In vivo infusion of UTP and uridine to the deafened guinea pig inner ear: effects on response thresholds and neural survival.

Nucleotides and nucleosides are known to function as neurotransmitters and neuromodulators but have recently been shown to have a trophic effect on neurons. It has previously been shown, in an animal model for cochlear implants, that local infusion of neurotrophic factors intervenes with the degenerative processes occurring after deafening and protects the auditory spiral ganglion neurons so that electrical responsiveness is maintained. Here we test the hypothesis that nucleosides and nucleotides have a similar effect on the acutely damaged inner ear. Pigmented guinea pigs received a cochlear implant electrode for measuring electrically evoked auditory brainstem responses and a miniosmotic pump for delivering drugs directly to the cochlea. The animals were deafened by a 48-hr infusion with 10% neomycin, followed by 23 days of treatment with primarily UTP, uridine nucleotides, or as control artificial perilymph. Electrically evoked responses were measured weekly, and at the end of the experiment the cochleae were collected and processed for morphological analysis and spiral ganglion neuron counting. Both UTP- and uridine-treated groups showed significantly better response after 23 days of treatment compared with the control group. The densities of spiral ganglion neuron were significantly higher for both treated groups compared with the control group treated with artificial perilymph. The results demonstrate that UTP and uridine rescue auditory neurons and suggest that drugs acting on purinoceptors could be of clinical importance.

Expression and localization of K channels KCNQ2 and KCNQ3 in the mammalian cochlea.

KCNQ1 and KCNQ4 voltage-gated potassium channel subunits play key roles in hearing. Other members of the KCNQ family also encode slow, low voltage-activated K(+) M currents. We have previously reported the presence of M-like K(+) currents in sensory hair cells, and expression of Kcnq family genes in the cochlea. Here, we describe Kcnq2/3 gene expression and distribution of M channel subunits KCNQ2 and 3 in the cochlea. By using RT-PCR, we found expression of Kcnq2 in the modiolus and organ of Corti, while Kcnq3 expression was also detected in the cochlear lateral wall. Five alternative splice variants of the Kcnq2 gene, one of which has not been reported previously, were identified in the rat cochlea. KCNQ2 and KCNQ3 immunoreactivities were observed in spiral ganglion auditory neurons. In addition, the unmyelinated parts of the nerve fibers innervating hair cells and synaptic regions under hair cells showed KCNQ2 immunoreactivity. KCNQ3 immunoreactivity was also prominent in spiral ganglion satellite cells. These findings suggest that cochlear M channels play important roles in regulation of cellular excitability and maintenance of cochlear K(+) homeostasis in the auditory system.

Activation of JNK in the inner ear following impulse noise exposure.

Noise exposure is known to induce cell death signaling in the cochlea. Since c-Jun N-terminal kinase (JNK) signaling is known to induce both cell survival and apoptosis, the present study focused on early changes (within 24 h) after impulse noise exposure, inquiring whether cell death is always related to phosphorylation of JNK in the inner ear. Anesthetized adult albino rats were exposed to a single impulse noise exposure (194 kPa) and sacrificed 3 or 24 h later. Paraffin-embedded sections were examined for positive staining of phosphorylated JNK and the presence of cells with fragmented DNA (TUNEL staining). Positive TUNEL staining was observed at the spiral limbus and in the stria vascularis at 24 h following impulse noise exposure, but no correlation with JNK activation was found at these locations. In the hearing organ (organ of Corti) and in the lateral wall, TUNEL-reactive cells were observed at 24 h following trauma. This was preceded by p-JNK staining at 3 h, indicating JNK-activated cell death in these regions. Finally, p-JNK reactivity was observed in the spiral ganglion with no correlation to TUNEL staining within the time frame of this study. These results suggest that JNK activation following impulse noise exposure may not always be related to cell death, and conversely, some cells may die through JNK-independent signaling.

Spatiotemporal loss of K+ transport proteins in the developing cochlear lateral wall of guinea pigs with hereditary deafness.

Genetic deafness is one of the most common human genetic birth defects. To understand the molecular mechanisms underlying human hereditary deafness, deaf animal strains have proved to be invaluable models. The German waltzing guinea pig is a new strain of animals with unidentified gene mutation(s), displaying recessively inherited cochleovestibular impairment. Histological investigations of the homozygous animals (gw/gw) revealed a collapse of the endolymphatic compartment and malformation of stria vascularis. RT-PCR showed a significant reduction in expression of the strial intermediate cell-specific gene Dct and the tight-junction gene Cldn11 in the embryonic day (E)40 and adult gw/gw cochlear lateral wall. Immunohistochemical analysis of the gw/gw cochlea showed loss of the tight junction protein CLDN11 in strial basal cells from E40, loss of the potassium channel subunit KCNJ10 in strial intermediate cells from E50, and loss of the Na-K-Cl cotransporter SLC12A2 in strial marginal cells from E50. In addition, a temporary loss of the gap junction protein GJB2 (connexin 26) between fibrocytes in the spiral ligament of the E50 gw/gw cochlea was observed. The barrier composed of tight junctions between strial basal cells was disrupted in the gw/gw cochlea as indicated by a biotin tracer permeability assay. In conclusion, spatiotemporal loss of K+ transport proteins in the cochlear lateral wall is caused by malformation of the stria vascularis in the developing German waltzing guinea pig inner ear. This new animal strain may serve as a good model for studying human genetic deafness due to disruption of inner ear ion homeostasis.

Survival, synaptogenesis, and regeneration of adult mouse spiral ganglion neurons in vitro.

The inner ear spiral ganglion is populated by bipolar neurons connecting the peripheral sensory receptors, the hair cells, with central neurons in auditory brain stem nuclei. Hearing impairment is often a consequence of hair cell death, e.g., from acoustic trauma. When deprived of their peripheral targets, the spiral ganglion neurons (SGNs) progressively degenerate. For effective clinical treatment using cochlear prostheses, it is essential to maintain the SGN population. To investigate their survival dependence, synaptogenesis, and regenerative capacity, adult mouse SGNs were separated from hair cells and studied in vitro in the presence of various neurotrophins and growth factors. Coadministration of fibroblast growth factor 2 (FGF-2) and glial cell line-derived neurotrophic factor (GDNF) provided support for long-term survival, while FGF-2 alone could strongly promote neurite regeneration. Fibroblast growth factor receptor FGFR-3-IIIc was found to upregulate and translocate to the nucleus in surviving SGNs. Surviving SGNs formed contacts with other SGNs after they were deprived of the signals from the hair cells. In coculture experiments, neurites extending from SGNs projected toward hair cells. Interestingly, adult mouse spiral ganglion cells could carry out both symmetric and asymmetric cell division and give rise to new neurons. The authors propose that a combination of FGF-2 and GDNF could be an efficient route for clinical intervention of secondary degeneration of SGNs. The authors also demonstrate that the adult mammalian inner ear retains progenitor cells, which could commit neurogenesis.

Malformation of stria vascularis in the developing inner ear of the German waltzing guinea pig.

Auditory function and cochlear morphology have previously been described in the postnatal German waltzing guinea pig, a strain with recessive deafness. In the present study, cochlear histopathology was further investigated in the inner ear of the developing German waltzing guinea pig (gw/gw). The lumen of the cochlear duct diminished progressively from embryonic day (E) 35 to E45 and was absent at E50 because of the complete collapse of Reissner's membrane onto the hearing organ. The embryonic stria vascularis, consisting of a simple epithelium, failed to transform into the complex trilaminar tissue seen in normal animals and displayed signs of degeneration. Subsequent degeneration of the sensory epithelium was observed from E50 and onwards. Defective and insufficient numbers of melanocytes were observed in the developing gw/gw stria vascularis. A gene involved in cochlear melanocyte development, Pax3, was markedly reduced in lateral wall tissue of the cochlea of both E40 and adult gw/gw individuals, whereas its expression was normal in the skin and diaphragm muscle of adult gw/gw animals. The Pax3 gene may thus be involved in the pathological process but is unlikely to be the primary mutated gene in the German waltzing guinea pig. TUNEL assay showed no signs of apoptotic cell death in the developing stria vascularis of this type of guinea pig. Thus, malformation of the stria vascularis appears to be the primary defect in the inner ear of the German waltzing guinea pig. Defective and insufficient numbers of melanocytes might migrate to the developing stria vascularis but fail to provide the proper support for the subsequent development of marginal and basal cells, thereby leading to stria vascularis malformation and dysfunction in the inner ear of the German waltzing guinea pig.

Auditory function and cochlear morphology in the German waltzing guinea pig.

The German waltzing guinea pig is a new strain of animals with a recessively inherited inner ear defect resulting in deafness and severe vestibular dysfunction. Measurements of auditory brainstem responses (ABRs) demonstrated that the homozygotes (gw/gw) are deaf while the heterozygotes (gw/+) have normal hearing. In the gw/gw cochlea, a collapse of Reissner's membrane leads to the absence of scala media. Melanin pigment accumulation was clearly observed in the gw/gw stria vascularis, and both the height and width of stria vascularis were significantly reduced. Ultrastructural observations further detailed the disorganization of stria vascularis in the gw/gw animals: marginal cells lacked basolateral infoldings; intermediate cells (melanocytes) were scarce and degenerated; and basal cells were difficult to identify. The level of degeneration of the organ of Corti varied between individual gw/gw animals. The density of spiral ganglion neurons was significantly decreased in old (1-2 years of age) gw/gw animals. In contrast, no pathological changes were observed in the cochleae of gw/+ animals. Our data suggest that the degeneration originates in the stria vascularis (most likely in the melanocytes), and that this is the primary cause for inner ear defects in the German waltzing guinea pig. Here, we describe the auditory function and cochlear morphology in this spontaneously mutated guinea pig strain.

Molecular analyses of KCNQ1-5 potassium channel mRNAs in rat and guinea pig inner ears: expression, cloning, and alternative splicing.

CONCLUSION: Expression of neuronal Kcnq gene family transcripts in the inner ear provides further evidence for cochlear M-type currents and for complex molecular heterogeneities of voltage-gated potassium channels composed of various KCNQ subunits and/or alternative splice variants. Furthermore, important roles in regulation of cellular excitability in the auditory system, and hearing disorders related to (hyper)excitability, e.g. tinnitus, are implied. BACKGROUND: Voltage-gated potassium channels play key roles in hearing, as evidenced by deafness resulting from disruption of genes encoding, for example, KCNQ1 or KCNQ4 subunits. Other members of the Kcnq gene family (Kcnq2, 3, and 5) are the molecular correlates of M currents, which regulate neuronal excitability. The expression of the latter has not previously been thoroughly investigated in the inner ear. OBJECTIVE: The aim of this study was to identify genetic correlates of M currents, previously identified in cochlear hair cells by electrophysiological methods. MATERIALS AND METHODS: Expression of Kcnq genes was investigated by reverse transcription-polymerase chain reaction (RT-PCR) using subtype-specific primers with total RNA isolated from whole guinea pig or rat cochlea as template. PCR products were confirmed by direct DNA sequencing. RESULTS: All members of the Kcnq family were expressed in guinea pig and rat cochlea. Cochlear expression of Kcnq2 exhibited two alternatively spliced forms, lacking exons 8, 15a, and 8, 12a, 15a, respectively. Novel molecular sequence data, e.g. guinea pig Kcnq cDNA sequences, were deposited in GenBank (AY684985-AY684990).

Developmental expression and localization of KCNJ10 K+ channels in the guinea pig inner ear.

The inward rectifier Kir4.1, composed of KCNJ10 K channel subunits, plays an essential role in inner ear K homeostasis. We have investigated the developmental expression and localization of KCNJ10 (Kir4.1) in the guinea pig inner ear using semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Kcnj10 was expressed at low levels from embryonic day 30 (E30), increased from E45, and persisted from E50 to adulthood. KCNJ10 channel protein was detected in spiral ganglion satellite cells of the basal turn at E40, and at E45 its expression proceeded with a base-to-apex gradient along the cochlear spiral. KCNJ10 protein was enriched in the myelin sheath around the cochlear nerve between E40 and E45 and disappeared gradually with age. In the strial intermediate cells, KCNJ10 channel expression was first observed at E50, and lagged behind that of the spiral ganglion. In addition, KCNJ10 channel protein was expressed and localized in vestibular transitional cells. Differential expression of KCNJ10 channel protein suggests roles for KCNJ10 channels in inner ear development and onset of auditory function.

An M-like potassium current in the guinea pig cochlea.

Potassium M currents play a role in stabilizing the resting membrane potential. These currents have previously been identified in several cell types, including sensory receptors. Given that maintaining membrane excitability is important for mechano-electrical transduction in the inner ear, the presence of M currents was investigated in outer hair cells isolated from the guinea pig hearing organ. Using a pulse protocol designed to emphasize M currents with the whole-cell patch-clamp technique, voltage- and time-dependent, non-inactivating, low-threshold currents (the hallmarks of M currents) were recorded. These currents were significantly reduced by cadmium chloride. Results from RT-PCR analysis indicated that genes encoding M channel subunits KCNQ2 and KCNQ3 are expressed in the guinea pig cochlea. Our data suggest that guinea pig outer hair cells express an M-like potassium current that, following sound stimulation, may play an important role in returning the membrane potential to resting level and thus regulating outer hair cell synaptic mechanisms.

ATP-gated ion channels assembled from P2X2 receptor subunits in the mouse cochlea.

Extracellular ATP has several neuro-humoral actions on cochlear physiology, many of which involve P2X receptor-mediated signal transduction. The present study extends the molecular physiology of P2X receptor gene expression in the cochlea to the principal platform for transgenic studies, the mouse model. P2X receptor subunits, which assemble to form ATP-gated ion channels, were localised in cryosections and whole-mount tissues from the adult mouse cochlea using a specific antiserum and immunoperoxidase histochemistry. Whole-cell voltage clamp recordings functionally correlated immunolocalisation of ATP-gated ion channels in isolated hair cells and supporting cells. P2X immunoreactivity was widespread throughout the epithelial lining of the cochlea (except vascular stria); spiral ganglion neurons, organ of Corti supporting cells, and outer hair cell (OHC) stereocilia exhibited strong P2X immunolabelling. Localisation of ATP-gated ion channels on the endolymphatic surface (cuticular plates and stereocilia) of outer hair cells was confirmed electrophysiologically. In contrast, Deiters' cells exhibited an even distribution of both immunolabelling over the whole cell membrane and inward currents could be evoked by localised ATP application anywhere on these cells. In both OHC and Deiters' cells, the slowly-desensitising inward currents were blocked by the P2X-selective antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), compatible with P2X subunits contributing to the ATP-gated ion channels. Our immunohistochemical and functional localisation of P2X receptors in the mouse cochlea extends previous studies to verify and characterise extracellular ATP signalling in the cochlea and extends support for P2X receptor-mediated regulation of endolymphatic ionic homeostasis, sound transduction, auditory neurotransmission and cochlear mechanics.