Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion cells and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning. We found that action potentials are intrinsically generated by immature IHCs of altricial rodents and that apical IHCs exhibit bursting activity as opposed to more sustained firing in basal cells. We show that the efferent neurotransmitter ACh, by fine-tuning the IHC’s resting membrane potential (Vm), is crucial for the bursting pattern in apical cells. Endogenous extracellular ATP also contributes to the Vm of apical and basal IHCs by activating SK2 channels. We hypothesize that the difference in firing pattern along the cochlea instructs the tonotopic differentiation of IHCs and auditory pathway.
In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both ␣9 and ␣10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88 -100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16 -P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10 -P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that ␣9 mRNA is expressed in adult IHCs but that ␣10 mRNA disappears after the onset of hearing. In the present study, antibodies to ␣10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of ␣10, but not ␣9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.
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.
The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (⌬C m ) in low-frequency (apical region, ϳ300 Hz) and high-frequency (basal, ϳ30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mM extracellular Ca 2ϩ and body temperature). With maturation, the Ca 2ϩ efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca 2ϩ cooperativity of exocytosis despite the smaller ⌬C m in apical cells. After maturation, ⌬C m in high-frequency IHCs increased linearly with the Ca 2ϩ current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca 2ϩ sensor otoferlin or the spatial coupling between Ca 2ϩ channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.
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.
Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.
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.
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