- Email: [email protected]
Calibration of acoustic transients
BR A IN RE S E A RCH 1 0 91 ( 20 0 6 ) 2 7 –3 1
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Calibration of acoustic transients Robert Burkard ⁎ Center for Hearing and Deafness, 137 Cary Hall, University of Buffalo, Buffalo, NY 14214, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
This article reviews the appropriate stimulus parameters (click duration, toneburst
Accepted 27 February 2006
envelope) that should be used when eliciting auditory brainstem responses from mice.
Available online 24 April 2006
Equipment specifications required to calibrate these acoustic transients are discussed. Several methods of calibrating the level of acoustic transients are presented, including the
Keywords:
measurement of peak equivalent sound pressure level (peSPL) and peak sound pressure
Auditory brainstem response
level (pSPL). It is hoped that those who collect auditory brainstem response thresholds in
ClickS
mice will begin to use standardized methods of acoustic calibration, so that hearing
Toneburst
thresholds across mouse strains obtained in different laboratories can more readily be
Acoustic calibration
compared. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The ABR is one of a family of auditory-evoked potentials that can be recorded relatively non-invasively from surface or subdermal electrodes. The ABR is largely unaffected by subject variables, such as attention and arousal, and is typically only minimally affected by anesthesia. In contrast, it is strongly affected by stimulation variables, including stimulus level, rate, background noise level, stimulus spectrum, and stimulus envelope. As the ABR is typically smaller in amplitude than the background noise floor, multiple stimuli must be time-domain signal averaged in order to obtain a response at a favorable signal-to-noise ratio. The ABR is also affected by recording variables, such as bioamplifier filtering and electrode placement. The reader unfamiliar with the ABR is referred to textbooks dedicated to auditory-evoked potentials in general or ABRs in particular (Moore, 1983; Glattke, 1983; Jacobson, 1985, 1994; Hall, 1992; Hood, 1998). A review of the human ABR literature will reveal that stimulation and recording parameters have been, to some extent, standardized. This standardization has facilitated using the ABR for clinical purposes, including
threshold estimation, site-of-lesion testing and intraoperative monitoring (see Sininger and Cone-Wesson, 2002; Don and Kwong, 2002; Martin and Mishler, 2002). Standardization of stimulation and recording parameters has not been achieved for ABR studies in non-human species. With the strong current interest in the study of hearing loss in various strains of mutant mice (e.g., see Willott, 2001), such standardization would clearly facilitate across-laboratory comparison of mouse ABR thresholds across strains of mutant mice. This article will not discuss the many different stimulation and recording parameters than can influence the mouse ABR. Rather, it will be limited to a consideration of several aspects of the acoustic stimulus used to elicit the auditory brainstem response (ABR) in mice. This review assumes familiarity with basic acoustical principles. Those who find themselves in need of a review of acoustics are referred to general texts on the topic, such as Durrant and Lovrinic (1995); Harris (1998); Haughton (2002); Luce (1993); Prout and Bienvenue (1990); Rosen and Howell (1991); Speaks (1996) and/or Yost (2000). In this article, particular attention will be paid to the calibration of the acoustic stimulus. This article will not discuss real-ear calibration, nor will it
⁎ Fax: +1 716 829 2980. E-mail address: [email protected]. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.132
28
BR A IN RE S EA RCH 1 0 91 ( 20 0 6 ) 2 7 –31
consider calibration issues unique to mouse otoacoustic emissions.
2. Some general issues when generating and calibrating acoustic transients When calibrating the acoustic stimuli used to obtain the auditory brainstem response (ABR) in mice, several challenges arise. The first is that ABRs are elicited with acoustic transients, and hence, one must use instantaneous peak measures (such as peak SPL: pSPL) or relate the baseline-topeak or peak-to-peak amplitude of the transient to the baseline-to-peak or peak-to-peak amplitude of a sine wave (obtaining peak equivalent SPL: peSPL). Sound level meters with fast or slow exponential time-weighting scales (see Yeager and Marsh, 1998), which are commonly used for longduration stimuli, are inadequate for quantifying the level of acoustic transients. A second issue is the small size of the ear canal of mice. If using some form of insert earphone in the mouse, the use of a commercially available coupler, such as the 2-cm3 coupler that is commonly used to calibrate insert earphones for audiometric testing in humans, is inappropriate. This is because this volume far exceeds that of the mouse ear canal. The size, shape, and how rigid the walls of the cavity are have an effect on its acoustic properties. Thus, the acoustic properties of a 2-cm3 volume are not likely to approximate the properties of the real mouse ear canal. Third, the mouse hears at frequencies well above the upper cutoff frequency of humans (see, e.g., Fay, 1988), and it is inappropriate to use earphones and microphones that are appropriate for presenting and calibrating stimuli for human use.
3.
Acoustic calibration of clicks
If we are obtaining an ABR from a human subject, we might present a pulse with a duration of 100 μs to produce a ‘click’ through an Etymotic ER2 insert earphone, couple it to a 2-cm3 coupler that is terminated with a 1” condenser microphone, to a sound level meter. Can you use this stimulation and calibration protocol when interested in obtaining mouse ABRs? You can, but these parameters are suboptimal for several reasons. First, as mentioned above, the 2-cm3 volume does not represent a reasonable approximation to the mouse ear-canal volume (or its acoustical properties). In addition, you should not use a 100-μs click, because the first spectral zero of a pulse is the inverse of its duration, or at 10 kHz for the 100-μs click (Speaks, 1996; Rosen and Howell, 1991), which is well below the upper bandwidth of mouse hearing (Fay, 1988). The time-domain waveform and spectrum of a 100-μs pulse is shown in Fig. 1, showing the dips in the spectrum near 10 and 20 kHz. It is important to understand that the properties of the transducer, and any coupler used, will affect the spectrum of the acoustic signal. Further, the auditory system of the animal species under investigation, including the outer, middle and inner ear properties, also ultimately affect the resulting ABR. It is true that the spectrum level (i.e., the SPL of a stimulus in a 1Hz band) decreases as the duration of a click decreases (and the upper cutoff frequency of the electrical signal increases). Nonetheless, ABR thresholds to relatively low-level brief duration clicks have been reported in the literature. For example, using 10-μs clicks, Burkard and Moss (1994) found that most of the 6 big brown bats (Eptesicus fuscus) showed an identifiable ABR response to click levels of 40-dB pSPL.
Fig. 1 – The time-domain (upper panel) and frequency-domain (lower panel) representation of a 100-μs click. Note the spectral zeroes near 10 kHz and 20 kHz. Reprinted from Burkard and Secor (2002) with permission of Lippincott, Williams and Wilkins.
29
BR A IN RE S E A RCH 1 0 91 ( 20 0 6 ) 2 7 –3 1
Even if we used a more appropriate click duration (say 20 μs, with the first spectral zero at 50 kHz), the Etymotic ER2 earphone starts attenuating sounds above ~12 kHz and hence would still not be optimal for testing the hearing of mice, who have good hearing well above 30 kHz (Fay, 1988). Even if the pulse was short enough to produce a wide-bandwidth electrical stimulus, the transducer passband was appropriate, and the coupler was a volume better representing that of the mouse ear canal, we would still be filtering the acoustic output because the 1” condenser microphone starts rolling off at ~10 kHz. As the upper cutoff of condenser microphones generally increase as the microphone diameter decreases (see Johnson et al., 1998), a ¼-in. or 1/8-in. microphone should generally be used for calibrating the higher frequency transients often used to elicit the mouse ABR. In my own laboratory, for rodent studies, I use either a B & K type 4938 1/4-in. pressure microphone or a B & K type 4138 1/8-in. pressure microphone. The upper cutoff frequency (−3 dB cutoff) is between 80 and 90 kHz for the ¼-in. microphone, and between 150 and 200 kHz for the 1/8-in. microphone. A number of other manufacturers sell equivalent condenser microphones, including Larson Davis, GRAS, and ACO Pacific. Even if the microphone used had an adequate passband, the processing performed by sound level meters made for human audiofrequencies can start dropping off above ~20 kHz. Finally, many sound level meters cannot appropriately measure the pSPL of acoustic transients, as this requires the ability to measure and ‘hold’ the peak SPL (called a ‘peak hold’ mode). It is important for the reader with limited experience in acoustic calibration of transients to understand that ‘impulse’ time weighting has a much longer time constant (35-ms time constant for sounds increasing over time, and 1500-ms time constant for sounds decreasing over time) than does the peak mode (see Yeager and Marsh, 1998), and thus, the impulse function is inappropriate for measuring the level of the very brief duration transients used to obtain the ABR. Instrumentation is available to produce a click with an appropriate bandwidth (up to let us say ~50 kHz) and to perform a meaningful acoustic calibration of the stimulus. The acoustic stimulus can be transduced using commercially available speakers or earphones, such as the Tucker-Davis ES1 speaker or EC1 insert earphone, which, according to specifications, have upper cutoff frequencies well above 50 kHz. If using a speaker, some attempt must be made to reduce echoes in the testing environment using some type of sound-absorbing foam to line the booth, should the booth be relatively small in size. You should measure the distance between the speaker diaphragm and the middle of the mouse's head, so you can correct for the acoustic delay (0.1 ms for every 3.4 cm). If using a pressure microphone, the microphone should be presented at grazing incidence to the direction of the plane progressive wave (i.e., microphone diaphragm should be at a right angle to the direction of the sound wave). Note that if the angle between the sound wave and the microphone differs appreciably from 90°, then this can have an impact on the voltage out of the microphone (and hence the pSPL measured), and this effect tends to increase with increasing frequency. If using a free-field microphone, frontal incidence is appropriate (i.e., the microphone diaphragm is pointed directly towards the speaker).
Performing a real-ear calibration in a mouse ear canal offers a number of technical challenges that go beyond the scope of this manuscript. Pearce et al. (2001) discuss the use of a real-ear mouse ear canal as a coupler, which will no doubt, on average, better approximate the SPL at the tympanic membrane than will a tube of a specified volume. If using an insert earphone and a coupler with a specified volume, you should choose a ‘coupler’ with a small acoustic volume (e.g., 0.05–0.1 cm3), with a diameter that allows the insert earphone tube to be inserted in one end and a condenser pressure microphone placed in the opposite end. I find it convenient to use a 0.5-cm3 syringe, with the ends cut off, so that you can read off the scale and you can reliably place both the insert earphone and a 1/8-in. diameter condenser microphone at either end, with a known volume between them. You should use a microphone with an upper cutoff frequency at or above 40–50 kHz. As stated above, the upper cutoff frequency of a condenser microphone varies inversely with microphone diameter, and thus, you will need to use either a 1/4-in. or 1/8-in. microphone. I prefer the 1/8in. microphone, as this fits nicely inside a 0.5-cm3 syringe. Condenser microphones come prepolarized, but many require a conditioning amplifier that provides a DC bias voltage (often 200 V); the conditioning amplifiers may also provide output gain. Usually, a preamplifier connects the microphone to the conditioning amplifier. Armed with such a setup, one can then measure dB pSPL. You can either use the microphone sensitivity or use an acoustic calibrator that produces a known SPL. Let us say the sensitivity of the microphone is 1 mV/Pa. This means that if we present 1 Pa of sound pressure to the diaphragm of the microphone, we produce 1 mV of voltage at the microphone output. As 1 Pa is equal to 94 dB SPL (dB SPL = 20 log P/0.00002 Pa), we should amplify this by, e.g., 100 times (40 dB), so we produce 100 mV/Pa. This voltage amplification allows an accurate reading on an oscilloscope or voltmeter (i.e., the voltages for moderate SPL values are above the noise floor of these instruments). The formula for calculating SPL in this example would be: dB SPL ¼ 20logðV=100 mVÞ þ 94 Alternatively, you might have an acoustic calibrator that puts out 114 dB SPL. As 114 dB SPL is 10 Pa (114 dB SPL = 20log(10Pa/0.00002 Pa), then putting this coupler on the microphone with a sensitivity of 1 mV/Pa and the 100× gain on the amplifier would deliver 1 V (RMS), because: 1 mV=Pa 100ðgainÞ ¼ 100 mV; 114 dB SPL ¼ 10 Pa; so 100 mV 10 ¼ 1000 mV ðor 1 VÞ Now let us calibrate. We present an acoustic click, and we measure on an oscilloscope a ‘click’ with an initial positive voltage of 50 mV, and a following negative voltage of 50 mV. To measure ‘peak SPL’, we take the largest ‘peak’ voltage in the transient (50 mV), and put it in the formula: dB SPL ¼ 20logð50 mV=100 mVÞ þ 94;
or 88 dB pSPL
When measuring peak equivalent SPL (peSPL), you use a microphone and a sound level meter that has an AC output that can be routed to an oscilloscope. Let us assume you have a microphone and sound level meter with an adequate upper cutoff frequency (e.g., 50 kHz). You present your 20-μs pulse,
30
BR A IN RE S EA RCH 1 0 91 ( 20 0 6 ) 2 7 –31
pass it through the transducer, coupler, microphone and sound level meter, and measure the peak (or, in some cases, the peak-to-peak) voltage on an oscilloscope that is connected to the AC output of the sound level meter (see Fig. 2). Then you route a continuous tone through the earphone and adjust the level of the tone until the peak voltage (or peak-to-peak voltage) is equal to the peak voltage (or peak-to-peak voltage) measured for the click stimulus (see the lower two waveforms of Fig. 3). Once adjusted, you read the SPL on the sound level meter to obtain the peSPL. Note that the peSPL using the peak voltage method will be 3 dB less than the pSPL. This is because the ratio of the peak to RMS voltage of a sine wave is 1.414, and 20 log1.414 = 3 dB. In our example, the peSPL would be 85 dB. Note further that the peSPL obtained using the peak-to-peak voltage method can be anywhere from 0 dB to 6 dB less than that obtained using the peak voltage approach. In our example, because the positive voltage and negative voltage are both 50 mV, the peSPL for both methods would be 85 dB SPL. Were the positive voltage 50 mV, and the following negative voltage 0 mV (i.e., the earphone was critically damped), the peSPL using the peak-to-peak method would have been 6 dB less than that recorded using the peak method, or 79 dB peSPL. For those interested in more detailed explanations of pSPL and peSPL, read Burkard (1984) and/or Burkard and Secor (2002).
4.
Acoustic calibration of tonebursts
I have been talking about measuring stimulus level for clicks, but we are often interested in more frequency-specific information when obtaining ABRs and use tonebursts. If you can turn your toneburst on for a long duration (say a half second or more), while keeping the peak voltage constant, then you can measure the SPL on a sound level meter. Simply route the earphone through your ‘mouse’ coupler, an appropriate microphone to a sound level meter, and read the SPL. For the fast meter mode, the meter time constant is 125 ms (Yeager and Marsh, 1998). As 3 time constants is long enough time for the meter to approximate the steady-state SPL, if you can turn the stimulus on for at least 375 ms (i.e., 3 × 125 ms), you should obtain a good estimate of the SPL. This reading then becomes the peSPL of the toneburst. For your information, the time constant for the slow meter mode is 1 s (Yeager and Marsh, 1998), and hence, it will take a stimulus of at least 3 s to approximate the steady-state SPL on the display. Should you not be able to extend the duration of the toneburst sufficiently for an accurate reading, you can route the AC
Fig. 3 – Measurements used to determine the peak equivalent SPL, using the baseline-to-peak procedure (middle waveform) and the peak-to-peak (lower waveform) procedure. The upper waveform shows the time-domain waveform of a click stimulus. Reprinted from Burkard and Secor (2002) with permission of Lippincott, Williams and Wilkins.
output of the sound level meter to an oscilloscope. You can measure the peSPL as described for the click above, except that you will use a toneburst instead of the click. Note that if the plateau time of the toneburst is at least one cycle, then the peSPL using the peak voltage and peak-to-peak voltage methods will usually be identical. Instead of a sound level meter, you can, of course, use an appropriate coupler, microphone, preamplifier and conditioning amplifier. Using such equipment, you can measure (using an oscilloscope) the peak voltage of the toneburst. You can use the same formula as used for clicks (above), which basically uses the microphone sensitivity to convert voltage to pressure and then converts pressure to pSPL using the familiar dB SPL formula. There is no direct empirical data about the optimal envelope parameters for tonebursts in mice. In humans, Suzuki and Horiuchi (1981) used ramp stimuli to identify how much of the rise time contributes to the ABR (in terms of its contribution to ABR peak latency, amplitude, threshold).
Table 1 – Relationship between frequency and time for tonebursts with 2-cycle rise times and a 1-cycle plateau times Frequency (kHz)
Fig. 2 – A block diagram of instrumentation used to calibrate acoustic transients.
5 10 20 40
Risetime (ms)
Duration (ms)
0.4 0.2 0.1 0.05
1 0.5 0.25 0.125
BR A IN RE S E A RCH 1 0 91 ( 20 0 6 ) 2 7 –3 1
Their results suggest that a 1–2 cycle rise time is optimal. If we accept that a 2-cycle rise time is optimal (note: most instrumentation requires that the fall time be equal to the rise time), and that we should include a 1-cycle plateau, then the following rise times and total durations shown in Table 1 for 5- to 40-kHz tonebursts are recommended. It may not be possible to generate such brief rise times and total durations (depending on software and/or hardware used). In that case, it is reasonable to use a constant stimulus envelope. In these cases, a reasonably brief rise time (perhaps a half ms or less, with an equal duration fall time) should be brief enough to elicit an ABR (which is, after all, an onset response), while still being reasonably narrow in spectrum for the higher frequency stimuli used to elicit the mouse ABR (usually no less than several kilohertz), even if no plateau time is used.
5.
Summary
The reason to standardize acoustic calibration procedures is so that data collected at different clinics (or different laboratories) are directly comparable. This is not the case with the available mouse ABR data. Perhaps mouse thresholds obtained within a laboratory can be compared (although in some instances this is probably not true), but it is difficult if not impossible to compare mouse ABR thresholds across laboratories. This is because the specifications for acoustic calibration in mouse ABR articles are often incomplete, described in a confusing manner, or done inappropriately. It is not really possible to state with any certainty whether a given strain of mouse hears better or worse than another strain of mouse, unless the data were obtained within a study (or in some cases, in the same laboratory).
REFERENCES
Burkard, R., 1984. Sound pressure level measurement and spectral analysis of brief acoustic transients. Electroenceph. clin. Neurophysiol. 57, 83–91. Burkard, R., Moss, C., 1994. The brainstem auditory evoked response (BAER) in the Big Brown Bat to clicks and FM sweeps. J. Acoust. Soc. Am. 96, 801–810. Burkard, R., Secor, C., 2002. Overview of AEPs, In: Katz, J. (Ed.), Handbook of Clinical Audiology, 5th ed. Lippincott, Williams and Wilkins, Baltimore, pp. 233–248. Don, M., Kwong, B., 2002. Auditory brainstem response: differential diagnosis, In: Katz, J. (Ed.), Handbook of Clinical
31
Audiology, 5th ed. Lippincott, Williams and Wilkins, Baltimore, pp. 274–297. Durrant, J., Lovrinic, J., 1995. Bases of Hearing Science, 3rd ed. Williams and Wilkins, Baltimore. Fay, R., 1988. Hearing in Vertebrates: A Psychophysics Databook. Hill-Fay Associates: Winnetka, Illinois. Glattke, T., 1983. Short-Latency Auditory Evoked Potentials. Fundamental Bases and Clinical Applications. Pro-Ed, Austin. Hall, J., 1992. Handbook of Auditory Evoked Responses. Allyn and Bacon, Boston. Harris, C., 1998. Handbook of Acoustical Measurements and Noise Control, 3rd ed. Acoustical Society of America, Woodbury, NY. Haughton, P., 2002. Acoustics for Audiologists. Academic, New York. Hood, L., 1998. Clinical Applications of the Auditory Brainstem Response. Singular, San Diego. Jacobson, J., 1985. The Auditory Brainstem Response. College-Hill Press, San Diego. Jacobson, J., 1994. Principles and Applications in Auditory Evoked Potentials. Allyn and Bacon, Boston. Johnson, D., Marsh, A., Harris, C., 1998. Acoustic measurement instruments, In: Harris, C. (Ed.), Handbook of Acoustical Measurements and Noise Control, 3rd ed. Acoustical Society of America, Woodbury, pp. 5.1–5.21. Luce, R., 1993. Sound and Hearing. A Conceptual Introduction. Lawrence Earlbaum, Hillsdale. Martin, W., Mishler, T., 2002. Intraoperative monitoring of auditory evoked potentials and facial nerve electromyography, In: Katz, J. (Ed.), Handbook of Clinical Audiology, 5th ed. Lippincott, Williams and Wilkins, Baltimore, pp. 323–348. Moore, E., 1983. Bases of Auditory Brain-Stem Evoked Responses. Grune and Stratton, New York. Pearce, M., Richter, C., Cheatham, M., 2001. A reconsideration of sound calibration in the mouse. J. Neurosci. Meth. 106, 57–67. Prout, J., Bienvenue, G., 1990. Acoustics for You. Robert E. Krieger, Malabar. Rosen, S., Howell, P., 1991. Signals and Systems for Speech and Hearing. Academic Press, London, UK. Sininger, Y., Cone-Wesson, B., 2002. Threshold prediction using auditory brainstem response and steady-state evoked potentials with infants and young children, In: Katz, J. (Ed.), Handbook of Clinical Audiology, 5th ed. Lippincott, Williams and Wilkins, Baltimore, pp. 298–322. Speaks, C., 1996. Introduction to Sound: Acoustics for the Hearing and Speech Sciences, 2nd ed. Singular, San Diego CA. Suzuki, T., Horiuchi, K., 1981. Rise time of pure-tone stimuli in brain stem response audiometry. Audiol. 20, 101–112. Willott, J., 2001. Handbook of Mouse Auditory Research. From Behavior to Molecular Biology. CRC Press, New York. Yeager, D., Marsh, A., 1998. Sound levels and their measurement, In: Harris, C. (Ed.), Handbook of Acoustical Measurements and Noise Control, 3rd ed. Acoustical Society of America, Woodbury, pp. 11.1–11.18. Yost, W., 2000. Fundamentals of Hearing. An Introduction, 4th ed. Academic Press, New York.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Calibration of acoustic transients Robert Burkard ⁎ Center for Hearing and Deafness, 137 Cary Hall, University of Buffalo, Buffalo, NY 14214, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
This article reviews the appropriate stimulus parameters (click duration, toneburst
Accepted 27 February 2006
envelope) that should be used when eliciting auditory brainstem responses from mice.
Available online 24 April 2006
Equipment specifications required to calibrate these acoustic transients are discussed. Several methods of calibrating the level of acoustic transients are presented, including the
Keywords:
measurement of peak equivalent sound pressure level (peSPL) and peak sound pressure
Auditory brainstem response
level (pSPL). It is hoped that those who collect auditory brainstem response thresholds in
ClickS
mice will begin to use standardized methods of acoustic calibration, so that hearing
Toneburst
thresholds across mouse strains obtained in different laboratories can more readily be
Acoustic calibration
compared. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The ABR is one of a family of auditory-evoked potentials that can be recorded relatively non-invasively from surface or subdermal electrodes. The ABR is largely unaffected by subject variables, such as attention and arousal, and is typically only minimally affected by anesthesia. In contrast, it is strongly affected by stimulation variables, including stimulus level, rate, background noise level, stimulus spectrum, and stimulus envelope. As the ABR is typically smaller in amplitude than the background noise floor, multiple stimuli must be time-domain signal averaged in order to obtain a response at a favorable signal-to-noise ratio. The ABR is also affected by recording variables, such as bioamplifier filtering and electrode placement. The reader unfamiliar with the ABR is referred to textbooks dedicated to auditory-evoked potentials in general or ABRs in particular (Moore, 1983; Glattke, 1983; Jacobson, 1985, 1994; Hall, 1992; Hood, 1998). A review of the human ABR literature will reveal that stimulation and recording parameters have been, to some extent, standardized. This standardization has facilitated using the ABR for clinical purposes, including
threshold estimation, site-of-lesion testing and intraoperative monitoring (see Sininger and Cone-Wesson, 2002; Don and Kwong, 2002; Martin and Mishler, 2002). Standardization of stimulation and recording parameters has not been achieved for ABR studies in non-human species. With the strong current interest in the study of hearing loss in various strains of mutant mice (e.g., see Willott, 2001), such standardization would clearly facilitate across-laboratory comparison of mouse ABR thresholds across strains of mutant mice. This article will not discuss the many different stimulation and recording parameters than can influence the mouse ABR. Rather, it will be limited to a consideration of several aspects of the acoustic stimulus used to elicit the auditory brainstem response (ABR) in mice. This review assumes familiarity with basic acoustical principles. Those who find themselves in need of a review of acoustics are referred to general texts on the topic, such as Durrant and Lovrinic (1995); Harris (1998); Haughton (2002); Luce (1993); Prout and Bienvenue (1990); Rosen and Howell (1991); Speaks (1996) and/or Yost (2000). In this article, particular attention will be paid to the calibration of the acoustic stimulus. This article will not discuss real-ear calibration, nor will it
⁎ Fax: +1 716 829 2980. E-mail address: [email protected]. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.132
28
BR A IN RE S EA RCH 1 0 91 ( 20 0 6 ) 2 7 –31
consider calibration issues unique to mouse otoacoustic emissions.
2. Some general issues when generating and calibrating acoustic transients When calibrating the acoustic stimuli used to obtain the auditory brainstem response (ABR) in mice, several challenges arise. The first is that ABRs are elicited with acoustic transients, and hence, one must use instantaneous peak measures (such as peak SPL: pSPL) or relate the baseline-topeak or peak-to-peak amplitude of the transient to the baseline-to-peak or peak-to-peak amplitude of a sine wave (obtaining peak equivalent SPL: peSPL). Sound level meters with fast or slow exponential time-weighting scales (see Yeager and Marsh, 1998), which are commonly used for longduration stimuli, are inadequate for quantifying the level of acoustic transients. A second issue is the small size of the ear canal of mice. If using some form of insert earphone in the mouse, the use of a commercially available coupler, such as the 2-cm3 coupler that is commonly used to calibrate insert earphones for audiometric testing in humans, is inappropriate. This is because this volume far exceeds that of the mouse ear canal. The size, shape, and how rigid the walls of the cavity are have an effect on its acoustic properties. Thus, the acoustic properties of a 2-cm3 volume are not likely to approximate the properties of the real mouse ear canal. Third, the mouse hears at frequencies well above the upper cutoff frequency of humans (see, e.g., Fay, 1988), and it is inappropriate to use earphones and microphones that are appropriate for presenting and calibrating stimuli for human use.
3.
Acoustic calibration of clicks
If we are obtaining an ABR from a human subject, we might present a pulse with a duration of 100 μs to produce a ‘click’ through an Etymotic ER2 insert earphone, couple it to a 2-cm3 coupler that is terminated with a 1” condenser microphone, to a sound level meter. Can you use this stimulation and calibration protocol when interested in obtaining mouse ABRs? You can, but these parameters are suboptimal for several reasons. First, as mentioned above, the 2-cm3 volume does not represent a reasonable approximation to the mouse ear-canal volume (or its acoustical properties). In addition, you should not use a 100-μs click, because the first spectral zero of a pulse is the inverse of its duration, or at 10 kHz for the 100-μs click (Speaks, 1996; Rosen and Howell, 1991), which is well below the upper bandwidth of mouse hearing (Fay, 1988). The time-domain waveform and spectrum of a 100-μs pulse is shown in Fig. 1, showing the dips in the spectrum near 10 and 20 kHz. It is important to understand that the properties of the transducer, and any coupler used, will affect the spectrum of the acoustic signal. Further, the auditory system of the animal species under investigation, including the outer, middle and inner ear properties, also ultimately affect the resulting ABR. It is true that the spectrum level (i.e., the SPL of a stimulus in a 1Hz band) decreases as the duration of a click decreases (and the upper cutoff frequency of the electrical signal increases). Nonetheless, ABR thresholds to relatively low-level brief duration clicks have been reported in the literature. For example, using 10-μs clicks, Burkard and Moss (1994) found that most of the 6 big brown bats (Eptesicus fuscus) showed an identifiable ABR response to click levels of 40-dB pSPL.
Fig. 1 – The time-domain (upper panel) and frequency-domain (lower panel) representation of a 100-μs click. Note the spectral zeroes near 10 kHz and 20 kHz. Reprinted from Burkard and Secor (2002) with permission of Lippincott, Williams and Wilkins.
29
BR A IN RE S E A RCH 1 0 91 ( 20 0 6 ) 2 7 –3 1
Even if we used a more appropriate click duration (say 20 μs, with the first spectral zero at 50 kHz), the Etymotic ER2 earphone starts attenuating sounds above ~12 kHz and hence would still not be optimal for testing the hearing of mice, who have good hearing well above 30 kHz (Fay, 1988). Even if the pulse was short enough to produce a wide-bandwidth electrical stimulus, the transducer passband was appropriate, and the coupler was a volume better representing that of the mouse ear canal, we would still be filtering the acoustic output because the 1” condenser microphone starts rolling off at ~10 kHz. As the upper cutoff of condenser microphones generally increase as the microphone diameter decreases (see Johnson et al., 1998), a ¼-in. or 1/8-in. microphone should generally be used for calibrating the higher frequency transients often used to elicit the mouse ABR. In my own laboratory, for rodent studies, I use either a B & K type 4938 1/4-in. pressure microphone or a B & K type 4138 1/8-in. pressure microphone. The upper cutoff frequency (−3 dB cutoff) is between 80 and 90 kHz for the ¼-in. microphone, and between 150 and 200 kHz for the 1/8-in. microphone. A number of other manufacturers sell equivalent condenser microphones, including Larson Davis, GRAS, and ACO Pacific. Even if the microphone used had an adequate passband, the processing performed by sound level meters made for human audiofrequencies can start dropping off above ~20 kHz. Finally, many sound level meters cannot appropriately measure the pSPL of acoustic transients, as this requires the ability to measure and ‘hold’ the peak SPL (called a ‘peak hold’ mode). It is important for the reader with limited experience in acoustic calibration of transients to understand that ‘impulse’ time weighting has a much longer time constant (35-ms time constant for sounds increasing over time, and 1500-ms time constant for sounds decreasing over time) than does the peak mode (see Yeager and Marsh, 1998), and thus, the impulse function is inappropriate for measuring the level of the very brief duration transients used to obtain the ABR. Instrumentation is available to produce a click with an appropriate bandwidth (up to let us say ~50 kHz) and to perform a meaningful acoustic calibration of the stimulus. The acoustic stimulus can be transduced using commercially available speakers or earphones, such as the Tucker-Davis ES1 speaker or EC1 insert earphone, which, according to specifications, have upper cutoff frequencies well above 50 kHz. If using a speaker, some attempt must be made to reduce echoes in the testing environment using some type of sound-absorbing foam to line the booth, should the booth be relatively small in size. You should measure the distance between the speaker diaphragm and the middle of the mouse's head, so you can correct for the acoustic delay (0.1 ms for every 3.4 cm). If using a pressure microphone, the microphone should be presented at grazing incidence to the direction of the plane progressive wave (i.e., microphone diaphragm should be at a right angle to the direction of the sound wave). Note that if the angle between the sound wave and the microphone differs appreciably from 90°, then this can have an impact on the voltage out of the microphone (and hence the pSPL measured), and this effect tends to increase with increasing frequency. If using a free-field microphone, frontal incidence is appropriate (i.e., the microphone diaphragm is pointed directly towards the speaker).
Performing a real-ear calibration in a mouse ear canal offers a number of technical challenges that go beyond the scope of this manuscript. Pearce et al. (2001) discuss the use of a real-ear mouse ear canal as a coupler, which will no doubt, on average, better approximate the SPL at the tympanic membrane than will a tube of a specified volume. If using an insert earphone and a coupler with a specified volume, you should choose a ‘coupler’ with a small acoustic volume (e.g., 0.05–0.1 cm3), with a diameter that allows the insert earphone tube to be inserted in one end and a condenser pressure microphone placed in the opposite end. I find it convenient to use a 0.5-cm3 syringe, with the ends cut off, so that you can read off the scale and you can reliably place both the insert earphone and a 1/8-in. diameter condenser microphone at either end, with a known volume between them. You should use a microphone with an upper cutoff frequency at or above 40–50 kHz. As stated above, the upper cutoff frequency of a condenser microphone varies inversely with microphone diameter, and thus, you will need to use either a 1/4-in. or 1/8-in. microphone. I prefer the 1/8in. microphone, as this fits nicely inside a 0.5-cm3 syringe. Condenser microphones come prepolarized, but many require a conditioning amplifier that provides a DC bias voltage (often 200 V); the conditioning amplifiers may also provide output gain. Usually, a preamplifier connects the microphone to the conditioning amplifier. Armed with such a setup, one can then measure dB pSPL. You can either use the microphone sensitivity or use an acoustic calibrator that produces a known SPL. Let us say the sensitivity of the microphone is 1 mV/Pa. This means that if we present 1 Pa of sound pressure to the diaphragm of the microphone, we produce 1 mV of voltage at the microphone output. As 1 Pa is equal to 94 dB SPL (dB SPL = 20 log P/0.00002 Pa), we should amplify this by, e.g., 100 times (40 dB), so we produce 100 mV/Pa. This voltage amplification allows an accurate reading on an oscilloscope or voltmeter (i.e., the voltages for moderate SPL values are above the noise floor of these instruments). The formula for calculating SPL in this example would be: dB SPL ¼ 20logðV=100 mVÞ þ 94 Alternatively, you might have an acoustic calibrator that puts out 114 dB SPL. As 114 dB SPL is 10 Pa (114 dB SPL = 20log(10Pa/0.00002 Pa), then putting this coupler on the microphone with a sensitivity of 1 mV/Pa and the 100× gain on the amplifier would deliver 1 V (RMS), because: 1 mV=Pa 100ðgainÞ ¼ 100 mV; 114 dB SPL ¼ 10 Pa; so 100 mV 10 ¼ 1000 mV ðor 1 VÞ Now let us calibrate. We present an acoustic click, and we measure on an oscilloscope a ‘click’ with an initial positive voltage of 50 mV, and a following negative voltage of 50 mV. To measure ‘peak SPL’, we take the largest ‘peak’ voltage in the transient (50 mV), and put it in the formula: dB SPL ¼ 20logð50 mV=100 mVÞ þ 94;
or 88 dB pSPL
When measuring peak equivalent SPL (peSPL), you use a microphone and a sound level meter that has an AC output that can be routed to an oscilloscope. Let us assume you have a microphone and sound level meter with an adequate upper cutoff frequency (e.g., 50 kHz). You present your 20-μs pulse,
30
BR A IN RE S EA RCH 1 0 91 ( 20 0 6 ) 2 7 –31
pass it through the transducer, coupler, microphone and sound level meter, and measure the peak (or, in some cases, the peak-to-peak) voltage on an oscilloscope that is connected to the AC output of the sound level meter (see Fig. 2). Then you route a continuous tone through the earphone and adjust the level of the tone until the peak voltage (or peak-to-peak voltage) is equal to the peak voltage (or peak-to-peak voltage) measured for the click stimulus (see the lower two waveforms of Fig. 3). Once adjusted, you read the SPL on the sound level meter to obtain the peSPL. Note that the peSPL using the peak voltage method will be 3 dB less than the pSPL. This is because the ratio of the peak to RMS voltage of a sine wave is 1.414, and 20 log1.414 = 3 dB. In our example, the peSPL would be 85 dB. Note further that the peSPL obtained using the peak-to-peak voltage method can be anywhere from 0 dB to 6 dB less than that obtained using the peak voltage approach. In our example, because the positive voltage and negative voltage are both 50 mV, the peSPL for both methods would be 85 dB SPL. Were the positive voltage 50 mV, and the following negative voltage 0 mV (i.e., the earphone was critically damped), the peSPL using the peak-to-peak method would have been 6 dB less than that recorded using the peak method, or 79 dB peSPL. For those interested in more detailed explanations of pSPL and peSPL, read Burkard (1984) and/or Burkard and Secor (2002).
4.
Acoustic calibration of tonebursts
I have been talking about measuring stimulus level for clicks, but we are often interested in more frequency-specific information when obtaining ABRs and use tonebursts. If you can turn your toneburst on for a long duration (say a half second or more), while keeping the peak voltage constant, then you can measure the SPL on a sound level meter. Simply route the earphone through your ‘mouse’ coupler, an appropriate microphone to a sound level meter, and read the SPL. For the fast meter mode, the meter time constant is 125 ms (Yeager and Marsh, 1998). As 3 time constants is long enough time for the meter to approximate the steady-state SPL, if you can turn the stimulus on for at least 375 ms (i.e., 3 × 125 ms), you should obtain a good estimate of the SPL. This reading then becomes the peSPL of the toneburst. For your information, the time constant for the slow meter mode is 1 s (Yeager and Marsh, 1998), and hence, it will take a stimulus of at least 3 s to approximate the steady-state SPL on the display. Should you not be able to extend the duration of the toneburst sufficiently for an accurate reading, you can route the AC
Fig. 3 – Measurements used to determine the peak equivalent SPL, using the baseline-to-peak procedure (middle waveform) and the peak-to-peak (lower waveform) procedure. The upper waveform shows the time-domain waveform of a click stimulus. Reprinted from Burkard and Secor (2002) with permission of Lippincott, Williams and Wilkins.
output of the sound level meter to an oscilloscope. You can measure the peSPL as described for the click above, except that you will use a toneburst instead of the click. Note that if the plateau time of the toneburst is at least one cycle, then the peSPL using the peak voltage and peak-to-peak voltage methods will usually be identical. Instead of a sound level meter, you can, of course, use an appropriate coupler, microphone, preamplifier and conditioning amplifier. Using such equipment, you can measure (using an oscilloscope) the peak voltage of the toneburst. You can use the same formula as used for clicks (above), which basically uses the microphone sensitivity to convert voltage to pressure and then converts pressure to pSPL using the familiar dB SPL formula. There is no direct empirical data about the optimal envelope parameters for tonebursts in mice. In humans, Suzuki and Horiuchi (1981) used ramp stimuli to identify how much of the rise time contributes to the ABR (in terms of its contribution to ABR peak latency, amplitude, threshold).
Table 1 – Relationship between frequency and time for tonebursts with 2-cycle rise times and a 1-cycle plateau times Frequency (kHz)
Fig. 2 – A block diagram of instrumentation used to calibrate acoustic transients.
5 10 20 40
Risetime (ms)
Duration (ms)
0.4 0.2 0.1 0.05
1 0.5 0.25 0.125
BR A IN RE S E A RCH 1 0 91 ( 20 0 6 ) 2 7 –3 1
Their results suggest that a 1–2 cycle rise time is optimal. If we accept that a 2-cycle rise time is optimal (note: most instrumentation requires that the fall time be equal to the rise time), and that we should include a 1-cycle plateau, then the following rise times and total durations shown in Table 1 for 5- to 40-kHz tonebursts are recommended. It may not be possible to generate such brief rise times and total durations (depending on software and/or hardware used). In that case, it is reasonable to use a constant stimulus envelope. In these cases, a reasonably brief rise time (perhaps a half ms or less, with an equal duration fall time) should be brief enough to elicit an ABR (which is, after all, an onset response), while still being reasonably narrow in spectrum for the higher frequency stimuli used to elicit the mouse ABR (usually no less than several kilohertz), even if no plateau time is used.
5.
Summary
The reason to standardize acoustic calibration procedures is so that data collected at different clinics (or different laboratories) are directly comparable. This is not the case with the available mouse ABR data. Perhaps mouse thresholds obtained within a laboratory can be compared (although in some instances this is probably not true), but it is difficult if not impossible to compare mouse ABR thresholds across laboratories. This is because the specifications for acoustic calibration in mouse ABR articles are often incomplete, described in a confusing manner, or done inappropriately. It is not really possible to state with any certainty whether a given strain of mouse hears better or worse than another strain of mouse, unless the data were obtained within a study (or in some cases, in the same laboratory).
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