Three-dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy.

Three-dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step-by-step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings.

Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing.

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion-matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two-photon action cross-sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six-colour neuronal tracing within the spinal cord and brain tissue.

EphB2 guides axons at the midline and is necessary for normal vestibular function.

Mice lacking the EphB2 receptor tyrosine kinase display a cell-autonomous, strain-specific circling behavior that is associated with vestibular phenotypes. In mutant embryos, the contralateral inner ear efferent growth cones exhibit inappropriate pathway selection at the midline, while in mutant adults, the endolymph-filled lumen of the semicircular canals is severely reduced. EphB2 is expressed in the endolymph-producing dark cells in the inner ear epithelium, and these cells show ultrastructural defects in the mutants. A molecular link to fluid regulation is provided by demonstrating that PDZ domain-containing proteins that bind the C termini of EphB2 and B-ephrins can also recognize the cytoplasmic tails of anion exchangers and aquaporins. This suggests EphB2 may regulate ionic homeostasis and endolymph fluid production through macromolecular associations with membrane channels that transport chloride, bicarbonate, and water.

Proprioceptor pathway development is dependent on Math1.

The proprioceptive system provides continuous positional information on the limbs and body to the thalamus, cortex, pontine nucleus, and cerebellum. We showed previously that the basic helix-loop-helix transcription factor Math1 is essential for the development of certain components of the proprioceptive pathway, including inner-ear hair cells, cerebellar granule neurons, and the pontine nuclei. Here, we demonstrate that Math1 null embryos lack the D1 interneurons and that these interneurons give rise to a subset of proprioceptor interneurons and the spinocerebellar and cuneocerebellar tracts. We also identify three downstream genes of Math1 (Lh2A, Lh2B, and Barhl1) and establish that Math1 governs the development of multiple components of the proprioceptive pathway.

Stem cells and molecular strategies to restore hearing.

Hearing loss is a costly and growing problem for the elderly population worldwide with millions of people being affected. There are currently two prosthetic devices available to minimize problems associated with the two forms of hearing loss: hearing aids that amplify sound to overcome middle ear based conductive hearing loss and cochlear implants that restore some hearing after neurosensory hearing loss. The current presentation provides information on the treatment of neurosensory hearing loss. Although the cochlear implant solution for neurosensory hearing loss is technologically advanced; it still provides only moderate hearing capacity in neurosensory deaf individuals. Inducible stem cells and molecular therapies are appealing alternatives to the cochlear implant and may provide more than a new form of treatment as they hold the promise for a cure. To this end, current insights into inducible stem cells that may provide cells for seeding the cochlea with the hope of new hair cell formation are being reviewed. Alternatively, similar to induction of stem cells, cells of the flat epithelium that remains after hair cell loss could be induced to proliferate and differentiate into hair cells. In either of these strategies, hair cell specific genes known to be essential for hair cell differentiation or maintenance such as ATOH1, POU4F3, GFI1, and miRNA-183 will be utilized with the hope of completely restoring hearing to all patients with hearing loss.

Effects of neurotrophin and neurotrophin receptor disruption on the afferent inner ear innervation.

Two neurotrophins and their two receptors appear to regulate the survival of vestibular and cochlear neurons in the developing ear. Mice lacking either brain derived neurotrophic factor (BDNF) or its associated receptor, Trk B, show a severe reduction in the number of vestibular neurons and a loss of all innervation to the semicircular canals. Mice lacking NT-3 or its receptor, Trk C, show a severe reduction of spiral neurons in the basal turn of the cochlea. Mice lacking both BDNF and NT-3 or Trk B and Trk C, reportedly lose all innervation to the inner ear. These two neurotrophins and their associated receptors are necessary for the normal afferent innervation of the inner ear.

The role of neurotrophic factors in regulating the development of inner ear innervation.

Several neurotrophins and their receptors regulate the survival of vestibular and cochlear neurons and probably also the efferent and autonomic neurons that innervate the inner ear. Mice lacking either brain-derived neurotrophic factor (BDNF) or its associated receptor, TrkB, lose all innervation to the semicircular canals and have reduced innervation of the outer hair cells in the apical and middle turns of the cochlea. Mice lacking neurotrophin-3 (NT-3) or its receptor, TrkC, lose many spiral ganglion cells predominantly in the basal turn of the cochlea. Nerve fibers from spiral ganglion cells in the middle turn extended to inner hair cells of the base. In mice lacking both BDNF and NT-3, or both TrkB and TrkC, there is a complete loss of innervation to the inner ear. Thus, these two neurotrophins and their associated receptors have been shown to be absolutely necessary for the normal development of afferent innervation of the inner ear. Current research efforts are testing the therapeutic potential for neurotrophins to treat hearing loss.

Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance.

The beta-galactosidase protein generated by the bacterial LacZ gene is widely used to map gene expression patterns. The ease of its use is only rivaled by green fluorescent protein, which can be used in combination with various other procedures such as immunocytochemistry, flow cytometry, or tract tracing. The beta-galactosidase enzymatic reaction potentially provides a more sensitive assay of gene expression than green fluorescent protein. However, the virtual impermeability and tendency to absorb light over a wide range limit the use of the most frequently used beta-galactosidase substrate, X-Gal, in combination with other fluorescent labeling procedures. Here, we provide details on a simple photoactivation procedure that transforms the light-absorbing X-Gal product, 5-bromo-4-chloro-3-indolyl (BCI) precipitate, into an intensely fluorescent product excited by 488 and 633 nm light. Photoactivation is achieved through exposure to 730 nm near-infrared light emitted from a femtosecond titanium-doped Sapphire laser. Photoactivation of BCI occurs in tissue sections suspended in buffered saline, glycerol, or even embedded in epoxy resin. A protocol for the use of BCI photoactivation is here provided. Importantly, the BCI photoactivated product is photoswitchable, displaying bistable photochromism. This permits the use of the fluorescent product in a variety of co-localization studies in conjunction with other imaging modalities. As with other bistable and photoswitchable products, the BCI reaction product shows concentration quenching at high density and can be degraded by continuous exposure to intense 730 nm illumination. Therefore, care must be taken in developing imaging strategies. Our findings have implications for the use of X-Gal in gene and protein detection and provide a novel substrate for high density digital information storage.

Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression.

Previous work suggested qualitatively different effects of neurotrophin 3 (NT-3) in cochlear innervation patterning in different null mutants. We now show that all NT-3 null mutants have a similar phenotype and lose all neurons in the basal turn of the cochlea. To understand these longitudinal deficits in neurotrophin mutants, we have compared the development of the deficit in the NT-3 mutant to the spatial-temporal expression patterns of brain-derived neurotrophic factor (BDNF) and NT-3, using lacZ reporters in each gene and with expression of the specific neurotrophin receptors, trkB and trkC. In the NT-3 mutant, almost normal numbers of spiral ganglion neurons form, but fiber outgrowth to the basal turn is eliminated by embryonic day (E) 13.5. Most neurons are lost between E13.5 and E15.5. During the period preceding apoptosis, NT-3 is expressed in supporting cells, whereas BDNF is expressed mainly in hair cells, which become postmitotic in an apical to basal temporal gradient. During the period of neuronal loss, BDNF is absent from the basal cochlea, accounting for the complete loss of basal turn neurons in the NT-3 mutant. The spatial gradients of neuronal loss in these two mutants appear attributable to spatial-temporal gradients of neurotrophin expression. Our immunocytochemical data show equal expression of their receptors, TrkB and TrkC, in spiral sensory neurons and thus do not relate to the basal turn loss. Mice in which NT-3 was replaced by BDNF show a qualitative normal pattern of innervation at E13.5. This suggests that the pattern of expression of neurotrophins rather than their receptors is essential for the spatial loss of spiral sensory neurons in NT-3 null mutants.

Diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon, NeuroVue Red and NeuroVue Green and their use for double and triple labeling of neuronal profile.

We describe here diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon (near infrared), NeuroVue Red and NeuroVue Green. Using pair-wise comparisons between the new dyes and existing dyes (DiI, DiA, DiD, DiO, PKH2, PKH26) applied to the left and the right side of fixed spinal cord preparations, we show that NeuroVue Maroon (excitation maximum 647 nm) surpasses all other dyes in this study in signal to noise ratio. We also present data showing the utility of these new dyes for both double labeling and triple labeling in combination with each other or existing lipophilic tracers. Using mice bearing the PLP-eGFP transgene, we demonstrate that either NeuroVue Maroon or NeuroVue Red can readily be combined with eGFP labeling. Double labeling experiments using NeuroVue Red and eGFP allowed us to demonstrate that every fiber in the neonatal ear is surrounded by developing Schwann cells.

How does the urodele ear develop?

An overview is provided of the structural and molecular events causing the transformation of undifferentiated epidermal cells together with the underlying mesenchyme to become the complex, three-dimensional ear. While tremendous progress has been achieved in a few model systems, enough is not yet known about the comparative embryology of ear development to provide causal explanations of the adult structural differences among species. It is hoped that the changes in selector and/or structural genes, as well as changes in the spatiotemporal induction of structural gene activation, and possible changes in the interaction between the various embryonic sources which contribute to the ear, will soon be understood. The most promising new avenue for research appears to be studies which combine classical transplantation tissue experiments with modern gene expression analyses and modern in vitro assays of the role of putative morphogens or trophic factors. It is emphasized that it is not understood what is missing in the developmental program of those salamanders which have lost a basilar papilla. Direct comparison of gene expression patterns and xenoplastic transplantations in salamanders of comparable stages which either do or do not develop this organ should help to clarify the molecular events that have led to this major evolutionary novel feature of the vertebrate ear.

Distribution of BDNF and trkB mRNA in the otic region of 3.5 and 4.5 day chick embryos as revealed with a combination of in situ hybridization and tract tracing.

We have used a recently developed technique which combines fluorescent tract tracing and in situ hybridization to study co-localization of neurotrophin mRNA and neurotrophin receptor mRNA expression simultaneously with the pattern of innervation in the developing chick ear. Efferent and afferent fibersfrom the VII/VIIIth cranial nerves were retrogradely and anterogradely filled with Dextran amines conjugated to Texas red and the brain stem was incubated for 2 hours in tissue culture medium. The tissue was subsequently fixed, sectioned frozen, mounted and subjected to in situ hybridization analysis using probes for brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, trkB. The results show that afferent and efferent fibers to the ear innervate areas of the developing otocyst which express BDNF mRNA. We also found that neurons in the stato-acoustic ganglion express high levels of trkB mRNA whereas the subset of facial motor neurons that is efferent to the ear only had no or very low levels of trkB mRNA. From our results we conclude that chicken otic efferent fibers preferentially project to areas with BDNF mRNA expression. The very low levels of trkB mRNA in these motor neurons compared to afferent neurons innervating the same region suggest that other factors, perhaps co-expressed with BDNF, may support efferents. A possible involvement of afferents in guiding efferents to specific areas of the ear is suggested.

Peripheral and central aspects of the acoustic and lateral line system of a bottom dwelling catfish, Ancistrus sp.

The topographical relationship between the swim bladder, the inner ear, and the otic lateral line was studied in the bottom dwelling catfish, Ancistrus sp. In addition, afferent and efferent subcomponents of the eighth and lateral line nerves were labelled with horseradish peroxidase (HRP) or with differently fluorescing dextran amines. The swim bladder of Ancistrus consists of two separate, transversely oriented parts of each of which is connected to the sinus impar of the inner ears via two Weberian ossicles and the perilymphatic sac. The osseous capsula of the ear has two foramina other than the nerve foramina. One is for the sinus impar. The other foramen, which also separates two fluid-filled spaces, exits where the horizontal canal of the ear contacts the otic lateral line. Both the otic and the postotic lateral line canal run deep below the epidermis. Each canal contains a neuromast that is innervated by the middle lateral line nerve. Further caudally, the otic lateral line canal gives rise to the postotic and finally to the truck canal whose nonossified anterior part travels through an ossified chamber that surrounds the swim bladder. Thus the anterior part of each trunk lateral line canal is in contact with a bipartite sound pressure receiver, the swim bladder. Anterior and posterior lateral line afferents terminate ipsilaterally throughout the neuropil of the electroreceptive lateral line nucleus and the mechanoreceptive nuclei medialis and caudalis of the medulla. Middle lateral line afferents terminate between the projection sites of anterior and posterior lateral line afferents. Some primary mechanosensory anterior lateral line nerve fibers continue into the ipsilateral eminentia granularis and the valvula cerebelli. In the electroreceptive lateral line projection, anterior lateral line fibers terminate more medially and posterior fibers more laterally. This somatotopy is not as clear-cut in the mechanosensory lateral line. Afferents of the sacculus and the lagena terminate predominantly in the saccular nucleus. Afferents of the utriculus, the horizontal canal, and the anterior vertical canal terminate in the magnocellular vestibular nucleus and in the medial octavolateral nucleus. The projection sites of the anterior part and the posterior part of the eighth nerve show little overlap. Eighth nerve projections to the valvula cerebelli are less prominent than the projections from the lateral line. Eighth nerve and lateral line nerve efferents arise from a common nucleus, the octavolateralis efferent nucleus. Axons of efferent cells may divide to supply two or more branches of the eighth nerve and some axons supply both lateral line and eighth nerve endorgans.(ABSTRACT TRUNCATED AT 400 WORDS)

Time course of structural changes in regenerating electroreceptors of a weakly electric fish.

We examined the regenerating electroreceptors of the weakly electric fish Sternopygus by light and electron microscopy to search for possible structural correlates of known physiological changes that occur during regeneration (Zakon: J. Neurosci. 6(11):3297-3308, 1986) and to compare them with developing electroreceptors in larval fish (Vischer: Brain Behav. Evol. 33:223-236). Nine days after removal of a patch of cheek skin, new skin had filled the wound and undifferentiated precursor cell clusters were located in the epidermis just above the dermis. Nerve fibers were present near most, but not all, cell clusters. A few recognizable tuberous and ampullary precursor organs were seen at this time. Tuberous organs were composed of a group of large cells surrounded by smaller cells without a lumen and showed the beginning of a cellular plug. Ampullary organs appeared as a ball of cells with a small lumen opening into a nascent canal. Degenerating cells were found within organs, and sometimes entire organs degenerated. These were not innervated. By 2 weeks the large cells of the tuberous organ were developing into sensory cells, while the smaller cells were forming the capsule wall and the underlying basal cells. The characteristic tuberous organ canal filled with loosely packed epidermal cells was evident. The sensory cells of the ampullary organs were visible within the epithelial layer at the base of the lumen, and the large synaptic discs were beginning to form. The sensory cells and postsynaptic terminals contained numerous vesicles. The presynaptic vesicles, which appear in normal receptor cells, remained throughout regeneration and presumably underlie transmitter release. The postsynaptic vesicles appeared transiently in large numbers but declined to adult values by 4 weeks. We presume that these may serve a trophic role. By 3 weeks, organs generally appeared mature and began dividing into daughter organs. The formation of individual receptor organs during regeneration is similar to that observed in development. Receptor organs continued dividing until the appropriate number of organs per afferent was reached for the size of the fish. Although the organization of the receptors appeared generally normal, there were a few anomalies. Some afferents sent sprouts into the epidermis, and, as a result of such sprouting, some of these afferents innervated multiple organs over a greater distance than normal. This was first seen early in regeneration and persisted for as long as 5 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Projection patterns of lateral-line afferents in anurans: a comparative HRP study.

Primary projections of the anterior and posterior lateral-line nerves were traced by means of transganglionic transport of horseradish peroxidase in species belonging to five of the six anuran superfamilies. Both anterior and posterior lateral-line nerve afferents each enter the brain via a single root which divides into two or more bundles. These bundles carry fibers from neuromasts only. No separate dorsal fascicle and no ampullary organs as in urodeles and gymnophions have been found. All bundles join in the neuropil of the nucleus intermedius to form ascending and descending fascicles. Two distinct fascicles are found in species showing little collateralization. No fasciculation is found in species with an elaborate telodendritic arborization. Afferents of the anterior lateral-line nerve run ventromedially and those of the posterior lateral-line nerve dorsolaterally within the ipsilateral nucleus intermedius neuropil. Rostrally they terminate in the vicinity of the eminentia granularis and caudally in the vicinity of the calamus scriptorius. The metamorphic changes in the alar plate do not support the hypothesis of Larsell ('34) as to a change in function of second-order lateral-line neurons into second-order auditory neurons. The rostral part of the nucleus intermedius shows numerous degenerating neurons at metamorphic climax whereas the caudal part becomes part of the nucleus caudalis of adult anurans. Besides the members of the Pipoidea, there is at least the genus Bombina which retains parts of the lateral-line system. The term "dorsal island," its relevance for any part of the anuran brain, and the possible relation between absence of electroreception and the development of the nucleus dorsolateralis are discussed.

Development of two morphological types of retinopetal fibers in chick embryos, as shown by the diffusion along axons of a carbocyanine dye in the fixed retina.

Centrifugal fibers to the retinas of chick embryos and hatched chicks have been examined and traced following staining by diffusion along their axonal membranes of the carbocyanine dye DiI in fixed tissue. In the older embryos and hatched chicks, the report of Dogiel (Arch. Mikrosk. Anat. 44:622-648, 1895) has been confirmed that there are two very different morphological types of centrifugal fiber. The restricted type ends as a relatively thick fiber, lacking varicosities, that runs for a short distance in the most sclerad level of the inner plexiform layer before terminating in a pericellular nest overlying the flask-shaped body of a single amacrine cell. Thin filaments occasionally leave the pericellular net, apparently to terminate on adjacent cells. The widespread type also runs in the most sclerad level of the inner plexiform layer, but it is thin, varicose, and highly branched, and its terminal arbor may span more than 1 mm, remaining at the same level. Both types of terminal arbor issue from parent axons in the optic fiber layer of the retina. A single parent axon gives either a single terminal fiber of the restricted type or several terminals of the widespread type, but never a mixture of the two. It is argued that the restricted and widespread types originate respectively from the neurons of the contralateral isthmo-optic nucleus and from the "ectopic" neurons scattered outside the isthmo-optic nucleus. In development, the centrifugal fibers reach the retina between E9 and E10 and initially run radially in the optic fiber layer, parallel to the retinofugal fibers but avoiding the dorsal retina. They dive into the inner plexiform layer at about E12. By E13, the terminal arbors are forming, and the widespread and restricted types can already be distinguished. The widespread type continues to increase its territory until about E18, and then appears to remain stable, whereas the restricted type attains its maximum ramification between E13 and E15 and then contracts. Prior to the retraction, the terminal territories of the restricted type fibers overlap, which may provide the anatomical basis for the interaxonal competition that apparently contributes to neuronal death in the isthmo-optic nucleus between E13 and E16. Axons of ganglion cells exhibit transient side branches between E11 and E13; these never reach as deep as the level where the centrifugal fibers run.

Organization of the six motor nuclei innervating the ocular muscles in lamprey.

The topography of motoneurons supplying each of the six ocular muscles of the lamprey, Lampetra fluviatilis, was studied by selective application of HRP to the cut nerves of identified muscles. In addition, the distributions of motoneuron populations to both eyes were studied simultaneously with fluorescein and rhodamine coupled dextran-amines (FDA and RDA) applied to cut ocular muscle nerves of either side. The motoneuron pool of the caudal oblique muscle is represented bilaterally in the trochlear (N IV) motor nucleus. The dorsal rectus muscle is innervated from a contralateral group of oculomotor (N III) motoneurons and the remaining four muscles exclusively from the ipsilateral side (N III and N VI). The inferior and posterior rectus muscles are both innervated by the abducens nerve. In contrast to all jawed vertebrates, only three eye muscles (the dorsal rectus, rostral rectus, and rostral oblique) are innervated by the oculomotor nerve in lampreys (N III). Lampreys have a motor nucleus similar to the accessory abducens nucleus previously described only in tetrapods. They lack the muscle homologous to the nasal rectus muscle of elasmobranchs and the medial rectus muscle of osteognathostomes. The distribution of the dendrites of different groups of motoneurons was studied and is considered in relation to inputs from tectum and the different cranial nerves.

Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear.

Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA-3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA-3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA-3 heterozygous and GATA-3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild-type littermates. This suggests that GATA-3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA-3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA-3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA-3 and the effects of null mutations on the ear suggest involvement of GATA-3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear.

Characterization of G-protein betagamma expression in inner ear.

Heterotrimeric guanine nucleotide binding proteins (G-proteins) are composed of a diverse set of alpha, beta, and gamma subunits, which couple cell surface receptors to intracellular effectors, such as adenylyl cyclase, phospholipase Cbeta, and ion channels. Both the Galpha and the Gbetagamma dimers mediate effector activity and are believed to contribute to the complexity of the signaling pathway. Molecular and immunocytochemical techniques were employed to determine diversity of Gbeta and Ggamma subunit expression in the murine inner ear. PCR-based assessment of lambdaZAP unidirectional cDNA libraries, representing the cochlea and inner ear hair cells, indicated all five known Gbeta subunits were present in the cochlea, while only a subset of Ggamma isoforms were found. New or novel G-protein beta and gamma subunits were not detected. cDNAs representing Gbeta1-4 and Ggamma2, Ggamma3, Ggamma5, Ggamma8olf subunit transcripts were isolated. In addition, cDNAs corresponding to the Gbeta5 and Ggamma11 isoforms exhibited restricted expression to inner and outer hair cells, respectively. Antisera specific for Gbeta3, Gbeta4, Ggamma3, Ggamma5 and Ggamma11 stained spiral ganglion and neurosensory hair cells. A unique finding was the variable topological distribution of Ggamma3 in the spiral ganglion cells along the cochlear axis. Collectively, our results demonstrate a complementary as well as differential distribution pattern for Gbeta and Ggamma isoforms exists in the inner ear. The co-localization of various G-protein isoforms within the same cell type suggests specific combinatorial Gbeta and Ggamma subunit associations may preferentially be formed. Thus, the detection of multiple subunits presumably reflects the extent of the functional diversity of inner ear signaling pathways and should provide specificity of G-protein mediated pathways.

Longitudinal gradients of KCNQ4 expression in spiral ganglion and cochlear hair cells correlate with progressive hearing loss in DFNA2.

Mutations in the human KCNQ4 gene were recently found by Kubisch et al. [Cell 96 (1999) 437-446] to cause a non-syndromic, autosomal dominant, progressive hearing loss, DFNA2. The mouse Kcnq4 orthologue was previously localized to the outer hair cells (OHCs) of the inner ear, suggesting the pathophysiological effects were due to dysfunctional OHCs. Yet, OHC dysfunction does not provide a plausible explanation for the progressive nature of the frequency specific hearing loss. We have re-examined and extended the expression analyses of KCNQ4 in the murine inner ear using RT-PCR and whole mount in situ hybridization. Our results confirmed that the rat KCNQ4 orthologue is expressed in both inner and outer hair cells. Reciprocal longitudinal gradients were found in inner hair cells (IHCs) and OHCs. The strongest expression of KCNQ4 in IHCc was in the base of the cochlea and in the apex for OHCs. Similar to the IHCs, a basal to apical gradient was present in the spiral sensory neurons. IHCs mediate hearing via their afferent sensory neurons, whereas OHCs function as active cochlear amplifiers. The complete absence of OHCs leads only to severe sensitivity reduction, but not complete hearing loss. Our data suggest that the primary defect leading to initial high frequency loss and subsequent progressive hearing loss for all frequencies may be due to spiral ganglion and/or IHC dysfunction, rather than an OHC aberration.