What Is Electromotility? - The History of Its Discovery and Its Relevance to Acoustics

William E. Brownell Experiments on an sensory cell revealed that it converts electri- Postal: cal energy directly into mechanical energy at acoustic frequencies. Department of Otolaryngology- Head and Neck Introduction Baylor College of Thirty-five years ago, the members of the Physiological and Psychological Acous- One Baylor Plaza tics Technical Committee of the Acoustical Society of America were at a pivotal Houston, Texas 77030 juncture in understanding . The original description of the ability of the USA ears to produce sound had been published in The Journal of the Acoustical Soci- ety of America a few years earlier. Remarkable differences in neural circuitry and Email: structural specializations in the hearing organ (Figures 1-4) were being described. [email protected] It was a propitious time to take a close look at the outer hair cell (OHC), which was then the most structurally and functionally mysterious cell in the ear. We dis- covered that OHCs undergo rapid changes of cell shape in response to electrical stimulation, something we now call OHC electromotility (http://acousticstoday. org/OHCEM1).1 OHC electromotility revolutionized the hearing sciences by re- vealing the active process responsible for the sounds coming from the ear. The history behind the discovery, the subsequent biophysical investigations, and the role of electromotility in hearing are the topics of this narrative.

Origins: Mammalian High-Frequency Hearing and the Outer Hair Cell The ability to detect and analyze high-frequency sounds that other animals could not hear is likely to have provided early mammals a strong survival advantage (Allman, 1999). The mammalian (Figures 1 and 2) houses a mechanosen- sitive sensory hair cell that is structurally and functionally different from the sen- sory hair cells found in other vertebrates. The defining characteristic of the newly evolved OHCs is their electromotility. The role of OHC electromotility is linked to the nature of sound that Pythagoras and his fellow Greeks, inspired by stringed musical instruments, reasoned was a vibration. They introduced the concept of the octave that eventually led to quantification and a physical understanding of tone. The study of acoustics progressed from these early beginnings, but exploration on the biological basis of hearing lagged. Two millennia passed before the structural organization of the mammalian inner ear began to emerge. Its structure provided clues as to how the ear works, particularly after it was realized that the inner ear is fluid filled.

Finding the Organ of Corti: Hair Cells in a Fluid-Filled Cavity The inner ear is difficult to study because it is small and encased in bone. Gabri- ello Fallopio described the openings between the middle ear and the cochlea in a

1 This is the first video recording of outer hair cell electromotility. An explanation of the video and links to other videos are provided in the supplementary material at the end of the article.

20 | Acoustics Today | Spring 2017 | volume 13, issue 1 ©2017 Acoustical Society of America. All rights reserved. Figure 2. Pathway of sound to the cochlea in humans. The external ear collects sound and directs it to the eardrum. Sound vibrations are conveyed across the middle ear by three tiny middle ear bones that match the acoustic impedance of air with the impedance of the fluids that fill the inner ear. The three middle ear bones and the outer hair cells (OHCs) are found in mammals. The bones and the OHCs are both required for high-frequency hearing.

Figure 1. Location of the spiral-shaped cochlea in the human head. It is found in a cavity that forms deep in the skull during development, Kölliker had pioneered techniques to harden, section, and behind and below the level of the eyes. The cavity also contains the stain body tissue before looking at it through the micro- sensory organs of balance (vestibular organs) that have their own scope. Corti used these techniques in Kölliker’s laboratory sensory hair cells and communicate to the brain via a bundle of nerve to examine tissue he dissected from the inner ear of humans fibers that terminate in different parts of the brain than do the au- and other mammals (Corti, 1851). He described the sensory ditory nerve fibers that emerge from the cochlea. The left and right are mirror images of one another. They spiral around their epithelium in the cochlea, which was soon named after him 3 central axes in opposite directions. The direction of the spiral of the (the organ of Corti). His drawings depicted the sensory cells right cochlea is the same as for a right-handed screw while the spiral that became known as “hair cells” because each had a tuft of of the left cochlea is left-handed. The drawing shows the left ear. enlarged microvilli at one end (see Figure 4 for greater detail of the stereociliary tuft). More than a century later, the func- 1561 publication. Two centuries later, Domenico Cotugno, a tion of the tuft was identified, and it was demonstrated that Neapolitan surgeon and humanist, published an anatomical bending the bundle of stereocilia modulates the flow of ions dissertation (Cotugno 1775)2 based on his scientific obser- into the cell (Wersäll et al., 1965; Harris et al., 1970). This, in vations. He expanded on the findings of two 17th-century turn, regulates the release of neurotransmitters that activate inner ear anatomists, Antonio Maria Valsalva and Guichard nerve fibers making contact at synapses located at the op- Joseph Duverney, correcting speculation by the latter about posite end of the hair cell. where high and low frequencies were processed in the co- At the beginning of the 20th century, it had been established chlea. Cotugno found fluid in the inner ear of freshly har- that the organ of Corti contained two types of hair cells. vested skulls, in contrast to Fallopio and earlier anatomists They were named based on their location relative to the cen- who examined desiccated cadaver skulls and assumed that tral axis of the cochlear spiral. Inner hair cells (IHCs) make the inner ear was filled with air. The fluid environment is up a single spiraling row of mechanoreceptor cells located relevant to understanding the role of OHC electromotility in over the thin bony plate to which the axial margin of the bas- hearing. But first, a greater understanding about the cellular ilar membrane is attached (Figure 3). Three rows of OHCs organization of the inner ear and identification of its hair are located further away from the central axis. OHCs have a cells was needed. distinctly cylindrical shape and are not in intimate contact Improvements in microscopic methods during the 19th cen- with supporting cells for most of their length. The large fluid tury led to Alfonso Corti’s detailed description of the cel- spaces surrounding the lateral walls of the OHCs are bio- lular organization of the mammalian hearing organ. Albert logically unusual. Cells in most organs (heart, brain, kidney,

2 Available online at https://goo.gl/PtvBFW. 3 Book available online at https://goo.gl/WAIsnS.

Spring 2017 | Acoustics Today | 21 Outer Hair Cell Electromotility

Figure 3. A cross section of the organ of Corti showing inner hair cells (IHCs) and OHCs with their stereociliary bundles. An OHC ste- reociliary bundle is circled. The central axis of the cochlear spiral is to the left of the drawing. An IHC is located over the bony (osseus) spiral lamina and is tightly enveloped with supporting cells. Sound- Figure 4. The apical end of an OHC showing organization of the evoked vibrations will be reduced at this location relative to those oc- membranes and cytoskeletal structures. Left: a cylindrical OHC curring under the OHCs located nearer the middle of the compliant has been sliced parallel to its long axis along a plane defined by the basilar membrane. The mottled dark circle in the hair cells is the cell dashed line in the insert at the top right. Top right: a view of the nucleus. Note the large fluid spaces around the OHCs created by the OHC looking down on the stereocilia bundle that has a typical W absence of adjacent supporting cells that surround the IHCs. Audi- shape. Each small circle represents a single stereocilium, while the tory nerve fibers (eighth cranial nerve) contact hair cells at the end large circle represents a structure that disappears during develop- opposite their mechanosensory stereociliary bundle. More than 15 ment. Bottom right: high-power view of the lateral wall showing its fibers contact the base of each IHC while a few course laterally and three layers. The outermost layer is the plasma membrane and the each contacts more than 15 OHCs. The fibers innervating the IHCs innermost layer is made up of the subsurface cisterna. Sandwiched in come from type 1 spiral ganglion cells (SGC1), whereas those inner- between is the cortical lattice composed of circumferentially oriented vating the OHCs come from type 2 SGCs (SGC2). actin filaments and axially oriented spectrin (spectrin filaments are the thinner filaments). The plasma membrane and cortical lattice muscle) are within 10-30 nm of one another. OHCs, in con- form nanoscale motor elements that generate force at acoustic fre- trast, are separated from adjacent cells by as much as 1 µm. quencies, resulting in electromotility. The elegant colonnade appearance of the organ of Corti is due to the large and unique spacing between OHCs, but the In the late 1960s, Heinrich Hans Spoendlin (Figure 5) reason for the large extracellular spaces was puzzling. completed a laborious EM study of the auditory nerve fi- ber innervation pattern at the base of the IHCs and OHCs Structural Evidence for Differences (Spoendlin, 1966). The results provided the first compelling Between Inner and Outer Hair Cells evidence that the OHCs might be doing something entirely The invention of the electron microscope (EM) in the ear- different than the IHCs. He meticulously reconstructed the ly 20th century provided a powerful new tool to examine auditory nerve fiber innervation pattern at the base of both the fine structure of cells because it could observe objects the IHCs and OHCs by examining hundreds of ultrathin that were smaller than the wavelength of light. By midcen- serial-section electromicrographs per hair cell. Each audi- tury, EM investigations had revealed structural similari- tory nerve fiber that enters the organ of Corti arises from ties between all hair cells and revealed unique specializa- a single cell located in what is called the spiral ganglion tions in OHCs. One of these was the presence of flattened (located closer to the spiral axis of the cochlea). He found membrane-bound organelles near the OHC cell membrane that 90-95% of the fibers came from a population of spiral at the same location where the lateral wall was exposed to ganglion cells (SGCs) that he called type 1 SGCs (SGC1). the large extracellular spaces (Figure 3). The membranes of The remainder were smaller fibers arising from type 2 SGCs these “subsurface cisternae” (Figure 4) invariably appeared (SGC2). SGC1 fibers were connected exclusively to the IHCs crisp and flat, whereas the nearby OHC cell membrane was (about 15 fibers per IHC), whereas each SGC2 fiber passed diaphanous and rippled. radially near the basilar membrane, then turned basely and

22 | Acoustics Today | Spring 2017 proceeded more than 500 µm before making contact with a convenient summary of work in the field up to that point. up to 20 OHCs. Before Spoendlin’s observations, both IHCs The tuning of the fibers was consistent across the entire and OHCs were assumed to carry out similar roles of mech- sample population. There were no surprising, broadly tuned anotransduction. The innervation pattern led to the sugges- fibers that may have originated from Spoendlin’s SGC2s. It tion, however, that most, if not all, hearing information was was also noted that the auditory nerve fiber response was coming from the IHCs. The big question raised by this dis- more highly tuned than the traveling wave patterns that von covery was what were the Békésy had described. The neural tuning was closer to what OHCs doing? was needed to explain the frequency selectivity of mamma- lian hearing. Passive Hearing Is Not Consistent Active Hearing: with Auditory Early Speculations and Evidence Nerve Fiber Tuning An active process in the cochlea had been hypothesized by Investigations on cochle- Thomas Gold (1948), a Viennese-born English polymath ar mechanics during the (Figure 6). He realized that viscous damping in the fluid late 19th and first half of environment of the cochlea was incompatible with the ex- the 20th century by scien- quisite frequency-resolving powers of human hearing. He tists such as Hermann von postulated that an active, essentially piezoelectric mecha- Helmholtz and Georg von nism that converted elec- Békésy led to a widely ac- trical to mechanical energy cepted model for how the could provide feedback to Figure 5. Photograph of the Swiss vibrations of sound were counteract viscous damp- -scientist and head of analyzed by the cochlea. ing from cochlear fluids. otolaryngology at the University of Innsbruck Heinrich Hans Spo- von Békésy used a light mi- His hypothesis was largely endlin (1927-1991). Spoendlin croscope and stroboscopic ignored, in part, because of provided the first structural evi- illumination to observe opposition by von Békésy. dence that most, if not all, neu- acoustically driven pat- Evidence that the cochlea ral acoustic information from the terns of movement in the was active began to mount ear came via IHCs. His discovery basilar membrane of cadav- set the stage for accepting OHCs in the 1970s. Brian John- ers (von Békésy, 1960). He as the cochlear amplifier. Photo- stone in Perth, WA, Aus- confirmed the tonotopic or- graph provided by Professor Jo- tralia, and Bill Rhode in ganization of high- to low- seph B. Nadol. Madison, WI, pioneered frequency vibrations going Figure 6. Photograph of Thomas the use of the Mössbauer from the base to the apex of the cochlea. He also described Gold (1920-2004) who, over a effect to measure the move- the traveling wave that resulted from hydraulic coupling. long active career, studied bio- ment of a gamma radia- von Békésy was awarded the Nobel Prize for his work and physics, astronomy, aerospace en- tion source placed on the helped establish a view of the cochlea as an elegant, but es- gineering, and geophysics. Gold was born in and moved basilar membrane in living sentially passive, device for converting the mechanical en- with his family to England where animals (Johnstone and ergy of sound into electrical signals to the brain. he studied at Cambridge. He con- Boyle, 1967; Rhode, 1971). Neurophysiological investigations of sensory coding by tributed to the British war effort By the mid-1970s, they had by working on improvements to the auditory nerve began with the advent of vacuum tube established that, in the best radar. While working with R. J. amplifiers and kymograph recordings. The introduction of Pumphrey after the war, Gold preparations, mechanical oscilloscopes and digital recording techniques facilitated concluded that an electrome- tuning approached that neurophysiological research. A research monograph on the chanically active mechanism was of single auditory nerve response characteristics of single auditory nerve fibers was necessary to counteract viscous fibers. When the animal published by Nelson Kiang and colleagues in the mid-sixties damping by cochlear fluids. Pho- died, the motion rapidly tograph provided by Professor (Kiang et al., 1965). It was a compendium of sensory coding deteriorated to the passive David Kemp. in the 8th nerve using well-controlled stimuli and provided tuning that von Békésy had Spring 2017 | Acoustics Today | 23 Outer Hair Cell Electromotility

measured in cadaver ears. Shyam Khanna in New York City microscope. Daniel was poised over the electronics looking soon confirmed these observations using laser interferom- for the tell-tale voltage shift that would indicate we were in- etry (Khanna and Leonard, 1982). side the cell. I was responsible for dissecting the cochlea and isolating the cells, after which I would sit behind the others The most direct demonstration of a mechanism in the co- and record the results in the lab notebook. chlea that could generate mechanical energy was the dis- covery of otoacoustic emissions by David Kemp in London A common technique at the time was to “buzz” the electrode late in the 1970s. He initially reported the presence of sound when its tip was positioned on the cell membrane. The buzz coming from the cochlea, now called otoacoustic emissions, was achieved by throwing the amplifier headstage into elec- after stimulation with a brief sound (Kemp, 1978). He then trical oscillation. For reasons that are still not clear, the buzz showed that some ears produce sound without being stimu- would push the tip of the electrode through the membrane. lated (for historical details, see Kemp, 2008). This was direct The pivotal moment occurred when Daniel buzzed and I evidence of Gold’s active process. Measuring otoacoustic could see Charlie’s back and shoulders jerk in a classic star- emissions has become an important tool for assessing hear- tle response to what he had seen through the microscope. ing loss, even in newborns and unresponsive patients. Steve Still looking though the microscope, Charlie called for an- Neely, Duck Kim, Egbert de Boer, David Mountain, and oth- other buzz, and his body responded with another somewhat ers began to include a source of mechanical energy in their smaller response. At that point, Charlie turned around and theoretical models of cochlear mechanics. In 1983, Hallowell said “We have a problem.” Daniel and I then took turns look- Davis, a senior hearing scientist at the Central Institute for ing at OHC electromotility as Charlie buzzed. Proving that the Deaf in St. Louis, MO, coined the term “cochlear ampli- the electrically evoked length change was not an artifact was fier” for the active process. The cellular basis of the cochlear the “problem” to which Charlie was referring. amplifier was a puzzle that was soon resolved. The remainder of my sabbatical was given over to OHC electromotility. We eliminated potential artifacts and fur- The First Observation of ther characterized the phenomenon. It was only after I es- Outer Hair Cell Electromotility tablished that hyperpolarization caused elongation and de- The discovery of OHC electromotility took place late in polarization caused shortening was I confident that OHC the morning on December 3, 1982, in the medical school electromotility was not an artifact. For several months, we laboratories of Daniel Bertrand and Charles Bader at the had no equipment for recording the conspicuous move- University of Geneva where I was taking a sabbatical from ments and visiting scientists were asked to sign our lab book the University of Florida. Charlie and Daniel had pioneered acknowledging that they saw the movements. I eventu- techniques to dissect, isolate, and record photoreceptors to ally captured the movements using video microscopy (see characterize ionic movement across their membranes. We http://acousticstoday.org/OHCEM1), eliminating the need had met at a photoreceptor meeting in Sicily a year earlier. for affidavits, and we published our findings (Brownell 1983; I attended the meeting because of the many structural and Brownell et al., 1985). functional similarities between vertebrate photoreceptors and hair cells. I had described these similarities and pro- The OHC was quickly identified as Gold’s piezoelectric-like posed isolated hair cell studies in a review paper that was energy source. The modelers now had a cellular locus for the published the same year as the discovery (Brownell, 1982). cochlear amplifier. The fact that OHC axial length changes Several months were required to optimize the primary cell would lose energy if they contacted adjacent cells explained culture procedures for hair cells. OHCs proved easier to iso- the large extracellular spaces around the OHCs. I had specu- late, most likely because of the absence of supporting cells lated about an electromechanical mechanism in my review on their lateral walls. We began voltage-clamp attempts in paper (Brownell, 1982) that was based on the synaptic activ- October. The homemade optics, amplifiers, and data-collec- ity in the base of the OHC. Looking through the microscope tion programs required a team effort to pull off the experi- and later examining the videotapes revealed that the active ments. We were using micropipettes to make intracellular process was in the lateral wall, thereby suggesting a function recordings (whole cell patch-clamp techniques were just be- for the structurally unique organization of the lateral wall ing developed). The standard routine was for Charlie to ad- (Figure 4). Other work going on at the same time identified vance the electrode while looking at it and the cell through a the battery that powers the process.

24 | Acoustics Today | Spring 2017 What Powers Outer Hair Cell Electromotility? - The Silent Current My laboratory in Gainesville, FL, had completed an in vivo current-density analysis of the cochlea before I left for Ge- neva. Paul Manis, Michael Zidanic, George Spirou, and I measured evoked currents in the fluid spaces of the cochlea (Brownell et al., 1983). We adapted techniques and analytic tools that Paul had developed for his study of neural orga- nization in the auditory brainstem (Manis and Brownell, 1983). Our examination of the inner ear revealed that acous- tic stimuli that moved the organ of Corti downward toward Figure 7. Cross section of the cochlea showing the flow of ions making the scala tympani (Figure 7) resulted in decreased ion flow, up the silent current. There are three fluid-filled chambers. The scala whereas upward movements toward the scala media resulted vestibuli and scala tympani contain a conventional extracellular flu- id called , whereas the scala media contains endolymph in an increased ion flow. The modulation in ion flow through that is high in potassium and low in sodium. The stria vascularis an OHC in either direction was about 500 pA and repre- maintains an endolymphatic potential and electrochemical gradient sents one of the largest known cell currents. It had previ- that drives the silent current (arrows). The metabolically active stria ously been proposed that the electroanatomy of the cochlea vascularis pumps potassium into the middle cochlear compartment. could result in a steady current across the organ of Corti The potassium passes through the hair cells into the scala tympani that we called the “silent current,” in analogy to a current in and then back to the stria vascularis. There is also a passive route for potassium through the cells, forming the boundary between the the retina called the dark current. We interpreted our initial scala media and scala vestibuli before returning to the stria vascu- findings as modulation of the silent current resulting from laris through the perilymph. The silent current is the power source for deflection of the OHC stereocilia. Because our recordings OHC electromotility. were AC coupled, we had not measured the actual steady- state silent current. Michael took on the challenge and sev- the cell membrane to show that electromotility was based eral years later reported his DC-coupled recordings (Zidanic on the voltage across and not the current through the mem- and Brownell, 1990) confirming that the silent current was brane (Santos-Sacchi and Dilger, 1988). This and the speed similar to what we had estimated in 1982. The silent current of the electromechanical transduction indicated that the en- is the power source for cochlear transduction (Figure 7). It ergy was produced by a membrane-based motor. is the source of a steady flow of ions through the OHC even in silence. The changes in membrane potential generated in The OHC is able to undergo rapid shape changes because it OHCs when the current is modulated drive electromotility. does not have the typical arrangement of rigid cytoskeletal proteins that maintains the shape in other types of cells. It Ultrasonic Force Generation, is, instead, a cellular hydrostat composed of an elastic outer Turgor Pressure, Cortical Lattice, shell enclosing a modestly pressurized core (approximately and Membrane-Based Motor 1-2 kPa). Most of the other cells in the body have no pres- Video was too slow to capture OHC length changes faster sure. The cytosolic turgor pressure and the tensile proper- than the 30 per second frame rate. A succession of tech- ties of the lateral wall together form a hydraulic skeleton niques revealed that OHC electromechanical force produc- that facilitates shape changes and hydraulic force transmis- tion occurred at rates consistent with hearing. Eventually, sion at high frequencies. OHC electromotility diminishes Tony Gummer’s lab in Tübingen, , developed a and vanishes if the cell becomes flaccid (Brownell 1983, way to measure isometric force production with a calibrat- 1990; Brownell et al., 1985; Santos-Sacchi, 1991; Shehata et ed probe while electrically stimulating and showed that the al., 1991). Most cells burst when their internal pressure is OHC generates force at frequencies approaching 100 kHz increased by even a small amount. The reinforcement pro- (Frank et al., 1999). Before that, Joe Santos-Sacchi at the vided by the lateral wall (Figure 4) and more specifically the Medical College of New Jersey used a variety of ion-channel cortical lattice (Oghalai et al., 1998) prevents this from hap- toxins and manipulated the electrochemical gradients across pening in OHCs.

Spring 2017 | Acoustics Today | 25 Outer Hair Cell Electromotility

Displacement Currents and Gold (1948) had proposed a piezoelectric-like energy source the Motor Gets a Protein that was necessary to compensate for viscous damping re- Passive OHC length changes resulting from mechanical sulting from the fluid environment that Cotugno (1775) had stretch or electrically evoked length changes produce a “dis- originally discovered. Spoendlin (1966) provided an impor- placement” current. The electrically evoked current is ca- tant clue by showing that the OHCs did something other pacitive in nature because it is out of phase with the stimu- than convey information to the brain. OHC electromotility lus voltage. This reactive (nonohmic) current is vulnerable proved to be the piezoelectric-like source of mechanical en- to manipulations such as aspirin or loss of turgor pressure ergy that acts as the cochlear amplifier to compensate for that modify electromotility (Santos-Sacchi, 1991; Shehata et viscous damping in the fluid-filled cochlea. The end result al., 1991). The magnitude of the displacement current varies is greater sensitivity and sharper tuning. Although we now with the membrane potential in a manner similar to the re- know with reasonable certainty how the OHC contributes lationship between length change and membrane potential. to hearing, we still don’t know the role of SGC2 fibers that The displacement current was found to require a membrane could, in principle, convey information to the brain directly protein. from the OHCs. And so, as is usually true in science, puzzles Peter Dallos and his colleagues found a protein important remain, and their resolution will bring even more puzzles. for electromotility at the the turn of the century (Zheng et Acknowledgments al., 2000). The protein was discovered using a differential The discovery of OHC electromotility was made possible expression assay that showed that a protein was strongly by sabbatical funding from the University of Florida and expressed in OHCs. When they introduced the protein into research funding from Hoffman LaRoche. My subsequent other cell types, the large displacement currents charac- investigations on the mechanisms involved and the writ- teristic of OHCs were also found. The investigators called ing of this narrative have been funded by the citizens of the the protein “prestin” from the Italian word “presto,” which United States in the form of Research Grants R01-DC-00354 indicates a rapid tempo in a muscial composition. Genetic and R01-DC-02775 from the National Institute on Deafness manipulations that removed the protein altogether resulted and Other Communication Disorders, National Institutes in severe hearing loss and decreased otoacoustic emisisons of Health. My wife Nancy and editor Arthur Popper made (Liberman et al., 2002). These observations demonstrated many contributions to the style and delivery of the story. that prestin plays an important role in the OHC electrome- chanical motor. The hearing science community was ecstatic Biosketch with the protein. The ability to insert prestin into the membranes of other cell Bill Brownell is a professor at the Bay- types and to manipulate its molecular structure provided lor College of Medicine, Houston, TX, further evidence for the strength of the membrane protein where he holds the Jake and Nina Ka- interactions that underlie the OHC electromechanical mo- min Chair of Otorhinolaryngology. Pre- tor mechanism (Rajagopalan et al., 2006, 2007; Zhang et viously held appointments were at the al., 2007). The precise role of the protein and its interaction University of Florida, Gainesville, FL, with the motor elements in the OHC lateral wall remain to and the Johns Hopkins School of Medi- be identified. Tremendous strides have been made in char- cine, Baltimore, MD. He spent two years (1965-1966) teach- acterizing the structural and molecular components that ing science and in Nigeria with the United make up the motor elements, and we are getting closer to States Peace Corps between his undergraduate training in a complete biophysical understanding of OHC electromotility. physics and graduate training in physiology at the Univer- New tools have been introduced that allow direct measurement sity of Chicago, Chicago, IL. He is a Fellow of the Acoustical of the length changes at acoustic frequencies in living animals Society of America and a past president of the Association (Gao et al., 2014; Ren et al., 2016). Analysis of these organ-level for Research in Otolaryngology. His main research focus is results will help clarify the cellular role of OHC electromotility. on the electromechanics of hearing.

26 | Acoustics Today | Spring 2017 References Pereira, F. A. (2006). Essential helix interactions in the anion transporter domain of prestin revealed by evolutionary trace analysis. Journal of Neu- Allman, J. M. (1999). Evolving Brains. Scientific American Library, New York. roscience 26, 12727-12734. Brownell, W. E. (1982). Cochlear transduction: An integrative model and Rajagopalan, L., Greeson, J. N., Xia, A., Liu, H., Sturm, A., Raphael, R. M., review. Hearing Research 6, 335-360. Davidson, A. L., Oghalai, J. S., Pereira, F. A., and Brownell, W. E. (2007). Brownell, W. E. (1983). Observation on a motile response in isolated hair Tuning of the outer hair cell motor by membrane cholesterol. Journal of cells. In Webster, W. R., and Aiken, L. M. (Eds.), Mechanisms of Hearing. Biological Chemistry 282, 36659-36670. Monash University Press, Melbourne, VIC, Australia, pp. 5-10. Ren, T., He, W., and Kemp, D. (2016). Reticular lamina and basilar mem- Brownell, W. E. (1990). Outer hair cell electromotility and otoacoustic emis- brane vibrations in living mouse cochleae. Proceedings of the National sions. Ear and Hearing 11, 82-92. Academy of Sciences of the United States of America 113, 9910-9915. Brownell, W. E., Manis, P. B., Zidanic, M., and Spirou, G. A. (1983). Acous- Rhode, W. S. (1971). Observations of the vibration of the basilar membrane tically evoked radial current densities in scala tympani. The Journal of the in squirrel monkeys using the Mossbauer technique. The Journal of the Acoustical Society of America 74, 792-800. Acoustical Society of America 49, 1218-1231. Brownell, W. E., Bader, C. R., Bertrand, D., and de Ribaupierre, Y. (1985). Santos-Sacchi, J. (1991). Reversible inhibition of voltage-dependent outer Evoked mechanical responses of isolated cochlear outer hair cells. Science hair cell motility and capacitance. Journal of Neuroscience 11, 3096- 227, 194-196. 3110. Corti, A. (1851). Recherches sur l'organe de l'ouïe des mammifères. Santos-Sacchi, J., and Dilger, J. P. (1988). Whole cell currents and mechani- Zeitschrift für wissenschaftlicke Zoologie3, 109-169. cal responses of isolated outer hair cells. Hearing Research 35, 143-150. Cotugno, D. (1775). De Aquaeductibus Auris Humanae Internae Anatomica Dis- Shehata, W. E., Brownell, W. E., and Dieler, R. (1991). Effects of salicylate sertation. Typographia Sanctae Tomae Aquinatis, Neopoli et Bononiae. on shape, electromotility and membrane characteristics of isolated outer Frank, G., Hemmert, W., and Gummer, A. W. (1999). Limiting dynamics hair cells from guinea pig cochlea. Acta Otolaryngology 111, 707-718. of high-frequency electromechanical transduction of outer hair cells. Pro- Spoendlin, H. (1966). The organization of the cochlear receptor. Fortschr ceedings of the National Academy of Sciences of the United States of America Hals Nasen Ohrenheilkd 13, 1-227. 96, 4420-4425. von Békésy, G. (1960). Experiments in Hearing (Translated by E. G. Wever). Gao, S. S., Wang, R., Raphael, P. D., Moayedi, Y., Groves, A. K., Zuo, J., Ap- McGraw-Hill Book Company, New York. plegate, B. E., and Oghalai, J. S. (2014). Vibration of the organ of Corti Wersäll, J., Flock, A., and Lundquist, P. G. (1965). Structural basis for direc- within the cochlear apex in mice. Journal of Neurophysiology, 112, 1192- tional sensitivity in cochlear and vestibular sensory receptors. Cold Spring 1204. Harbor Symposium on Quantitative Biology 30, 115-132. Gold, T. (1948). Hearing. II. The physical basis of the action of the cochlea. Zhang, R., Qian, F., Rajagopalan, L., Pereira, F. A., Brownell, W. E., and An- Proceedings of the Royal Society B Biological Sciences 135, 492-498. vari, B. (2007). Prestin modulates mechanics and electromechanical force Harris, G. G., Frishkopf, L. S., and Flock, A. (1970). Receptor potentials of the plasma membrane. Biophysical Journal 93, L07-L09. from hair cells of the lateral line. Science 167, 76-79. Zheng, J., Shen, W., He, D. Z., Long, K. B., Madison, L. D., and Dallos, P. Johnstone, B. M., and Boyle, A. J. (1967). Basilar membrane vibration ex- (2000). Prestin is the motor protein of cochlear outer hair cells. Nature amined with the Mossbauer technique. Science 158, 389-390. 405, 149-155. Kemp, D. T. (1978). Stimulated acoustic emissions from within the human audi- Zidanic, M., and Brownell, W. E. (1990). Fine structure of the intracochlear tory system. The Journal of the Acoustical Society of America 64, 1386-1391. potential field. I. The silent current.Biophysical Journal 57, 1253-1268. Kemp, D. T. (2008). Otoacoustic emissions: Concepts and origins. In Man- ley, G. A., Popper, A. N., and Fay, R. R. (Eds.), Active Processes and Oto- Supplementary Material acoustic Emissions. Springer-Verlag, New York, pp. 1-38. The first video recording of outer hair cell electromotility recorded on a Khanna, S. M., and Leonard, D. G. (1982). Basilar membrane tuning in the VCR in April 1983 is presented at http://acousticstoday.org/OHCEM1. A cat cochlea. Science 215, 305-306. glass pipette electrode has been positioned at the basal end of one of the Kiang, N. Y.-S., Watanabe, T., Thomas, E. C., and Clark, L. F. (1965).Dis - OHCs in a cluster of OHCs joined together at their apex. The pipette is the charge Patterns of Single Fibers in the Cat's Auditory Nerve. MIT Press, sharply conical object pointing up from the bottom of the field of view. Cambridge, MA. The audio indicates the presentation of an extracellular electrical pulse. Liberman, M. C., Gao, J., He, D. Z., Wu, X., Jia, S., and Zuo, J. (2002). Prestin After a few pulses the stimulated cell begins to shorten with sufficient is required for electromotility of the outer hair cell and for the cochlear force to move the entire cluster. There is a progressive cell shortening with amplifier.Nature 419, 300-304. repeated stimulation. The evoked movements are large enough to damage Manis, P. B., and Brownell, W. E. (1983). Synaptic organization of eighth the cell and it undergoes a volume increase from water influx. Note the nerve afferents to cat dorsal cochlear nucleus. Journal of Neurophysiology sudden lengthening about 2/3 of the way through the video. This is due to 50, 1156-1181. a volume decrease after which the cell fails to respond to electrical stimula- Oghalai, J. S., Patel, A. A., Nakagawa, T., and Brownell, W. E. (1998). Fluo- tion because of the loss of internal hydrostatic (turgor) pressure. rescence-imaged microdeformation of the outer hair cell lateral wall. Jour- nal of Neuroscience 18, 48-58. Two other videos of OHC electromotility in response to musical record- Rajagopalan, L., Patel, N., Madabushi, S., Goddard, J. A., Anjan, V., Lin, F., ings are available on line at Shope, C., Farrell, B., Lichtarge, O., Davidson, A. L., Brownell, W. E., and http://acousticstoday.org/OHCEM2 and http://acousticstoday.org/OHCEM3.

Spring 2017 | Acoustics Today | 27