J Neurophysiol 113: 3531–3542, 2015. First published March 25, 2015; doi:10.1152/jn.00869.2014. Activity-dependent regulation of prestin expression in mouse outer hair cells Yohan Song, Anping Xia, Hee Yoon Lee, Rosalie Wang, Anthony J. Ricci, and John S. Oghalai Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California Submitted 4 November 2014; accepted in final form 19 March 2015 Song Y, Xia A, Lee HY, Wang R, Ricci AJ, Oghalai JS. patch-clamp recording conditions (Kakehata and Santos-Sac- Activity-dependent regulation of prestin expression in mouse outer chi, 1995 and 1996; Santos-Sacchi 1991; Santos-Sacchi et al. hair cells. J Neurophysiol 113: 3531–3542, 2015. First published 1998a). Thus, measuring the NLC is a common way of assess- March 25, 2015; doi:10.1152/jn.00869.2014.—Prestin is a membrane protein necessary for outer hair cell (OHC) electromotility and normal ing the amount of functional prestin within an OHC (Abe et al. 2007; Oliver and Fakler 1999;). hearing. Its regulatory mechanisms are unknown. Several mouse Downloaded from models of hearing loss demonstrate increased prestin, inspiring us to There are many ways to modulate the voltage dependence of investigate how hearing loss might feedback onto OHCs. To test force production by prestin protein within the plasma mem- whether centrally mediated feedback regulates prestin, we developed brane, such as changing the surrounding lipid environment, a novel model of inner hair cell loss. Injection of diphtheria toxin (DT) intracellular Ca2ϩ level, membrane tension, membrane poten- into adult CBA mice produced significant loss of inner hair cells tial, and ionic composition of the intracellular and extracellular without affecting OHCs. Thus, DT-injected mice were deaf because they had no afferent auditory input despite OHCs continuing to compartments (Kakehata and Santos-Sacchi 1995; Keller et al. receive normal auditory mechanical stimulation and having normal 2014; Rajagopalan et al. 2007; Ratnanather et al. 1996; Santos- http://jn.physiology.org/ function. Patch-clamp experiments demonstrated no change in OHC Sacchi et al., 1998b and 2001; Wu and Santos-Sacchi 1998). prestin, indicating that loss of information transfer centrally did not However, the basic mechanisms of how prestin levels within alter prestin expression. To test whether local mechanical feedback the plasma membrane are regulated remain unclear. We know C1509G regulates prestin, we used Tecta mice, where the tectorial that prestin has an extremely high density within the lateral membrane is malformed and only some OHCs are stimulated. OHCs wall plasma membrane. It has a charge density of ϳ6,000– connected to the tectorial membrane had normal prestin levels, Ϫ ␮ 2 Ϫ whereas OHCs not connected to the tectorial membrane had elevated 10,000 e / m (where e is the electron charge) (Belyantseva prestin levels, supporting an activity-dependent model. To test et al. 2000; Mahendrasingam et al. 2010), and an estimated whether the endocochlear potential was necessary for prestin regula- 60–75% of the lateral wall surface area is composed of by 10.220.33.4 on September 23, 2016 tion, we studied TectaC1509G mice at different developmental ages. intramembranous particles, thought to be composed of or OHCs not connected to the tectorial membrane had lower than normal include prestin (Forge 1991; Kumano et al. 2010; Wang et al. prestin levels before the onset of the endocochlear potential and 2010). In addition, prestinϪ/Ϫ OHCs transfected with prestin higher than normal prestin levels after the onset of the endocochlear driven by the constitutively active cytomegalovirus promoter potential. Taken together, these data indicate that OHC prestin levels have a similar prestin density to untransfected wild-type OHCs are regulated through local feedback that requires mechanoelectrical (Xia et al. 2008). These data suggest that prestin production is transduction currents. This adaptation may serve to compensate for variations in the local mechanical environment. not limited at the level of transcription but argue instead that mechanical considerations physically limit the amount of pres- cochlea; hair cell; hearing; prestin; electromotility; mechanics tin within the plasma membrane. However, there is growing evidence that prestin levels can increase in states of hearing loss. For example, prestin mRNA SOUND-INDUCED VIBRATIONS of the organ of Corti deflect the expression is increased after salicylate administration (Yu et al. sensory hair cell stereociliary bundle, creating a receptor po- 2008), after noise exposure (Chen 2006; Mazurek et al. 2007; tential within the soma. In response, outer hair cells (OHCs) Xia et al. 2013), and in the presence of a malformed tectorial generate force, a process termed electromotility (Brownell et membrane (Liu et al. 2011; Xia et al. 2010). These observa- al. 1985). These forces amplify the cochlear traveling wave and tions suggest the possibility that hearing loss stimulates OHCs increase organ of Corti vibration, producing the exquisite to upregulate prestin, perhaps in an effort to compensate for auditory sensitivity and frequency selectivity characteristic of decreased hearing sensitivity. However, all of these previously normal mammalian hearing. Prestin (SLC26A5), a tetrameric published reports created hearing loss in animals by affecting protein present within the lateral wall plasma membrane of OHCs in some way. Thus, it remains unclear whether higher OHCs, is necessary for electromotility (Cheatham et al. 2004; brain centers recognize a state of hearing loss and provide Dallos et al. 2008; Hallworth and Nichols 2012; Liberman et efferent feedback to residual OHCs via the medial olivoco- al. 2002; Wang et al. 2010). Prestin is often modeled as having chlear bundle (Fig. 1, A and B). Alternatively, signaling could two states so that voltage-dependent stochastic transitions fit a occur locally within the epithelium to regulate prestin levels Boltzmann function and produce electromotility (Dallos et al. through a direct reduction in the OHC stereociliary bundle 1993; Iwasa 1994). These transitions are associated with stimulation and/or reciprocal synapses that have been found in charge movement across the membrane that can be measured type II nerve fibers that innervate multiple OHCs within a row as a voltage-dependent non-linear capacitance (NLC) under (Francis and Nadol Jr. 1993; Spoendlin 1969). In the present Address for reprint requests and other correspondence: J. S. Oghalai, Dept. study, we sought to clearly differentiate between efferent of Otolaryngology-Head and Neck Surgery, Stanford Univ., 300 Pasteur Drive, feedback and local feedback as regulators of prestin level using Edwards R113, Stanford, CA 94305-5739 (e-mail: [email protected]). two different mouse models that allowed us to isolate the www.jn.org 0022-3077/15 Copyright © 2015 the American Physiological Society 3531 3532 ACTIVITY-DEPENDENT REGULATION OF PRESTIN Downloaded from http://jn.physiology.org/ by 10.220.33.4 on September 23, 2016 Fig. 1. Experimental concept and mouse models. A: illustration of a normal organ of Corti. The tectorial membrane (TM), inner hair cell (IHC), outer hair cell (OHC) rows (1, 2, and 3), basilar membrane (BM), afferent auditory nerves (blue arrow), medial olivocochlear efferent nerves (green arrow) are shown. The TM stimulates OHC stereociliary bundles in all three rows of OHCs in control mice. B: potential mechanisms of prestin regulation. OHCs amplify the vibration of the organ of Corti in synchrony with its stereociliary bundle stimulation. This drives IHC stimulation and an afferent auditory response. Higher brain centers could then modulate efferent neural activity, which effects a change in OHC prestin levels. We broke this feedback loop using mice with IHC loss. Alternatively, OHC bundle stimulation could directly regulate prestin within individual OHCs or along a row via reciprocal synapses in type II nerve fibers. We selectively blocked OHC bundle stimulation using TectaC1509G mice. C: mice injected with diphtheria toxin (DT inj) demonstrated severe IHC loss and thus lacked an afferent neural response. OHC stimulation was unaffected. D: TectaC1509G mice had altered TM anatomy. The TM only stimulates first-row OHCs in TectaC1509G/ϩ mice (top). None of the OHCs are stimulated in TectaC1509/C1509 mice (bottom). source of the feedback. Our findings indicate that prestin is F2 mice were crossed with wild-type CBA mice for three generations regulated locally within the epithelium in an activity-dependent to reduce the 129/Sv/Ev background to 12.5%. All mice studied were manner. F6–F7 generation littermates in this background. Auditory brain stem responses and distortion product otoacoustic emissions. Mice were anesthetized using ketamine (100 mg/kg) and METHODS xylazine (10 mg/kg), and supplemental doses were given until paw areflexia was achieved. Auditory brain stem responses (ABRs) and The Institutional Animal Care and Use Committee of Stanford distortion product otoacoustic emissions (DPOAEs) were measured as University approved the study protocol. previously described (Cho et al. 2013; Oghalai 2004a; Xia et al. Diphteria toxin mouse model. This mouse model has not been 2007). Briefly, the ABR signal was measured with a bioamplifier previously published. We used 5-wk-old wild-type CBA/CaJ mice (DP-311, Warner Instruments, Hamden, CT) from needle elec- (stock no. 000654, The Jackson Laboratory, Bar Harbor, ME). These trodes placed at the ventral surface of the tympanic bulla and the mice have stable auditory thresholds from postnatal day (P)12 to P21 skull vertex with a ground electrode placed in the hindleg. The (Ohlemiller et al. 2010). Intraperitoneal injections of diphteria toxin sound stimulus frequency ranged from 4 to 70 kHz. At each (DT) (no. 15042B1, List Biological Laboratories, San Jose, CA), each frequency, the sound intensity was raised in 10-dB steps from 10 at a dosage of 50 ng/g, were administered for 3 days in a row. to 80 dB sound pressure level (SPL). After 260 repetitions were Uninjected mice of the same age were used as controls. averaged, ABR responses for each frequency were interpolated, TectaC1509G mouse model.