Research reportMouse outer hair cells lacking the α9 ACh receptor are motile
Introduction
In the mammalian hearing organ, the organ of Corti, sensory receptor cells have fully differentiated into two separate populations. One row of inner hair cells (IHCs) resides on the modiolar side of the tunnel of Corti; three rows of outer hair cells (OHCs) on the lateral side. Although IHCs receive 90–95% of the afferent innervation [37], [49], OHCs are innervated predominantly by efferents that originate in the brainstem [50], [56]. In general terms, the efferent system innervating the organ of Corti arises from two main pools of neurons. Their relationship to the medial superior olive, either lateral or medial, has given rise to their nomenclature, i.e., the lateral and medial efferent systems. The medial efferent fibers form chemical synapses at the bases of OHCs, with acetylcholine (ACh) as their primary neurotransmitter [1], [29], [45]. In contrast, lateral efferents form axo-dendritic synapses with afferent fibers beneath IHCs [56].
Acetylcholine receptors on OHCs have an unusual pharmacology exhibiting characteristics of both nicotinic- and muscarinic-receptor types (for reviews, see Refs. [3], [13], [15], [22]). Recent evidence suggests that the α9/α10 nicotinic receptor complex may underlie this unusual pharmacology and mediate efferent action on OHCs [9], [10]. Activation of ACh receptors on isolated OHCs by ACh application induces rapid calcium influx through ionotropic receptors, followed by a large outward K+ current via nearby Ca2+-activated K+ channels [16], [17], [28]. This outward K+ current, associated with “SK”-type potassium channels [58], hyperpolarizes the cell. It is also known that electrical stimulation of medial olivocochlear fibers diminishes cochlear sensitivity [2], [18], [57]. This action by ACh is thought to reflect a modulation of the contribution by OHCs to active cochlear mechanics [4], [20], [38], [47].
One way to investigate the possible role of efferent innervation on hair cell development and function is to use a gene-targeting strategy to generate various knockout animals that either lack hair cell innervation or neurotransmitter receptors on outer hair cell bodies. In this study, we measured OHC motility in vitro, as well as compound action potentials in vivo, from a mouse strain homozygous for a null mutation of the α9 gene [54]. The motility measurements were used to determine whether OHC motor function develops normally in α9 knockout mice; the CAP measurements to determine if cochlear sensitivity is affected. Given that Vetter et al. [54] stated that CAP thresholds and distortion-product otoacoustic emissions were normal in mice lacking α9, it would be assumed that OHC motility would also be normal. Nonetheless, this assumption requires experimental verification. In addition, the Vetter et al. study only showed CAP thresholds at 16 kHz. Hence, it is useful to provide these results over the entire range of mouse hearing. Finally, at least one study [55] has suggested that loss of efferent nerve fibers leads to abnormal development of OHCs and to reductions in sensitivity and frequency selectivity.
Section snippets
Materials and methods
Because several mouse strains develop age-related hearing loss at high frequencies [60], all experiments were performed on mice between 4 and 9 weeks of age. Data were collected from homozygous mutants (−/−) and from wild-type (+/+) controls. These animals were obtained from designated knockout and wild-type breedings undertaken at the Salk Institute. In order to increase the number of controls, additional animals of the 129S6/SvEv genetic background were acquired from a commercial source
CAP measurements
The recording of synchronized,discharges from the auditory nerve [19], [41], [52] reveals aspects of neural function. Hence, compound action potential (CAP) thresholds were used to measure frequency specific activity, thereby providing a measure of the “audiogram” [7], [30]. These results are shown in Fig. 1 where CAP threshold curves for two mice lacking the α9 ACh receptor are plotted with open symbols and solid lines. For comparison, mean CAP threshold curves for wild-type 129S6/SvEvTac mice
Discussion
It has been suggested that during development neurotransmitters can serve as regulatory signals prior to their conventional role in neurotransmission (for review see Ref. [32]. It is, therefore, possible that efferent fibers exert some influence on the functional maturation of OHCs through the release of ACh and its mediation by the α9 receptor subunit. By measuring the motility of OHCs isolated from a mouse strain homozygous for a null mutation of the α9 gene, we demonstrated that OHCs were
Conclusion
The data reported here suggest that efferent regulation, mediated by α9, is not necessary for the development of OHC motor function or for the maturation of cochlear sensitivity. Because the CAP measurements provided here were obtained between 1.7 and 50 kHz, this conclusion applies to cochlear responses recorded from most of the hearing range in this species. If ACh is regulating these aspects of maturation, then it is doing so through receptors other than those in which α9 plays a dominant
Acknowledgements
Work supported by grants DC00089 and DC00708 to P. Dallos, DC02764 to L. Madison, DC02871 to S.F. Heinemann, and DC 04696 to Z.Z. He, all from the NIDCD. The Adler Foundation also provided funds to S.F. Heinemann.
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