The prevalence of hearing problems in the Western world has, due to aging of the population, doubled over the past 30 years. Thereby, noise-induced hearing loss is an important factor that worsens over time in addition to age-related hearing loss. Hearing loss is usually measured as an elevation of a person's hearing thresholds, expressed in decibel (dB). However, recent animal studies have unraveled a type of permanent cochlear damage, without an elevation of hearing thresholds. This subtle damage is linked to a permanent and progressive degeneration of auditory fibers that occurs in association with damage of the inner hair cell synapse. Afferent neuronal degeneration has been suggested to be involved in hyperacusis (over sensitivity to sound) and tinnitus (a phantom sound percept). Hyperacusis and tinnitus are potentially devastating conditions that are still incurable. The main risk factors to develop tinnitus or hyperacusis are hearing loss, social stress and age. Both tinnitus and hyperacusis have been discussed in the context of a pathological increased response gain in subcortical brain regions as a reaction to deprivation of sensory input. Novel studies confirm the involvement of peripheral deafferentation for tinnitus and hyperacusis, but suggest that the disorder results from different brain responses to different degrees of deafferentation: while tinnitus may arise as a failure of the brain to adapt to deprived peripheral input, hyperacusis may result from an 'over-adaptive' increase in response gain. Moreover, moderate and high stress levels at the time of acoustic trauma have been suggested to play a pivotal role in the vulnerability of the cochlea to acoustic damage and therefore for the development of tinnitus and hyperacusis.

Increasing evidence shows that hearing loss is a risk factor for tinnitus and hyperacusis. Although both often coincide, a causal relationship between tinnitus and hyperacusis has not been shown. Currently, tinnitus and hyperacusis are assumed to be caused by elevated responsiveness in subcortical circuits. We examined both the impact of different degrees of cochlear damage and the influence of stress priming on tinnitus induction. We used (1) a behavioral animal model for tinnitus designed to minimize stress, (2) ribbon synapses in inner hair cells (IHCs) as a measure for deafferentation, (3) the integrity of auditory brainstem responses (ABR) to detect differences in stimulus-evoked neuronal activity, (4) the expression of the activity-regulated cytoskeletal protein, Arc, to identify long-lasting changes in network activity within the basolateral amygdala (BLA), hippocampal CA1, and auditory cortex (AC), and (5) stress priming to investigate the influence of corticosteroid on trauma-induced brain responses. We observed that IHC ribbon loss (deafferentation) leads to tinnitus when ABR functions remain reduced and Arc is not mobilized in the hippocampal CA1 and AC. If, however, ABR waves are functionally restored and Arc is mobilized, tinnitus does not occur. Both central response patterns were found to be independent of a profound threshold loss and could be shifted by the corticosterone level at the time of trauma. We, therefore, discuss the findings in the context of a history of stress that can trigger either an adaptive or nonadaptive brain response following injury.

Mutations in the DFNB31 gene encoding the PDZ scaffold protein whirlin are causative for hearing loss in man and mouse. Whirlin is known to be essential for the elongation process of the stereocilia of sensory hair cells in the inner ear, though its complete spatial and temporal expression patterns remained elusive. Here, we demonstrate that, in embryonic development, the gene is not only expressed in the inner ear, but also in the developing brain and the retina. Various isoforms of whirlin are widely and differentially expressed, and we provide evidence that whirlin directly associates with USH2A isoform b and VLGR1b, two proteins that we previously reported to be part of the Usher protein interactome. These proteins co-localize with whirlin at the synaptic regions of both photoreceptor cells and outer hair cells in the cochlea. These findings indicate that whirlin is part of a macromolecular PDZ protein scaffold that functions in the organization of the pre- and/or postsynaptic side of photoreceptor and hair cell synapses. Whirlin might be involved in synaptic adhesion through interaction with USH2A and VLGR1b as well as in synaptic development as suggested by its spatial and temporal expression patterns. In addition, we demonstrate that whirlin, USH2A and Vlgr1b co-localize at the connecting cilium and the outer limiting membrane of photoreceptor cells and in spiral ganglion neurons of the inner ear. Our data show that whirlin is connected to the dynamic Usher protein interactome and indicate that whirlin has a pleiotropic function in both the retina and the inner ear.

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. USH is clinically and genetically heterogeneous with at least 11 chromosomal loci assigned to the three USH types (USH1A-G, USH2A-C, USH3A). Although the different USH types exhibit almost the same phenotype in human, the identified USH genes encode for proteins which belong to very different protein classes and families. We and others recently reported that the scaffold protein harmonin (USH1C-gene product) integrates all identified USH1 molecules in a USH1-protein network. Here, we investigated the relationship between the USH2 molecules and this USH1-protein network. We show a molecular interaction between the scaffold protein harmonin (USH1C) and the USH2A protein, VLGR1 (USH2C) and the candidate for USH2B, NBC3. We pinpoint these interactions to interactions between the PDZ1 domain of harmonin and the PDZ-binding motifs at the C-termini of the USH2 proteins and NBC3. We demonstrate that USH2A, VLGR1 and NBC3 are co-expressed with the USH1-protein harmonin in the synaptic terminals of both retinal photoreceptors and inner ear hair cells. In hair cells, these USH proteins are also localized in the signal uptaking stereocilia. Our data indicate that the USH2 proteins and NBC3 are further partners in the supramolecular USH-protein network in the retina and inner ear which shed new light on the function of USH2 proteins and the entire USH-protein network. These findings provide first evidence for a molecular linkage between the pathophysiology in USH1 and USH2. The organization of USH molecules in a mutual 'interactome' related to the disease can explain the common phenotype in USH.

The large conductance voltage-and Ca 2؉ -activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming ␣-subunit coexpressed with the auxiliary ␤1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel ␣-(BK␣ ؊/؊ ) and ␤1-subunit (BK␤1 ؊/؊ ) knockout mice, we demonstrate normal hearing function and cochlear structure of BK␤1 ؊/؊ mice. During the first 4 postnatal weeks also, BK␣ ؊/؊ mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BK␣ ؊/؊ mice only from Ϸ8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.cochlea ͉ KCNQ4 C a 2ϩ -activated potassium (BK) channels are heterooctamers of four ␣-and four ␤-subunits. The pore-forming ␣-subunit (KCNMA1) is a member of the slo family of potassium channels (1), originally identified in Drosophila (2). Studies of BK channels from smooth muscle have identified an auxiliary ␤1-subunit (KCNMB1) whose presence in the channel complex confers an increased voltage and calcium sensitivity toward the poreforming ␣-subunit (3).In turtle and chick, there is evidence that differential splicing of the BK channel ␣-subunit in conjunction with a graded expression of the auxiliary ␤-subunit along the tonotopic axis provides the functional heterogeneity of BK channels that underlies electrical tuning (for review, see ref. 4).In inner hair cells (IHCs) of the mammalian organ of Corti, the predominant K ϩ conductance is a voltage-and Ca 2ϩ -activated K ϩ channel termed I K,f (5, 6). BK channel mRNA (7,8) and protein expression (8) were shown in IHCs, indicating that I K,f flows through BK channels. The presumed physiological roles of BK channels are (i) a decrease of the membrane time constant even at the resting potential and (ii) fast repolarization of the receptor potential. Both contribute to phase-locked receptor potentials up to high sound frequencies (6). In addition to IHCs, BK type Ca 2ϩ -activated K ϩ conductances have been measured in OHCs (9) and in efferent fibers onto outer hair cells (OHCs) (10). The role of BKs in either OHCs or efferents is still controversially discussed (9).Studying the expression of BK channel ␣-splice variants and ␤-isoforms in rat cochlea using in situ hybridization and PCR techniques revealed the strict coexpressio...

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.

Thyroid hormone deficiency before the onset of hearing causes irreversible damage to peripheral and central auditory systems. J. Neurophysiol. 83: 3101-3112, 2000. Both a genetic or acquired neonatal thyroid hormone (TH) deficiency may result in a profound mental disability that is often accompanied by deafness. The existence of various TH-sensitive periods during inner ear development and general success of delayed, corrective TH treatment was investigated by treating pregnant and lactating rats with the goitrogen methimazole (MMI). We observed that for the establishment of normal hearing ability, maternal TH, before fetal thyroid gland function on estrus days 17-18, is obviously not required. Within a crucial time between the onset of fetal thyroid gland function and the onset of hearing at postnatal day 12 (P12), any postponement in the rise of TH-plasma levels, as can be brought about by treating lactating mothers with MMI, leads to permanent hearing defects of the adult offspring. The severity of hearing defects that were measured in 3-to 9-mo-old offspring could be increased with each additional day of TH deficiency during this critical period. Unexpectedly, the active cochlear process, assayed by distortion product otoacoustic emissions (DPOAE) measurements, and speed of auditory brain stem responses, which both until now were not thought to be controlled by TH, proved to be TH-dependent processes that were damaged by a delay of TH supply within this critical time. In contrast, no significant differences in the gross morphology and innervation of the organ of Corti or myelin gene expression in the auditory system, detected as myelin basic protein (MBP) and proteolipid protein (PLP) mRNA using Northern blot approach, were observed when TH supply was delayed for few days. These classical TH-dependent processes, however, were damaged when TH supply was delayed for several weeks. These surprising results may suggest the existence of different TH-dependent processes in the auditory system: those that respond to corrective TH supply (e.g., innervation and morphogenesis of the organ of Corti) and those that do not, but require T3 activity during a very tight time window (e.g., active cochlear process, central processes).

CorrectionsBIOCHEMISTRY. For the article ''Differential effects of a centrally acting fatty acid synthase inhibitor in lean and obese mice,'' by Monica V. Kumar, Teruhiko Shimokawa, Tim R. Nagy, and M. Daniel Lane, which appeared in number 4, February 19, 2002, of Proc. Natl. Acad. Sci. USA (99, 1921-1925, the authors note the following. ''Under a licensing agreement between FASgen, Inc., and The Johns Hopkins University, Dr. Lane is entitled to a share of royalty received by the University on sales of products that embody the technology described in this article. (98, 10687-10691; First Published August 28, 2001; 10.1073͞pnas.181354398), the authors note the following. In the Introduction and Discussion of our paper, we failed to reference a recent article by Rousseau et al.(1), which demonstrated that single point mutations can significantly perturb the equilibrium between monomeric and domain-swapped dimeric p13suc1. Rational methods were used to redesign p13suc1 from a fully monomeric protein (dissociation constant of Ϸ900 mM) to a fully dimeric protein (dissociation constant of Ϸ100 nM). Fig. 3 appeared incorrectly. The correct version of the figure and its legend appear below.