Aspartate aminotransferase immunoreactivity in cochlea of guinea pig

The distribution of aspartate aminotransferase-like immunoreactivity in the cochlea of the guinea pig was studied at the light microscopy level. Indirect immunofluorescence histochemistry using antisera against cytoplasmic aspartate aminotransferase prepared from pig heart was applied to surface preparations of the organ of Corti and cryostat sections of the cochlea. In the modiolus, immunofluorescence was localized to spiral ganglion cells and myelinated fibers of the auditory nerve and intraganglionic spiral bundles. In the organ of Corti, immunofluorescence was seen in upper tunnel crossing fibers and at the base of outer hair cells, following a distribution similar to that of the efferent innervation of the outer hair cells. Weak immunofluorescence was seen in the inner spiral bundle and tunnel spiral bundle, but was not present in all preparations. Immunofluorescence was not seen in inner hair cells, nor at the base of inner hair cells, and may have been absent from outer hair cells.It is concluded that spiral ganglion cells and myelinated auditory nerve axons contain aspartate aminotransferase-like immunoreactivity; such immunoreactivity has previously been determined in auditory nerve endings in the cochlear nucleus. Olivocochlear neurons that innervate outer hair cells also contain such immunoreactivity while other cochlear efferents contain little or none.     

Purinergic regulation of sound transduction and auditory neurotransmission

In the cochlea, extracellular ATP influences the endocochlear potential, micromechanics, and neurotransmission via P2 receptors. Evidence for this arises from studies demonstrating widespread expression of ATP-gated ion channels (assembled from P2X receptor subunits) and G protein-coupled receptors (P2Y receptors). P2X2 receptor subunits are localized to the luminal membranes of epithelial cells and hair cells lining scala media. These ion channels provide a shunt pathway for K+ ion egress. Thus, when noise exposure elevates ATP levels in this cochlear compartment, the K+ conductance through P2X receptors reduces the endocochlear potential. ATP-mediated K+ efflux from scala media is complemented by a P2Y receptor G protein-coupled pathway that provides coincident reduction of K+ transport into scala media from the stria vascularis when autocrine or paracrine ATP signalling is invoked. This purinergic signalling likely provides a basis for a reactive homoeostatic regulatory mechanism limiting cochlear sensitivity under stressor conditions. Elevation of ATP in the perilymphatic compartment under such conditions is also likely to invoke purinergic receptor-mediated changes in supporting cell micromechanics, mediated by Ca2+ influx and gating of Ca2+ stores. Independent of these humoral actions, ATP can be classified as a putative auditory neurotransmitter based on the localization of P2X receptors at the spiral ganglion neuron-hair cell synapse, and functional verification of ATP-gated currents in spiral ganglion neurons in situ. Expression of P2X receptors by type II spiral ganglion neurons supports a role for ATP as a transmitter encoding the dynamic state of the cochlear amplifier.     

KCNQ1/KCNE1 K+ channel and P2Y4 receptor are co-expressed from the time of birth in the apical membrane of rat strial marginal cells

CONCLUSION: KCNQ1/KCNE1 K(+) channels and P2Y(4) receptors are expressed in the apical membrane of rat strial marginal cells from postnatal day 1 (P1) and maintained throughout development. OBJECTIVES: The purpose of the present study was to investigate the developmental expression of KCNQ1/KCNE1 K(+) channel and of P2Y(4), which is an important metabotropic regulator of KCNQ1/KCNE1 K(+) channel in strial marginal cells. MATERIALS AND METHODS: Sprague-Dawley rats at different stages of development (P1, P3, P5, P7, P14, and P21) were studied. The spiral ligament with the stria vascularis was detached from the cartilaginous or bony cochlea and prepared for a voltage-sensitive vibrating probe and immunohistochemistry. RESULTS: Chromanol 293B, a blocker of KCNQ1/KCNE1 K(+) channel, inhibited short-circuit currents (I ( sc )) from P1 to P21. Similarly, I ( sc ) were found to be decreased by uridine 5'-triphosphate at all ages. The antagonist profiles indicated that the apical P2Y receptor is P2Y(4) subtype. KCNQ1, KCNE1, and P2Y(4) were immunolocalized in the apical region of stria vascularis at P1.     

Time course of inner ear degeneration and deafness in mice lacking the Kir4.1 potassium channel subunit

The Kir4.1 gene (KCNJ10) encodes an inwardly rectifying K(+) channel subunit abundantly expressed in the CNS. Its expression in the mammalian inner ear has been suggested but its function in vivo in the inner ear is unknown. Because diverse human hereditary deafness syndromes are associated with mutations in K(+) channels, we examined auditory function and inner ear structure in mice with a genetically inactivated Kir4.1 K(+) channel subunit. Startle response experiments suggest that Kir4.1-/- mice are profoundly deaf, whereas Kir4.1+/- mice react like wild-type mice to acoustic stimuli. In Kir4.1-/- mice, the Reissner membrane is collapsed, the tectorial membrane is swollen, and type I hair cells and spiral ganglion neurons as well as their central processes degenerate over the first postnatal weeks. In the vestibular ganglia, neuronal cell death with apoptotic features is also observed. Immunostaining reveals that Kir4.1 is strongly expressed in stria vascularis of wild-type but not Kir4.1-/- mice. Within the spiral ganglion, Kir4.1 labeling was detected on satellite cells surrounding spiral ganglion neurons and axons. We conclude that Kir4.1 is crucial for normal development of the cochlea and hearing, via two distinct aspects of extracellular K(+) homeostasis: (1). in stria vascularis, Kir4.1 helps to generate the cochlear endolymph; and (2). in spiral and vestibular ganglia, Kir4.1 in surrounding glial cells helps to support the spiral and vestibular ganglion neurons and their projecting axons.     

K(+) cycling and its regulation in the cochlea and the vestibular labyrinth

Potassium (K(+)) plays a very important role in the cochlea. K(+) is the major cation in endolymph and the charge carrier for sensory transduction and the generation of the endocochlear potential. The importance of K(+) handling in the cochlea is marked by the discovery of several forms of hereditary deafness that are due to mutations of K(+) channels. Deafness results from mutations of KCNQ4, a K(+) channel in the sensory hair cells, as well as from mutations of the gap junction proteins GJB2, GJB3 and GJB6 that may facilitate cell-to-cell movements of K(+). Deafness results also from mutations of KCNQ1 or KCNE1, subunits of a K(+) channel that carries K(+) from strial marginal cells and vestibular dark cells into endolymph. Further, deafness results from mutations of KCNJ10, a K(+) channel that generates the endocochlear potential in conjunction with the high K(+) concentration in strial intermediate cells and the low K(+) concentration in the intrastrial fluid spaces. This review details recent advances in the understanding of K(+) transport and its regulation in the cochlea and the vestibular labyrinth.     

F-actin cleavage in apoptotic outer hair cells in chinchilla cochleas exposed to intense noise

Sensory transduction in the cochlea and the vestibular labyrinth depends on the cycling of K+. In the cochlea, endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells into perilymph via basolateral K+ channels including KCNQ4. K+ may be taken up by fibrocytes in the spiral ligament and transported from cell to cell via gap junctions into strial intermediate cells. Gap junctions may include GJB2, GJB3 and GJB6. K+ is released from the intermediate cells into the intrastrial space via the KCNJ10 K+ channel that generates the endocochlear potential. From the intrastrial space, K+ is taken up across the basolateral membrane of strial marginal cells via the Na+/2Cl-/K+ cotransporter SLC12A2 and the Na+/K+-ATPase ATP1A1/ATP1B2. Strial marginal cells secrete K+ across the apical membrane into endolymph via the K+ channel KCNQ1/KCNE1, which concludes the cochlear cycle. A similar K+ cycle exists in the vestibular labyrinth. Endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells via basolateral K+ channels including KCNQ4. Fibrocytes connected by gap junctions including GJB2 may be involved in delivering K+ to vestibular dark cells. Extracellular K+ is taken up into vestibular dark cells via SLC12A2 and ATP1A1/ATP1B2 and released into endolymph via KCNQ1/KCNE1, which concludes the vestibular cycle. The importance of K+ cycling is underscored by the fact that mutations of KCNQ1, KCNE1, KCNQ4, GJB2, GJB3 and GJB6 lead to deafness in humans and that null mutations of KCNQ1, KCNE1, KCNJ10 and SLC12A2 lead to deafness in mouse models.     

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.     

Expression of the P2X7 receptor subunit of the adenosine 5'-triphosphate-gated ion channel in the developing and adult rat cochlea

ATP-gated ion channels assembled from P2X(7) subunits have been implicated in ontogeny and cellular pathology. Here, the expression of the P2X(7) receptor subunit was studied in the embryonic (E14-E18 days) and postnatal (P0-adult) rat cochlea using immunohistochemistry. Strong P2X(7) immunolabelling was observed in the primary auditory neurons of the spiral ganglion from E18 to adult and in the fibres innervating the sensory inner and outer hair cells from birth to adult. Strong immunolabelling of P2X(7) receptor protein was also observed in the inner and outer hair cells over a limited developmental period, from birth to P6. Weak expression was observed in cochlear duct epithelium on E18 and in the supporting cells (footplates of pillar cells in adult and in B?ttcher's cells after birth). The immunolocalisation of P2X(7) receptors further implicates extracellular ATP in signalling process in cochlear ontogeny and in establishment and function of auditory neurotransmission. The P2X(7) receptors may be involved in signal transduction and modulation as well as in regulating cell death during development and in pathological conditions.     

Immunocytochemical localization of neurofilament subunits in the spiral ganglion of normal and neomycin-treated guinea pigs

Neurofilaments are a major component of the neuronal cytoskeleton and present in all neurons. The expression of subunits of neurofilaments has been shown to be altered by conditions such as development, aging, degeneration and regeneration of the neuron. In the present study, we determined 1) the distribution of neurofilament subunits in spiral ganglion cells of normal guinea pigs and 2) if this distribution is altered by hair cell degeneration. Immunocytochemical analyses were done with monoclonal antibodies to the 200,000 (NF 200), 160,000 (NF 160), and 68,000 (NF 68) daltons neurofilament subunits. In the normal guinea pig, type II spiral ganglion cells were intensely labeled with NF 200, NF 160, NF 68 antibodies, whereas type I cells were significantly labeled only with NF 200 antibody. Neurofilament subunit immunoreactivity was also localized in the auditory nerve and afferent and efferent fibers to the hair cells. To determine the effects of hair cell loss on neurofilament expression in spiral ganglion cells, guinea pigs were treated with neomycin at doses known to cause extensive hair cell damage. Type I and type II spiral ganglion cells responded differently to this treatment. Type II cells remained strongly immunoreactive after treatment although the number of such cells was reduced, especially in the longer surviving animals. NF 160 and NF 68 immunoreactivities increased gradually from base to apex in type I cells after neomycin treatment, while NF 200 immunoreactivity decreased in all turns.     

Delta/Notch-Like EGF-Related Receptor (DNER) is Expressed in Hair Cells and Neurons in the Developing and Adult Mouse Inner Ear

The Notch signaling pathway is known to play important roles in inner ear development. Previous studies have shown that the Notch1 receptor and ligands in the Delta and Jagged families are important for cellular differentiation and patterning of the organ of Corti. Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a novel Notch ligand expressed in developing and adult CNS neurons known to promote maturation of glia through activation of Notch. Here we use in situ hybridization and an antibody against DNER to carry out expression studies of the mouse cochlea and vestibule. We find that DNER is expressed in spiral ganglion neuron cell bodies and peripheral processes during embryonic development of the cochlea and expression in these cells is maintained in adults. DNER becomes strongly expressed in auditory hair cells during postnatal maturation in the mouse cochlea and immunoreactivity for this protein is strong in hair cells and afferent and efferent peripheral nerve endings in the adult organ of Corti. In the vestibular system, we find that DNER is expressed in hair cells and vestibular ganglion neurons during development and in adults. To investigate whether DNER plays a functional role in the inner ear, perhaps similar to its described role in glial maturation, we examined cochleae of DNER−/− mice using immunohistochemical markers of mature glia and supporting cells as well as neurons and hair cells. We found no defects in expression of markers of supporting cells and glia or myelin, and no abnormalities in hair cells or neurons, suggesting that DNER plays a redundant role with other Notch ligands in cochlear development.     

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.     

In Vitro and In Vivo Assessment of the Ability of Adeno‐Associated Virus–Brain‐Derived Neurotrophic Factor to Enhance Spiral Ganglion Cell Survival Following Ototoxic Insult

Objectives/Hypothesis Auditory dysfunction following ototoxic insult results from loss of cochlear hair cells. Secondary degeneration of auditory neurons ensues from withdrawal of neurotrophic support from hair cells and can be prevented with administration of neurotrophins. Administration of adeno‐associated virus containing the gene for brain‐derived neurotrophic factor will promote spiral ganglion neuron survival after the destruction of hair cells. Methods Prevention of aminoglycoside‐induced spiral ganglion neuron loss through the expression of brain‐derived neurotrophic factor mediated by means of the adeno‐associated virus was tested in vitro in cochlear explants and in vivo in mammalian cochlea. Results Neuronal survival was significantly enhanced in adeno‐associated virus–brain‐derived neurotrophic factor transfected rat cochlear explants compared with control samples (30% vs. 19%, P <.05) following exposure to aminoglycoside. Following deafening with aminoglycoside and loop diuretic and introduction of adeno‐associated virus–brain‐derived neurotrophic factor through osmotic minipump, the experimental group of animals infused with adeno‐associated virus–brain‐derived neurotrophic factor displayed enhanced spiral ganglion neuron survival in the basal turn of the cochlea when compared with the control group infused with adeno‐associated virus containing green fluorescent protein reporter gene. Conclusions Administration of adeno‐associated virus–brain‐derived neurotrophic factor enhances spiral ganglion neuron survival following ototoxic exposure in vitro and in vivo. These studies lay the groundwork for further exploration of its application as an adjunct therapy for patients undergoing cochlear implantation because the success of implantation depends directly on the population of neurons available for electrical stimulation.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

CONCLUSION: Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. OBJECTIVES: At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. MATERIALS AND METHODS: Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. RESULTS: Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

腺相关病毒转导EC-SOD对耳蜗药物性损害的保护作用

目的探讨腺相关病毒（adeno-associated virus,AAV）作为载体,介导分泌型超氧化物歧化酶（extracel-lular superoxide dismutase,EC-SOD）基因,转染耳蜗细胞后对卡那霉素致大鼠耳蜗损害的保护作用。方法构建AAV8血清型载体,携载绿荧光蛋白基因（enhanced green fluorescent protein,EGFP）或EC-SOD基因,体外感染细胞,蛋白电泳检测EC-SOD的表达;将AAV8-EGFP和AAV8-EC-SOD分别微注射到卡那霉素耳聋模型大鼠耳蜗内,检测听性脑干反应阈值变化,生化法检测耳蜗外淋巴液EC-SOD酶活性,免疫组织化学检测EC-SOD蛋白在耳蜗细胞内的表达,耳蜗毛细胞和螺旋神经节细胞染色检测细胞存活的数量。结果AAV8转导的绿荧光蛋白可在耳蜗内毛细胞、螺旋韧带细胞、内沟细胞等多种细胞内表达。体外蛋白电泳检测EC-SOD在细胞培养液中存在单体、二聚体、四聚体等;在AAV8-EC-SOD组注射载体的耳蜗外淋巴液中EC-SOD酶活性明显高于AAV8-EGFP组,转导的EC-SOD在耳蜗内的表达分布与绿荧光蛋白的表达相似。在卡那霉素耳聋模型鼠中,AAV8-EC-SOD组的ABR反应阈值变化比AAV8-EGFP组明显小,AAV8-EC-SOD组的耳蜗毛细胞和螺旋神经节细胞比AAV8-EGFP组的存活多,差异有显著统计学意义。结论AAV8型载体可转染耳蜗内多种细胞,AAV8型转导的EC-SOD可以拮抗氨基糖甙类抗生素对耳蜗的损害。     

Regenerated Synapses Between Postnatal Hair Cells and Auditory Neurons

Regeneration of synaptic connections between hair cells and spiral ganglion neurons would be required to restore hearing after neural loss. Here we demonstrate by immunohistochemistry the appearance of afferent-like cochlear synapses in vitro after co-culture of de-afferented organ of Corti with spiral ganglion neurons from newborn mice. The glutamatergic synaptic complexes at the ribbon synapse of the inner hair cell contain markers for presynaptic ribbons and postsynaptic densities. We found postsynaptic density protein PSD-95 at the contacts between hair cells and spiral ganglion neurons in newly formed synapses in vitro. The postsynaptic proteins were directly facing the CtBP2-positive presynaptic ribbons of the hair cells. BDNF and NT-3 promoted afferent synaptogenesis in vitro. Direct juxtaposition of the postsynaptic densities with the components of the preexisting ribbon synapse indicated that growing fibers recognized components of the presynaptic sites. Initiation of cochlear synaptogenesis appeared to be influenced by glutamate release from the hair cell ribbons at the presynaptic site since the synaptic regeneration was impaired in glutamate vesicular transporter 3 mutant mice. These insights into cochlear synaptogenesis could be relevant to regenerative approaches for neural loss in the cochlea.