Assessment of Cochlear Function in Mice: Distortion‐Product Otoacoustic Emissions

Glen K. Martin1, Barden B. Stagner2, Brenda L. Lonsbury‐Martin3

1 Jerry Pettis Memorial Veterans Medical Center and Loma Linda University, Loma Linda, California, 2 Jerry Pettis Memorial Veterans Medical Center, Loma Linda, California, 3 Loma Linda University, Loma Linda, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.21C
DOI:  10.1002/0471142301.ns0821cs34
Online Posting Date:  February, 2006
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Abstract

Distortion-product otoacoustic emissions (DPOAEs) can be measured in the ear canal following the presentation of two tones. These emissions are generated by the outer hair cells (OHCs) of the inner ear and they are reduced or absent when the OHCs are damaged by, for example, exposure to excessive noise or ototoxic drugs. Consequently, DPOAEs provide a powerful and noninvasive means to assess the robustness of OHC function. A detailed method is described for measuring DPOAEs to assess cochlear function in mice. Recommendations are given for the required equipment and instructions are presented for setting up a DPOAE system. Also, a protocol is outlined for measuring DPOAEs in mice and troubleshooting tips are provided. Examples of data analysis procedures following noise exposure in mice are included, as well. These methods are not only applicable to mice, but can be performed using essentially all small laboratory animals.

Keywords: distortion-product otoacoustic emissions; cochlear function; mice; hearing

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Computer Control and DPOAE Measurement
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Computer Control and DPOAE Measurement

 Materials
  • Mice
  • Anesthesia (e.g., ketamine/xylazine, Avertin, or a gaseous anesthetic such as isoflurane; appendix 4B)
  • Two channel, low-distortion frequency synthesizer (e.g., 33220A Function Generator, Agilent Technologies) or two-channel computer soundcard (100 Hz to 60 kHz frequency range; e.g., LynxTWO, Lynx Studio Technology)
  • High-frequency tweeters to produce pure tones from 100 Hz to at least 60 kHz (e.g., ES1/EC1 electrostatic speakers, Tucker-Davis Technologies)
  • Low-distortion amplifier for impedance-matching speakers to sound sources (e.g., HB-7 headphone buffer, Tucker-Davis Technologies)
  • Programmable attenuator to adjust the levels of the two tones (e.g., PA5 programmable attenuator, Tucker-Davis Technologies)
  • Specialized low-noise microphone/amplifier for measuring ear-canal sound pressure (e.g., ER-10B+ emissions microphone, Etymotic Research) fitted with sound-delivery tubes supplied by the vendor for delivering acoustic stimuli and an internal port for microphone pickup (DPOAE frequency range = 1 Hz to 35 kHz)
  • Sound-delivery tubes to connect high-frequency tweeters to microphone assembly (2-mm i.d.)
  • Sound-attenuation chamber: commercially available (Acoustic Systems or Industrial Acoustics Corporation) or laboratory-made sound-attenuation enclosure (see step )
  • Temperature-controlled heating pad or heating table (e.g., Harvard Apparatus)
  • Spectrum analyzer (e.g., 35670A FFT dynamic signal analyzer, Agilent Technologies) or computer soundcard (e.g., LynxTWO, Lynx Studio Technology) with spectral analysis software (range = 1 Hz to at least 50 kHz)
  • 1/4-in. high-frequency microphone (e.g., type 4136 pressure microphone, Bruel & Kjaer) and associated preamplifier (range = 1 Hz to 100 kHz)
  • Sound calibrator (QC-20 sound calibrator, Quest Technologies)
  • 1-ml syringes
  • Teflon tape
  • Personal computer equipped with an instrument controller card (e.g., NI-PC/104-GPIB, National Instruments) to control the frequency synthesizers and spectrum analyzer for a stand-alone setup or a soundcard with appropriate software to substitute for these instruments
  • Small pieces of silicon tubing to use as probe tips for the microphone assembly (ER3-34 infant silicon tips, Etymotic Research)
  • Small, curved forceps
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Figures

  •  FigureFigure 8.21C.1 Spectrum of the ear-canal signal from a mouse showing primary tones f1 and f2 and the several upper- (e.g., 2f2-f1) and lower-sideband (e.g., 3f1-2f2) DPOAEs commonly observed in rodent ears. It is clear that the 2f1-f2 DPOAE is the largest emission and for this reason it is the DPOAE most commonly studied. The NF is indicated by the arrow and, in this example, the NF is approximately –10 dB SPL.
    View Image
  •  FigureFigure 8.21C.2 Photograph of the setup for testing mouse DPOAEs. High-frequency tweeters are shown at right encased in conical housings so that small polyethylene tubes can be used to couple them to the emissions probe shown in the center foreground. Speaker sound tubes couple with tubing supplied with the emissions microphone (Etymotic Research). The small tubing connects directly to two sound ports that pass through the microphone. Preamplifier cable from the microphone can be seen leaving the probe at the right. The probe is fitted with small silicon tubing (ER3-34 infant silicon tips, Etymotic Research) to help seal the probe into the mouse ear canal. In this photograph, a self-retaining Codman surgical retractor at left holds the probe. However, the tension of speaker tubing can also hold the probe in place. When the probe is properly placed in the ear canal, the fit is often secure enough to be able to lift the head of the mouse off the heating table by pulling up on the probe.
    View Image
  •  FigureFigure 8.21C.3 Example of a “check-fit” window used for judging the quality of the placement of the probe in the ear canal. (A) A poor check-fit in that both primary tones are too low and do not converge on the 75-dB SPL reference line (dotted line) near the top of the window. Repositioning the probe (B) results in a good check-fit with both primaries extending to the reference line.
    View Image
  •  FigureFigure 8.21C.4 Photograph of a 1-ml syringe with the needle-end cut off, which is used as a standardized test cavity. The DPOAE microphone with silicon tip is positioned in the cutoff end and the plunger is adjusted to ~0.1 ml to approximate the volume of the mouse ear canal.
    View Image
  •  FigureFigure 8.21C.5 Example of common ways DPOAEs data can be analyzed and displayed. In the left column, mean (n = 10) DP-grams (DPOAE level in dB SPL) are plotted as a function of the f2 frequency for a particular tone pair evoking the emission. These plots show DPOAE levels for FVB/NJ mice obtained for three levels of primary-tone stimulation at 75 (top), 65 (middle), and 55 (bottom) dB SPL. The plots show before (solid squares) exposure, immediate (open circles) exposure, 1 day (open triangles) following exposure, and 7 days (open diamonds) following exposure to a 1-hr, 105-dB SPL octave band of noise (OBN) centered at 10 kHz (box at lower left). These plots show average DPOAE levels without any transformation of the raw data. Higher levels of stimulation at, e.g., 75 dB SPL, evoke larger DPOAEs (top left) than those produced by the 65-dB SPL (middle left) primaries. Lower levels of stimulation are frequently more sensitive to the effects of damage than higher levels of stimulation. For example, at 1 day postexposure (open triangles), successively lower primary-tone levels show less recovery, especially above 20 kHz. A number of peaks and valleys in the data are clearly evident with an especially prominent dip ~15 to 17 kHz in the 55-dB SPL plot (lower left). The prominent notch observed over this frequency range is clearly a calibration problem in the system due to standing waves that are present over this particular frequency region in the mouse ear canal. Although the authors have made several attempts to correct for this notch, it has been extremely difficult to consistently eliminate. For this reason, the authors prefer to use fixed calibrations rather than an in-the-ear procedure, where every mouse gets slightly different levels of stimulation, depending on the particular characteristics associated with that ear. The graphs in the right column show these same data plotted as differences from the preexposure baseline. The dashed line at the ‘0’ on the ordinate indicates no change from preexposure levels. Here, it is easier to appreciate the loss patterns for the various primary-tone test levels. For example, for 75-dB SPL primaries (top right), an orderly recovery occurred from essentially NF levels immediately postexposure, to almost full recovery at 7 days postexposure, with recovery about halfway for the in-between period of 1 day after the noise trauma. Solid vertical bars indicate ±1 SD for baseline DPOAEs, while gray vertical bars show variability (±1 SD) immediately postexposure. Stippled gray line in lower portion of each plot shows the NF of measurement system.
    View Image
  •  FigureFigure 8.21C.6 Diagram depicting the traveling-wave envelopes on the basilar membrane for f1, f2, and the 2f1-f2 DPOAE. Note that the 2f1-f2 DPOAE is lower in frequency (and level) than the primary tones, but is generated where the two stimuli overlap (shaded area) basal to the DPOAE characteristic frequency place. Because of the sharp apical cutoff of the f2 traveling wave, the DPOAE can only be generated at the f2 frequency place, or basal to this region.
    View Image

Videos

Literature Cited

Literature Cited
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    Huang, J.M., Berlin, C.I., Lin, S.T., and Keats, B.J., 1998. Low intensities and 1.3 ratio produce distortion product otoacoustic emissions which are larger in heterozygous (+/dn) than homozygous (+/+) mice. Hear. Res. 117:24-30.
    Jimenez, A.M., Stagner, B.B., Martin, G.K., and Lonsbury-Martin, B.L. 1999. Age-related loss of distortion product otoacoustic emissions in four mouse strains. Hear. Res. 138:91-105.
    Jimenez, A.M., Stagner, B.B., Martin, G.K., and Lonsbury-Martin, B.L. 2001. Susceptibility of DPOAEs to sound overexposure in inbred mice with AHL. J. Assoc. Res. Otolaryngol. 2:233-245.
    Kemp, D.T. 1978. Stimulated acoustic emissions from within the human auditory system. J. Acoust. Soc. Am. 64:1386-1391.
    Kemp, D.T. 1979a. Evidence of mechanical nonlinearity and frequency selective wave amplification in the cochlea. Arch. Otorhinolaryngol. 224:37-45.
    Kemp, D.T. 1979b. The evoked cochlear mechanical response and the auditory microstructure—Evidence for a new element in cochlear mechanics. Scand. Audiol. 9:35-47.
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    Knight, R.D. and Kemp, D.T. 2000. Indications of different distortion product otoacoustic emission mechanisms from a detailed f1,f2 area study. J. Acoust. Soc. Am. 107:457-473.
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    Lonsbury-Martin, B.L., McCoy, M.J., Whitehead, M.L., and Martin, G.K. 1993. Clinical testing of distortion-product otoacoustic emissions. Ear Hear. 14:11-22.
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    Lukashkin, A.N., Lukashkina, V.A., Legan, P.K., Richardson, G.P., and Russell, I.J. 2004. Role of the tectorial membrane revealed by otoacoustic emissions recorded from wild-type and transgenic Tecta(deltaENT/deltaENT) mice. J. Neurophysiol. 91:163-171.
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    Martin, G.K., Jassir, D., Stagner, B.B., Whitehead, M.L., and Lonsbury-Martin, B.L. 1998. Locus of generation for the 2f1-f2 vs 2f2-f1 distortion-product otoacoustic emissions in normal-hearing humans revealed by suppression tuning, onset latencies, and amplitude correlations. J. Acoust. Soc. Am. 103:1957-1971.
    Mills, D.M., and Rubel, E.W. 1994. Variation of distortion product otoacoustic emissions with furosemide injection. Hear. Res. 77:183-199.
    Mom, T., Bonfils, P., Gilain, L., and Avan, P. 2001. Origin of cubic difference tones generated by high-intensity stimuli: Effect of ischemia and auditory fatigue on the gerbil cochlea. J. Acoust. Soc. Am. 110:1477-1488.
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    Parham, K., Sun, X.M., and Kim, D.O. 1999. Distortion product otoacoustic emissions in the CBA/J mouse model of presbycusis. Hear. Res. 134:29-38.
    Parham, K., Sun, X.M., and Kim, D.O. 2001. Noninvasive assessment of auditory function in mice: Auditory brainstem response and distortion product otoacoustic emissions. In Handbook of Mouse Auditory Research from Behavior to Molecular Biology (J.F. Willott, ed.) pp. 37-58. CRC Press, New York.
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    Shera, C.A., and Guinan, J.J. Jr. 1999. Evoked otoacoustic emissions arise by two fundamentally different mechanisms: A taxonomy for mammalian OAEs. J. Acoust. Soc. Am. 105:782-798.
    Sirjani, D.B., Salt, A.N., Gill, R.M., and Hale, S.A. 2004. The influence of transducer operating point on distortion generation in the cochlea. J. Acoust. Soc. Am. 115:1219-1229.
    Sutton, L.A., Lonsbury-Martin, B.L., Martin, G.K., and Whitehead, M.L. 1994. Sensitivity of distortion-product otoacoustic emissions in humans to tonal over-exposure: Time course of recovery and effects of lowering L2. Hear. Res. 75:161-174.
    Vazquez, A.E., Luebke, A.E., Martin, G.K., and Lonsbury-Martin, B.L. 2001. Temporary and permanent noise-induced changes in distortion product otoacoustic emissions in CBA/CaJ mice. Hear. Res. 156:31-43.
    Vazquez, A.E., Jimenez, A.M., Martin, G.K., Luebke, A.E., and Lonsbury-Martin, B.L. 2004. Evaluating cochlear function and the effects of noise exposure in the B6.CAST+Ahl mouse with distortion product otoacoustic emissions. Hear. Res. 194:87-96.
    Whitehead, M.L., McCoy, M.J., Lonsbury-Martin, B.L., and Martin, G.K. 1995a. Dependence of distortion-product otoacoustic emissions on primary levels in normal and impaired ears. I. Effects of decreasing L2 below L1. J. Acoust. Soc. Am. 97:2346-2358.
    Whitehead, M.L., Stagner, B.B., Lonsbury-Martin, B.L., and Martin, G.K. 1995b. Effects of ear-canal standing waves on measurements of distortion-product otoacoustic emissions. J. Acoust. Soc. Am. 98:3200-3214.
    Whitehead, M.L., Stagner, B.B., McCoy, M.J., Lonsbury-Martin, B.L., and Martin, G.K. 1995c. Dependence of distortion-product otoacoustic emissions on primary levels in normal and impaired ears. II. Asymmetry in L1, L2 space. J. Acoust. Soc. Am. 97:2359-2377.
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 Key References
    Jimenez, et al., 1999. See above.
    Jimenez et al., 2001. See above.

Original peer-reviewed published articles reporting many of the details itemized in the Basic Protocol.

    Kemp, 2002. See above.

Current view of the generation and propagation of otoacoustic emissions.

    Parham, 1997. See above.
    Parham et al., 1999. See above.

Original peer-reviewed published articles reporting many of the details itemized in the Basic Protocol.

    Parham et al., 2001. See above.

Earlier review of research on DPOAEs in mice.

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