Compartmentalized and signal-selective gap junctional coupling in the hearing cochlea

Gap junctional intercellular communication (GJIC) plays a major role in cochlear function. Recent evidence suggests that connexin 26 (Cx26) and Cx30 are the major constituent proteins of cochlear gap junction channels, possibly in a unique heteromeric configuration. We investigated the functional and structural properties of native cochlear gap junctions in rats, from birth to the onset of hearing [ postnatal day 12 (P12)]. Confocal immunofluorescence revealed increasing Cx26 and Cx30 expression from P0 to P12. Functional GJIC was assessed by coinjection of Lucifer yellow (LY) and Neurobiotin (NBN) during whole-cell recordings in cochlear slices. At P0, there was restricted dye transfer between supporting cells around outer hair cells. Transfer was more extensive between supporting cells around inner hair cells. At P8, there was extensive transfer of both dyes between all supporting cell types. By P12, LY no longer transferred between the supporting cells immediately adjacent to hair cells but still transferred between more peripheral cells. NBN transferred freely, but it did not transfer between inner and outer pillar cells. Freeze fracture further demonstrated decreasing GJIC between inner and outer pillar cells around the onset of hearing. These data are supportive of the appearance of signal-selective gap junctions around the onset of hearing, with specific properties required to support auditory function. Furthermore, they suggest that separate medial and lateral buffering compartments exist in the hearing cochlea, which are individually dedicated to the homeostasis of inner hair cells and outer hair cells

Connexins and gap junctions in the inner ear - it's not just about K(+) recycling

Normal development, function and repair of the sensory epithelia in the inner ear are all dependent on gap junctional intercellular communication. Mutations in the connexin genes GJB2 and GJB6 (encoding CX26 and CX30) result in syndromic and non-syndromic deafness via various mechanisms. Clinical vestibular defects, however, are harder to connect with connexin dysfunction. Cx26 and Cx30 proteins are widely expressed in the epithelial and connective tissues of the cochlea, where they may form homomeric or heteromeric gap junction channels in a cell-specific and spatiotemporally complex fashion. Despite the study of mutant channels and animal models for both recessive and dominant autosomal deafness, it is still unclear why gap junctions are essential for auditory function, and why Cx26 and Cx30 do not compensate for each other in vivo. Cx26 appears to be essential for normal development of the auditory sensory epithelium, but may be dispensable during normal hearing. Cx30 appears to be essential for normal repair following sensory cell loss. The specific modes of intercellular signalling mediated by inner ear gap junction channels remain undetermined, but they are hypothesised to play essential roles in the maintenance of ionic and metabolic homeostasis in the inner ear. Recent studies have highlighted involvement of gap junctions in the transfer of essential second messengers between the non-sensory cells, and have proposed roles for hemichannels in normal hearing. Here, we summarise the current knowledge about the molecular and functional properties of inner ear gap junctions, and about tissue pathologies associated with connexin mutations

Identification of Persistent and Resurgent Sodium Currents in Spiral Ganglion Neurons Cultured from the Mouse Cochlea

In spiral ganglion neurons (SGNs), the afferent single units of the auditory nerve, high spontaneous and evoked firing rates ensure preservation of the temporal code describing the key features of incoming sound. During postnatal development, the spatiotemporal distribution of ion channel subtypes contributes to the maturation of action potential generation in SGNs, and to their ability to generate spike patterns that follow rapidly changing inputs. Here we describe tetrodotoxin (TTX)-sensitive Na+ currents in SGNs cultured from mice, whose properties may support this fast spiking behavior. A subthreshold persistent Na+ current (INaP) and a resurgent Na+ current (INaR) both emerged prior to the onset of hearing and became more prevalent as hearing matured. Navβ4 subunits, which are proposed to play a key role in mediating INaR elsewhere in the nervous system, were immunolocalized to the first heminode where spikes are generated in the auditory nerve, and to perisomatic nodes of Ranvier. ATX-II, a sea anemone toxin that slows classical Na+ channel inactivation selectively, enhanced INaP five-fold and INaR three-fold in voltage clamp recordings. In rapidly-adapting SGNs under current clamp, ATX-II increased the likelihood of firing additional action potentials. The data identify INaP and INaR as novel regulators of excitability in SGNs, and consistent with their roles in other neuronal types, we suggest that these nonclassical Na+ currents may contribute to the control of refractoriness in the auditory nerve

Divergent membrane properties of mouse cochlear glial cells around hearing onset

Spiral ganglion neurons (SGNs) are the primary afferent neurons of the auditory system, and together with their attendant glia, form the auditory nerve. Within the cochlea, satellite glial cells (SGCs) encapsulate the cell body of SGNs, whereas Schwann cells (SCs) wrap their peripherally- and centrally-directed neurites. Despite their likely importance in auditory nerve function and homeostasis, the physiological properties of auditory glial cells have evaded description. Here, we characterized the voltage-activated membrane currents of glial cells from the mouse cochlea. We identified a prominent weak inwardly rectifying current in SGCs within cochlear slice preparations (postnatal day P5-P6), which was also present in presumptive SGCs within dissociated cultures prepared from the cochleae of hearing mice (P14-P15). Pharmacological block by Ba2+ and desipramine suggested that channels belonging to the Kir4 family mediated the weak inwardly rectifying current, and post hoc immunofluorescence implicated the involvement of Kir4.1 subunits. Additional electrophysiological profiles were identified for glial cells within dissociated cultures, suggesting that glial subtypes may have specific membrane properties to support distinct physiological roles. Immunofluorescence using fixed cochlear sections revealed that although Kir4.1 is restricted to SGCs after the onset of hearing, these channels are more widely distributed within the glial population earlier in postnatal development (i.e., within both SGCs and SCs). The decrease in Kir4.1 immunofluorescence during SC maturation was coincident with a reduction of Sox2 expression and advancing neurite myelination. The data suggest a diversification of glial properties occurs in preparation for sound-driven activity in the auditory nerve

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions

In the present study we investigated the life cycle, trafficking, assembly and cell surface dynamics of a poorly characterized connexin family member, connexin 30 (Cx30; also known as GJB6), which plays a critical role in skin health and hearing. Unexpectedly, Cx30 localization at the cell surface and gap junctional intercellular communication was not affected by prolonged treatments with the endoplasmic reticulum (ER)-Golgi transport inhibitor brefeldin A or the protein synthesis inhibitor cycloheximide, whereas Cx43 (also known as GJA1) was rapidly cleared. Fluorescent recovery after photobleaching revealed that Cx30 plaques were rebuilt from the outer edges in keeping with older channels residing in the inner core of the plaque. Expression of a dominant-negative form of Sar1 GTPase led to the accumulation of Cx30 within the ER, in contrast to a report that Cx30 traffics via a Golgi-independent pathway. Co-expression of Cx30 with Cx43 revealed that these connexins segregate into distinct domains within common gap junction plaques, suggesting that their assembly is governed by different mechanisms. In summary, Cx30 was found to be an unusually stable, long-lived connexin (half-life >12 h), which may underlie its specific role in the epidermis and cochlea

The enigmatic root cell - Emerging roles contributing to fluid homeostasis within the cochlear outer sulcus.

Despite their curious morphology prompting numerous hypotheses of their normal function, the root cells lining the cochlear outer sulcus have long evaded physiological characterization. A growing body of evidence now suggests that they regulate the solute content of the endolymph and/or the perilymph, and may be essential in safe-guarding the global homeostasis of the cochlea. Immuno-labeling experiments have demonstrated polarized expression of key ion transport proteins, and recent electrophysiological recordings have identified specific membrane conductances. These studies have painted a clearer picture of how this unusual cell type may contribute to the maintenance of sound transduction, and how they may be central to pathological processes associated with various forms of hearing loss

Phosphoinositide Modulation of Heteromeric Kv1 Channels Adjusts Output of Spiral Ganglion Neurons from Hearing Mice

Spiral ganglion neurons (SGNs) relay acoustic code from cochlear hair cells to the brainstem, and their stimulation enables electrical hearing via cochlear implants. Rapid adaptation, a mechanism that preserves temporal precision, and a prominent feature of auditory neurons, is regulated via dendrotoxin-sensitive low-threshold voltage-activated (LVA) K(+) channels. Here, we investigated the molecular physiology of LVA currents in SGNs cultured from mice following the onset of hearing (postnatal days 12-21). Kv1.1- and Kv1.2-specific toxins blocked the LVA currents in a comparable manner, suggesting that both subunits contribute to functional heteromeric channels. Confocal immunofluorescence in fixed cochlear sections localized both Kv1.1 and Kv1.2 subunits to specific neuronal microdomains, including the somatic membrane, juxtaparanodes, and the first heminode, which forms the spike initiation site of the auditory nerve. The spatial distribution of Kv1 immunofluorescence appeared mutually exclusive to that of Kv3.1b subunits, which mediate high-threshold voltage-activated currents. As Kv1.2-containing channels are positively modulated by membrane phosphoinositides, we investigated the influence of phosphatidylinositol-4,5-bisphosphate (PIP2) availability on SGN electrophysiology. Reducing PIP2 production using wortmannin, or sequestration of PIP2 using a palmitoylated peptide (PIP2-PP), slowed adaptation rate in SGN populations. PIP2-PP specifically inhibited the LVA current in SGNs, an effect reduced by intracellular dialysis of a nonhydrolysable analog of PIP2. PIP2-PP also inhibited heterologously expressed Kv1.1/Kv1.2 channels, recapitulating its effect in SGNs. Collectively, the data identify Kv1.1/Kv1.2 heteromeric channels as key regulators of action potential initiation and propagation in the auditory nerve, and suggest that modulation of these channels by endogenous phosphoinositides provides local control of membrane excitability

Disruption of SorCS2 reveals differences in the regulation of stereociliary bundle formation between hair cell types in the inner ear

Behavioural anomalies suggesting an inner ear disorder were observed in a colony of transgenic mice. Affected animals were profoundly deaf. Severe hair bundle defects were identified in all outer and inner hair cells (OHC, IHC) in the cochlea and in hair cells of vestibular macular organs, but hair cells in cristae were essentially unaffected. Evidence suggested the disorder was likely due to gene disruption by a randomly inserted transgene construct. Whole-genome sequencing identified interruption of the SorCS2 (Sortilin-related VPS-10 domain containing protein) locus. Real-time-qPCR demonstrated disrupted expression of SorCS2 RNA in cochlear tissue from affected mice and this was confirmed bySorCS2 immuno-labelling. In all affected hair cells, stereocilia were shorter than normal, but abnormalities of bundle morphology and organisation differed between hair cell types. Bundles on OHC were grossly misshapen with significantly fewer stereocilia than normal. However, stereocilia were organised in rows of increasing height. Bundles on IHC contained significantly more stereocilia than normal with some longer stereocilia towards the centre, or with minimal height differentials. In early postnatal mice, kinocilia (primary cilia) of IHC and of OHC were initially located towards the lateral edge of the hair cell surface but often became surrounded by stereocilia as bundle shape and apical surface contour changed. In macular organs the kinocilium was positioned in the centre of the cell surface throughout maturation. There was disruption of the signalling pathway controlling intrinsic hair cell apical asymmetry. LGN and Gαi3 were largely absent, and atypical Protein Kinase C (aPKC) lost its asymmetric distribution. The results suggest that SorCS2 plays a role upstream of the intrinsic polarity pathway and that there are differences between hair cell types in the deployment of the machinery that generates a precisely organised hair bundle

Gap Junctional Coupling is Essential for Epithelial Repair in the Avian Cochlea

The loss of auditory hair cells triggers repair responses within the population of non-sensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process which is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the “basilar papilla”, BP) dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions, and supporting cells then replace hair cells via mitotic and/or conversion mechanisms. Here we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. FRAP experiments confirmed GJIC remained robust in gentamicin-damaged explants, though regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium, and supporting cells remained unexpanded. These observations suggest repair of the auditory epithelium shares common mechanisms across vertebrate species, and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration

Characterizing human vestibular sensory epithelia for experimental studies: new hair bundles on old tissue and implications for therapeutic interventions in ageing.

Balance disequilibrium is a significant contributor to falls in the elderly. The most common cause of balance dysfunction is loss of sensory cells from the vestibular sensory epithelia of the inner ear. However, inaccessibility of inner ear tissue in humans severely restricts possibilities for experimental manipulation to develop therapies to ameliorate this loss. We provide a structural and functional analysis of human vestibular sensory epithelia harvested at trans-labyrinthine surgery. We demonstrate the viability of the tissue and labeling with specific markers of hair cell function and of ion homeostasis in the epithelium. Samples obtained from the oldest patients revealed a significant loss of hair cells across the tissue surface, but we found immature hair bundles present in epithelia harvested from patients >60 years of age. These results suggest that the environment of the human vestibular sensory epithelium could be responsive to stimulation of developmental pathways to enhance hair cell regeneration, as has been demonstrated successfully in the vestibular organs of adult mice