Ulrike Zimmermann

Reversible contraction of isolated mammalian cochlear hair cells

Outer hair cells were isolated from the guinea pig cochlea using a micromechanical non-enzymatic procedure. Depolarization of outer hair cells in the presence of 25-125 mM K+ was accompanied by a longitudinal contraction of the isolated cells. A decrease of [K+] to 5.4 mM interrupted contraction and induced a relaxation. Individual hair cells were able to undergo as many as 5 cycles of contraction and relaxation. External Ca2+ was required for relaxation of the contracted hair cells. The contractile event led to the production of a visible cytoplasmic network between the supranuclear area and the cuticular plate.

Thyroid hormone is a critical determinant for the regulation of the cochlear motor protein prestin

The most impressive property of outer hair cells (OHCs) is their ability to change their length at high acoustic frequencies, thus providing the exquisite sensitivity and frequency-resolving capacity of the mammalian hearing organ. Prestin, a protein related to a sulfate/anion transport protein, recently has been identified and proposed as the OHC motor molecule. Homology searches of 1.5 kb of genomic DNA 5′ of the coding region of the prestin gene allowed the identification of a thyroid hormone (TH) response element (TRE) in the first intron upstream of the prestin ATG codon. PrestinTRE bound TH receptors as a monomer or presumptive heterodimer and mediated a triiodothyronine-dependent transactivation of a heterologous promotor in response to triiodothyronine receptors α and β. Retinoid X receptor-α had an additive effect. Expression of prestin mRNA and prestin protein was reduced strongly in the absence of TH. Although prestin protein typically was redistributed to the lateral membrane before the onset of hearing, an immature pattern of prestin protein distribution across the entire OHC membrane was noted in hypothyroid rats. The data suggest TH as a first transcriptional regulator of the motor protein prestin and as a direct or indirect modulator of subcellular prestin distribution.

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.

Tinnitus behavior and hearing function correlate with the reciprocal expression patterns of BDNF and Arg3.1/arc in auditory neurons following acoustic trauma

The molecular changes following sensory trauma and the subsequent response of the CNS are poorly understood. We focused on finding a molecular tool for monitoring the features of excitability which occur following acoustic trauma to the auditory system. Of particular interest are genes that alter their expression pattern during activity-induced changes in synaptic efficacy and plasticity. The expression of brain-derived neurotrophic factor (BDNF), the activity-dependent cytoskeletal protein (Arg3.1/arc), and the immediate early gene c-Fos were monitored in the peripheral and central auditory system hours and days following a traumatic acoustic stimulus that induced not only hearing loss but also phantom auditory perception (tinnitus), as shown in rodent animal behavior models. A reciprocal responsiveness of activity-dependent genes became evident between the periphery and the primary auditory cortex (AI): as c-Fos and BDNF exon IV expression was increased in spiral ganglion neurons, Arg3.1/arc and (later on) BDNF exon IV expression was reduced in AI. In line with studies indicating increased spontaneous spike activity at the level of the inferior colliculus (IC), an increase in BDNF and GABA-positive neurons was seen in the IC. The data clearly indicate the usefulness of Arg3.1/arc and BDNF for monitoring trauma-induced activity changes and the associated putative plasticity responses in the auditory system.

Two classes of outer hair cells along the tonotopic axis of the cochlea

The molecular basis of high versus low frequency hearing loss and the differences in the sensitivity of outer hair cells depending on their cochlear localization are currently not understood. Here we demonstrate the existence of two different outer hair cell phenotypes along the cochlear axis. Outer hair cells in low frequency regions exhibit early sensitivity for loss of Ca(v)1.3 (alpha1 subunit 1.3 forming the class D L-type voltage-gated Ca(2+) channel), while high frequency regions display a progressive susceptibility for loss of the Ca(2+)-activated large conductance K(+) (BK) channel. Despite deafness, young Ca(v)1.3-deficient mice displayed distortion-product otoacoustic emissions (DPOAEs), indicating functional outer hair cells in the higher frequency range of the cochlea. Considering that DPOAEs are also found in the human deafness syndrome DFNB9 caused by mutations in the synaptic vesicle protein otoferlin, we tested the expression of otoferlin in outer hair cells. Surprisingly, otoferlin showed a distinct tonotopic expression pattern at both the mRNA and protein level. Otoferlin-expressing, Ca(v)1.3 deletion-sensitive outer hair cells in the low frequency range could be clearly separated from otoferlin-negative, BK deletion-sensitive outer hair cells in the high frequency range. In addition, BK deletion led to a higher noise vulnerability in low frequency regions, which are normally unaffected by the BK deletion alone, suggesting that BK currents are involved in survival mechanisms of outer hair cells under noise conditions. Our findings propose new mechanisms and candidate genes for explaining high and low frequency hearing loss.

Transient expression of NMDA receptors during rearrangement of AMPA-receptor-expressing fibers in the developing inner ear

Abstract.A major reorganization of afferent and efferent nerve terminals, concomitant to significant neuronal cell loss and pruning of superfluous fibers, takes place during the development of the organ of Corti, prior to the onset of hearing. We examined the spatio/temporal distribution of subtype-specific AMPA- and N-methyl-d-aspartate (NMDA)-selective glutamate receptor proteins in postnatal inner ears from rats during this critical period. From the first postnatal day onwards, GluR2/3 receptor subtypes appeared in nerve endings of afferent fibers associated with inner and outer hair cells. During the following 2 weeks, GluR2/3 receptors were downregulated in exchange for GluR4 receptors. In parallel efferents projecting from the medial olivocochlear complex to the outer hair cells underwent synaptogenesis and efferents projecting from the lateral olivocochlear complex to the inner hair cells appeared to change contacts to the dendrites of afferents. Concomitant to these events, NMDA receptor subtypes NR1 and NR2A transiently appeared in hair cells as well as afferent and efferent fibers. Recently, we described a temporary expression of the neurotrophin receptor trkB in hair cells, coincident to the growth (GAP-43) and synaptogenesis (synaptophysin) of efferents. Here, we show that trkB was expressed together with NR1 receptors in hair cells in high spatio/temporal correlation with the rearrangement of afferents and efferents. Cochlea NMDA receptors may, therefore, be a part of the mechanism by which, in addition to neurotrophic activity, the mature phenotype of cochlea neurons is acquired through activity-dependent processes.

The reduced cochlear output and the failure to adapt the central auditory response causes tinnitus in noise exposed rats.

Tinnitus is proposed to be caused by decreased central input from the cochlea, followed by increased spontaneous and evoked subcortical activity that is interpreted as compensation for increased responsiveness of central auditory circuits. We compared equally noise exposed rats separated into groups with and without tinnitus for differences in brain responsiveness relative to the degree of deafferentation in the periphery. We analyzed (1) the number of CtBP2/RIBEYE-positive particles in ribbon synapses of the inner hair cell (IHC) as a measure for deafferentation; (2) the fine structure of the amplitudes of auditory brainstem responses (ABR) reflecting differences in sound responses following decreased auditory nerve activity and (3) the expression of the activity-regulated gene Arc in the auditory cortex (AC) to identify long-lasting central activity following sensory deprivation. Following moderate trauma, 30% of animals exhibited tinnitus, similar to the tinnitus prevalence among hearing impaired humans. Although both tinnitus and no-tinnitus animals exhibited a reduced ABR wave I amplitude (generated by primary auditory nerve fibers), IHCs ribbon loss and high-frequency hearing impairment was more severe in tinnitus animals, associated with significantly reduced amplitudes of the more centrally generated wave IV and V and less intense staining of Arc mRNA and protein in the AC. The observed severe IHCs ribbon loss, the minimal restoration of ABR wave size, and reduced cortical Arc expression suggest that tinnitus is linked to a failure to adapt central circuits to reduced cochlear input.

Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell

Otoferlin has been proposed to be the Ca(2+) sensor in hair cell exocytosis, compensating for the classical synaptic fusion proteins synaptotagmin-1 and synaptotagmin-2. In the present study, yeast two-hybrid assays reveal myosin VI as a novel otoferlin binding partner. Co-immunoprecipitation assay and co-expression suggest an interaction of both proteins within the basolateral part of inner hair cells (IHCs). Comparison of otoferlin mutants and myosin VI mutant mice indicates non-complementary and complementary roles of myosin VI and otoferlin for synaptic maturation: (i) IHCs from otoferlin mutant mice exhibited a decoupling of CtBP2/RIBEYE and Ca(V)1.3 and severe reduction of exocytosis. (ii) Myosin VI mutant IHCs failed to transport BK channels to the membrane of the apical cell regions, and the exocytotic Ca(2+) efficiency did not mature. (iii) Otoferlin and myosin VI mutant IHCs showed a reduced basolateral synaptic surface area and altered active zone topography. Membrane infoldings in otoferlin mutant IHCs indicated disturbed transport of endocytotic membranes and link the above morphological changes to a complementary role of otoferlin and myosin VI in transport of intracellular compartments to the basolateral IHC membrane.

Differential expression of otoferlin in brain, vestibular system, immature and mature cochlea of the rat

Mutations of the human otoferlin gene lead to an autosomal recessive nonsyndromic form of prelingual, sensorineural deafness (deafness autosomal recessive 9, DFNB9). Several studies have demonstrated expression of otoferlin in the inner ear and brain, and suggested a role of otoferlin in Ca2+‐triggered exocytosis. So far, otoferlin expression profiles were solely based on the detection of mRNA. Here, we analysed the expression of otoferlin protein and mRNA using immunohistochemistry, in situ hybridization and RT‐PCR in neonatal and mature Wistar rat tissue. In agreement with previous studies, otoferlin expression was found in the brain and in inner and vestibular hair cells. Otoferlin mRNA and protein was, however, also detected in mature outer hair cells of low‐frequency processing cochlear turns and in auditory nerve fibres. In outer, inner and vestibular hair cells, otoferlin was subcellularly localized at a considerable distance from the presumed active release sites. Double‐staining with the synaptic ribbon marker, C‐terminal binding protein 2 (CtBP2), or the presynaptic Ca2+‐channel, Cav1.3, both assumed to mark the sites of vesicle fusion and transmitter release, did not colocalize with otoferlin expression and thus do not necessarily support a selected role of otoferlin in Ca2+‐triggered exocytosis. The widespread distribution of otoferlin in neurons, nerve fibres and hair cells, and its subcellular distribution extending beyond the regions of synaptic vesicle fusion, i.e. coenrichment with the cytosolic Golgi matrix protein 130 (GM130) in inner hair cells or the early endosomal autoantigen 1 (EEA1) in outer hair cells support instead the idea of a more ubiquitous role of otoferlin in early/recycling endosome trans‐Golgi network dynamics.

Fast motility of isolated mammalian auditory sensory cells

Auditory sensory cells (hair cells) are responsible for sound transduction in the cochlea of the inner ear. In the presence of a longitudinal a.c. field isolated living outer hair cells showed reversible motile responses. They followed the stimulus up to at least 1 kHz. Control experiments in the presence of cytochalasin B, phalloidin and dinitrophenol excluded actomyosin as a molecular basis of the high frequency motility. The results suggest, that outer hair cells might amplify sound-induced oscillations in the inner ear and thus increase sensitivity and frequency selectivity of hearing.