice (24), the cell loss in the sensory epithelium, especially the loss of the outer hair cells and their supporting cells, may possibly be accompanied by epithelial breaches. These would be sufficient to account for the significant decrease of the endolymphatic K⁺ concentration that was concomitant to the epithelial degeneration in Cx30^(-/-) mice. Moreover, the absence of a partial collapse of the endolymphatic space, which is shown by the normal position of the Reissner's membrane in the mutant mice, argues in favour of a normal K⁺ secretion by the stria vascularis (27–30). In contrast, we show that the complete absence of EP in Cx30^(-/-) mice precedes by several days the first detectable cell loss in the sensory epithelium, which strongly argues in favour of a defect of the EP generation in the stria vascularis. The situation in the cochlea of young Cx30^(-/-) mice is reminiscent of the physiological situation in the vestibular labyrinth, which is characterized by a high endolymphatic K⁺ concentration but no electrical potential difference between the endolymphatic and perilymphatic compartments (31). Therefore, one attractive hypothesis is that the intrastrial space, i.e. the sealed liquid compartment that separates the basal and marginal cell layers of the stria vascularis and has been shown to be essential for production of the EP (32,33), is abnormal in the Cx30^(-/-) mice. Although light-microscopical and preliminary electron microscopical analysis of the stria vascularis in Cx30^(-/-) adult mice did not reveal gross cell anomalies (see Fig. 5), more subtle anomalies of the basal cell barrier that would lead to ion leak cannot be excluded.

Pathogenesis of the Cx30^(-/-) hearing loss
Several factors account for the severe hearing impairment of the Cx30^(-/-) mice. Firstly, from the age of hearing onset (around P12 in the mouse), these mice lack the endocochlear potential, which plays a key role in the driving force for K⁺ influx through the hair cell transducer channels. Secondly, cell apoptosis in the sensory epithelium, which occurs from P18 onward and leads to the loss of many hair cells, explains the aggravation of the hearing impairment in the adult animals. Thirdly, the low endolymphatic K⁺ concentration that was measured in adult mutant mice results in further decrease of the transducer current driving force, and thereby directly contributes to the profound deafness of Cx30^(-/-) mice.

Cx26 immunoreactivity of the cochlea is not modified in Cx30^(-/-) mice
Since colocalization of Cx26 and Cx30 has been demonstrated in the rat cochlea (6,9), we wondered whether the distribution of Cx26 is modified in the absence of Cx30. Cryosections of inner ears from young adult Cx30-deficient mice were incubated with anti-Cx26 for immunofluorescence analysis. The pattern of Cx26 immunoreactivity in wild-type and Cx30^(-/-) cochlea (Fig. 3C and D) was similar with respect to cell distribution and intensity of the staining.

Assuming that the Cx26 immunoreactivity in Cx30^(-/-) mice corresponds to functional Cx26 channels, the question arises why Cx26-containing channels cannot compensate for the Cx30 deficiency. One possible explanation lies in the channel properties. In heterologous expression studies, Cx30 gap junction channels have a similar single channel conductivity, a higher voltage sensitivity compared with Cx26 channels and, unlike the latter, they do not allow intercellular diffusion of Lucifer Yellow (25,34,35). In addition, electrophysiological experiments on cochlear supporting cells suggest that heterotypic channels with asymmetric voltage gating properties are present in the inner ear (36); these channels could contain both Cx26 and Cx30 (34). Alternatively, as proposed for other connexins (37,38), structural or adhesive properties specific to Cx30, rather than the channel function, may explain some of the cochlear anomalies in Cx30-deficient mice. Cx43 was not clearly expressed in the cochlea of both wild-type and Cx30-deficient mice, when analysed by immunohistochemistry (cf. Fig. 6). In previous studies, Cx43 had been shown to be weakly expressed in the rat cochlea (9).

View larger version (93K): [in this window] [in a new window]   Figure 6. Immunofluorescence analysis of Cx43 in the inner ear. (A) The lateral wall (stria vascularis and spiral ligament) of wild-type mice was stained immunohistochemically for Cx43. (B, corresponding phase contrast micrograph.) Specific staining could only be detected in the bone but not in lateral wall tissues. Staining for Cx43 in Cx30(-/-) inner ear was very similar as in wild-type and therefore is not illustrated. Scale bar: 10 µm.

 
Relevance of the Cx30 null mouse model to human deafness
Double heterozygous deaf individuals have recently been reported, who carry both a deleterious CX26 (GJB2) allele on one chromosome and a large deletion that truncates CX30 (GJB6) and extends 342 kb upstream of the gene (16). Because CX30 and CX26 are close to each other on chromosome 13q12, the possibility was put forward that the loss of the CX30 allele may not play a pathogenic role per se; as a corollary, the deletion was proposed to have a cis silencing effect on the adjacent CX26 allele, due to the loss of a hypothetical regulatory element located upstream of CX30. Even though the present mouse model does not allow us to exclude this possibility, the fact that the Cx30^(-/-) mice have severe hearing impairment suggests that the absence of CX30 is sufficient to account for the severe deafness in some patients who have been found to carry only the large deletion in a homozygous state (i.e. with two intact CX26 alleles) (16).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation of Cx30^(-/-) mice
The targeting vector consisted of the backbone pHM4 (18), containing the lacZ reporter gene and a neo cassette, and Cx30 flanking regions from a mouse genomic 129 Sv library in lambda fixII (Stratagene, La Jolla, CA, USA) (34). A 1.7 kb fragment of DNA upstream of the Cx30 start codon, synthesized by PCR, was inserted in front of the lacZ gene. Behind the neo gene regulated by a Pgk promoter, a 0.7 kb PCR product corresponding to the immediate 3'UTR of Cx30 was inserted, followed by a 4.5 kb HindIII/EcoRI restriction fragment further downstream of the Cx30 coding region. The targeting vector was linearized by NotI and electroporated into HM1 ES cells as described (39). Cells were grown under G418 (geniticin) selective pressure; resistant clones were tested for homologous recombination by Southern blot analysis of HindIII-digested genomic DNA, hybridized to a 0.7 kb SacI/HindIII fragment of the 5' region of Cx30, not part of the targeting vector. The probe labelled a 5.2 kb HindIII fragment of wild-type DNA and a 10.5 kb HindIII fragment of the targeted allele. Homologously recombined cells were injected into C57BL/6 blastocysts and transferred into pseudopregnant NMRI foster mothers. Chimeric mice were backcrossed to C57BL/6 mice and the heterozygous offspring was intercrossed to obtain homozygous Cx30^(-/-) mice, confirmed by Southern blot analysis of tail DNA. All experiments were performed with mice of 75% C57BL/6 and 25% 129 ola genetic background.

Northern blot analysis
Total RNA of mouse brain was extracted using TRIZOL (Gibco BRL, Germany) according to the manufacturer's instructions. RNA was separated by electrophoresis, blotted, and hybridized to a ³²P-labelled fragment of the Cx30 coding region (34).

Western blot analysis
Fifty micrograms of protein from mouse brain homogenates were separated by SDS–PAGE and the blotted membrane incubated with rabbit polyclonal anti-Cx30 (Zymed, San Francisco, CA, USA). Signals were visualized with the ECL system (Amersham Pharmacia Biotech, Freiburg, Germany).

Immunohistochemistry
Cx30^(-/-) and wild-type mice were deeply anaesthetized with ether and decapitated. The inner ears were removed, perilymphatically perfused in ice-cold methanol and left in methanol for 3 h. Inner ears were subsequently washed in phosphate-buffered saline (PBS) (8 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.15 M NaCl, pH 7.4), and decalcified in 10% Na₄EDTA in PBS at 4°C. Subsequently, specimens were immersed in 5% sucrose in PBS overnight and in 15% sucrose in PBS for 5 h, embedded in OCT (Miles Inc., Elkhart, IN, USA), frozen in liquid nitrogen and stored at -80°C. Inner ears were sectioned at 10 µm thickness in a Jung Frigocut 2800 E cryostat (Leica, Germany) and mounted on Biobond-coated (Ted Pella, Redding, CA, USA) glass slides. Following a 30 min preincubation in 1% bovine serum albumin (BSA)-containing PBS, sections were incubated with affinity-purified polyclonal rabbit anti-Cx26 (dilution 1:100) (Zymed, San Francisco, CA, USA), anti-Cx43 (40) or anti-Cx30 antibodies (9). The incubation was performed for 90 min in a humidified chamber at room temperature. Following incubation, slides were washed three times for 10 min in 0.1% BSA in PBS and incubated for 45 min at room temperature with fluorescein isothiocyanate-conjugated swine anti-rabbit antibodies (Dako, Hamburg, Germany) diluted 1:100 in 1% BSA-containing PBS. After three rinses for 10 min in PBS, sections were coverslipped in Vectashield mounting medium (Vector Laboratories Inc., Petersborough, UK) and examined under a fluorescence microscope (Zeiss Axiovert 100M, LSM510, confocal microscope, Germany).

Histological analyses
Inner ears of decapitated mice were removed, perilymphatically perfused with 10% paraformaldehyde (PFA) and left in the fixative overnight. They were washed in PBS and decalcified in 10% Na₄EDTA in PBS for 7 days. Tissues were dehydrated in increasing ethanol concentrations (30–100%) and placed in infiltration medium. Specimens were then embedded in Technovit 7100 (Heraeus Kulzer, Germany) and sectioned at 3 µm thickness on a microtome (Reichert-Jung, Germany). Sections were coloured with o-toluidine blue for 5 min, rinsed three times in distilled water, once in 96% ethanol and once in bidistilled water. Sections were incubated in 0.2% Fuchsin solution for 1 min and washed again in bidistilled water and ethanol. Then they were examined by light-microscopy (Zeiss, Oberkochen, Germany).

Electron microscopy
For analysis of the stria vascularis by transmission electron microscopy, five cochleae from 3-month-old Cx30^(-/-) mice were fixed in 2.5% glutaraldehyde in phosphate buffered solution (pH 7.4) for 24 h. The temporal bones were decalcified for 10 days in 10% EDTA, and afterwards immersed in 1% osmium tetroxide for 2 h.

Specimens were dehydrated in increasing alcohol concentrations (50–100%) and embedded in Epon. Ultrathin sections were stained with lead-citrate and uranyl-acetate for 5 min and analysed with a Zeiss electron microscope (type 902A).

Detection of apoptotic cells
Inner ears of Cx30^(-/-) and wild-type mice were dissected, fixed in 4% PFA in PBS for 1 h at 4°C, decalcified in 16.8% Na₄EDTA in PBS pH 7.4 at 4°C for 3 days, post-fixed in 4% PFA in PBS at 4°C for 48 h, dehydrated in ethanol and xylene, and embedded in paraffin. TUNEL assay was performed on 5 µm sections with fluorescein-labelled nucleotides, using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany). Apoptotic cells were identified by simultaneous transmission and fluorescence microscopy.

Audiometry and balance tests
The Preyer reflex is a flick backward of the pinna upon hearing a sound of 96 dB sound pressure level (SPL) at 20 kHz, delivered by a calibrated clickbox held 30 cm above the mouse's head. The absence of a Preyer reflex is consistent with an ABR threshold >80 dB (41).

Auditory brainstem responses (ABR) were recorded on Inactin-anaesthetized mice using the Tucker Davis Technologies system II, as described (42). The intensity of the stimulation (click stimulus, or 8, 16 and 32 kHz pure-tone stimuli) was calibrated for the mouse ear and varied from 30 to 100 dB SPL with steps of 5 dB. Vestibular function (balance) was tested as described (42).

Endolymphatic potential and K⁺concentration measurements
Mice were anaesthetized by intra-peritoneal injection of 0.1 mg/g thiobutabarbital sodium (Inactin, Sigma, Deisenhofen, Germany) and the cochlea exposed by a ventral approach. Access to the endolymphatic compartment (scala media) of the basal turn was gained by thinning the bone over the spiral ligament and making a small opening with a small pick. Double-barrelled glass microelectrodes were pulled from borosilicate capillaries. The inner surface of one barrel was siliconized by exposure to dimethyldichlorosilane vapour for 1 min followed by baking for 2 h at 200°C. On the day of use, the tip of the siliconized barrel (potassium) was filled with potassium ion-exchanger (World Precision Instruments, Berlin, Germany) and 0.5 M NaCl on top, and the non-siliconized (potential) barrel was filled with 0.5 M NaCl. Electrical contact to the electrolyte was made with Ag/AgCl wires. Electrodes were connected to a FD223 (World Precision Instruments) differential electrometer (input resistance 10¹⁵ ) from which EP and ion-dependent voltages were recorded. Electrodes were calibrated before and after each in vivo measurement, in KCl standards over a range of 0–150 mM. K⁺ concentrations were calculated from the respective voltages using an algebraic rearrangement of the Nicolski equation: V=V_i+Sxlog([K⁺]+Ax[Na⁺]), where V_i is an offset term, S is slope, and A is selectivity.

	ACKNOWLEDGEMENTS

 
We thank Petra Altenhoff, Joana Fischer, Thomas Hennek and Meike Weigel for their excellent technical assistance, Drs Klaus Kästner and Günther Schütz (Heidelberg) for the vector pHM4 and Dr Thomas Magin (Bonn) for HM1 cells. This work was supported by grants of the Deutsche Forschungsgemeinschaft through SFB 400, project E3, and the Fritz Thyssen Stiftung to K.W. and B.T., the Fonds der Chemischen Industrie to K.W., and the Association Française contre les Myopathies and Ministère de la Recherche (France) to C.P.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Institut für Genetik, Römerstr. 164, D-53117 Bonn, Germany. Tel: +49 228734210; Fax: +49 228734263; Email: genetik{at}uni-bonn.de

Present address: Deutsches Zentrum für Luft- und Raumfahrt, Südstr. 125, D-53175 Bonn, Germany.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 

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