Mouse ABR/DPOAE Lab

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Laboratory Exercise:

Noninvasive assessment of auditory function in mice: ABRs and DPOAEs

Place: Eaton-Peabody Laboratory, Library

Massachusetts Eye and Ear Infirmary (4th Floor) 243 Charles St. Boston Directions: http://www.meei.harvard.edu/info/dir.php

Date and Time: August 28, 2007 - 9:00 a.m. Lab Director: M. Charles Liberman (Charles_Liberman@meei.harvard.edu; 617-573-3745)

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1. Introduction 1.1 Main Objectives

The main objective of this laboratory is to understand the conceptual bases underlying, and the practical techniques involved in, noninvasive assessment of cochlear function. You will measure Auditory Brainstem Responses (ABRs) and Distortion Products Otoacoustic Emissions (DPOAEs) in anesthetized mice. Both techniques are also used in humans, with only minor modification. In the first part of the lab, you will all make baseline measurements of cochlear function using both methods. In the second part, each group will choose from a menu of possible "experiments" involving the effects of perturbations on the measurements you did on the first part. 1.2 Mouse as a model organism

Mice are widely used in research as models for studying the biological processes underlying health and disease. Although evolution separates mouse from human by over 50 million years, biologically and genetically they have many similarities. Their genome is now well characterized and can be manipulated to create gene mutations, knockouts, over-expressers, etc. that serve as models for many human diseases. 1.3 Statement about the use of animals

The laboratory exercise has been reviewed by Institutional Animal Care and Use Committees at both MIT and the Massachusetts Eye and Ear Infirmary. Copies of the approved animal use protocol and the phone number of the animal care office will be made available to any interested student.

2. Background 2.1 Auditory Brainstem Response (ABR)

The ABR is an evoked potential recorded from subdermal electrodes in the scalp by averaging the electrical response to many identical acoustic stimuli. Stimuli used to evoke an ABR are punctate in time: either tone pips or clicks. The ABR represents synchronized activity of several classes of neurons in the auditory pathway, and comprises several positive (P) and negative (N) waves (Figure 1). The use of a punctate stimulus helps us separate, in the time domain, different stages of the ascending auditory pathway, as the synchronous, stimulus-evoked electrical signals propagate up the system from cochlea to the brain. The first ABR peak (P1) is generated by the auditory nerve. Each of the next four peaks (P2-P5) has multiple sources, but all are dominated by cell types in the cochlear nucleus and superior olivary complex. The use of tone pips as an evoking stimulus, allows us to stimulate different regions of the inner ear and produce a type of audiogram, indicating the "threshold" for ABR response as a function of stimulus frequency. This gives us insight into the functional state of the cochlea along its entire spiral dimension. For this reason, ABR is widely used both in research and in the clinic. 2.2 Distortion Products Otoacoustic Emissions (DPOAEs)

Otoacoustic emissions (OAEs) are sounds measured in the ear canal, created in the healthy cochlea and propagated back through the middle ear to the ear canal, where they are measured non-invasively with a sensitive microphone.

Figure 1: ABR waveform from a cat

Time (msec)

0

0

20

42 6 8

Figure 2: Spectrum of ear canal sound pressure showing a DPOAE at 2f1-f2 in addition to the two primaries at f1 and f2.

f1=3.16

Noise

Floor

2f1 - f2

f2=3.82

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OAEs can occur spontaneously, or can be evoked by putting sounds into the ear. For OAEs to be measurable in the ear canal requires the normal amplification of cochlear vibration provided by electromotility of outer hair cells (OHCs). Thus, the size of evoked OAEs is a useful measure of OHC function. Evoked OAEs can be classified according to the stimulus used to elicit them: stimulus frequency OAEs (a pure tone); transient OAEs (a click); and distortion product OAEs (two continuous tones).

In this lab, we measure DPOAEs. When two tones (f1 and f2) of appropriate frequency separation and level are presented to the ear, distortions are created by non-linearities in transduction, for example, the saturating nonlinearity between stereocilia deflection and receptor current. The distortions created by this transducer non-linearity, and the resulting distorted receptor current waveform, drive the OHC motors thereby producing

mechanical vibrations at the distortion frequencies on the basilar membrane. This vibration causes pressure changes in the cochlear fluids at the distortion frequencies which drive the middle ear and produce sound in the ear canal by vibrating the eardrum.

In the ear, DPOAEs can be created at all sum and difference frequencies of f1 and f2, but the largest is at 2f1-f2. As shown in Figure 2, DPOAEs are best seen in the frequency domain: to produce this spectrum, the ear-canal sound pressure was averaged and a Fourier transform performed. Note that the frequencies of f1 and f2 are close (f2/f1 = 1.2). Thus, we can assess cochlear function along the cochlear spiral by measuring the "threshold" for DPOAE generation at a number of different primary-tone frequencies. Note that the "threshold" we find will be a strong function of the sensitivity of our measurement system. A measure of that sensitivity is the noise floor of the system used to generate the data in Figure 2, i.e. the lowest sound pressure detectable by the apparatus, was -20 dB SPL.

3. Overview of Measurement Hardware 3.1 Digital Stimulus Generation and Response Averaging

As summarized by the schematics in Figures 3 and 4, much of the hardware for ABR and DPOAE measurement is the same. Both use a computer workstation with an inboard "sound card" to create waveforms (Digital to analog , D-A output, conversion) and to digitize electric response waveforms (analog to digital, A-D input).

The D-A board creates a stimulus waveform, by repeatedly sending (to the Signal Out port) an array of values stored in the Memory Buffer. For example, the array can define a sinusoid, thereby creating a continuous tone (as for DPOAEs). Alternatively, the array can sandwich a few cycles of a sinusoid between two long arrays of zeroes, thereby creating a train of short tone bursts (as for ABRs). D-A boards usually have multiple output channels; thus, a second channel can be loaded with a short pulse waveform to be used as a Trigger Out, to indicate, for example, the precise time of tone-burst onset. As indicated in the schematics, this pulse can be used to

Figure 3: Schematic of hardware used to evoke, record and average the ABR

Figure 4: Schematic of hardware used to evoke, record and average the DPOAE

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trigger an Oscilloscope so that we can look at the stimulus or response waveform, and as Trigger In, to command data acquisition on the A-D board every time a stimulus is presented.

Response averaging of electric signals is used in measuring both ABRs and DPOAEs. This signal averaging is done on the A-D board, which digitizes a piece of the incoming response waveform every time a trigger pulse arrives. Each digitized waveform is an array of voltages, measured at regular time intervals after the trigger pulse (e.g. every 10 µsec). Each digitized waveform is sent to the CPU of the computer, where a running average can be computed by adding each new waveform array to the accumulating sum and dividing by the number of presentations.

For ABR measurement, averaging 1024 stimulus presentations is typically required to produce an adequate signal to noise ratio. The noise level will be roughly 1 µV. Sometimes, the response waveform is very noisy: usually from spontaneous activity in the nearby musculature, or (especially in mice) the EKG or movement artifacts associated with respiration. In such situations, an artifact-reject feature in the software is highly desirable. With such a feature, each digitized waveform is analyzed after acquisition: if the peak-to-peak amplitude is greater than a user-defined rejection-threshold (as might occur with a large muscle twitch), the trace is rejected (not added into the accumulating sum).

For DPOAE measurement, averaging of about 4 seconds of ear-canal sound pressure is required to lower the measurement noise floor to about -20 dB SPL. [Note that the noise floor will be different at different frequencies, due to the acoustic filtering of the probe tube and the inherent sensitivity of the microphone].

3.2 Sound Generation and Sound Pressure Measurement

The sound-generating Earphones and sound-measuring Microphone are housed in a Coupler to bring them both as close as possible to the animal's eardrum. As shown in Figure 5, the coupler is a metal block with a) two communicating channels drilled to mix sound from two earphones into one tube and b) a smaller concentric "Probe Tube" to provide a path for the ear-canal sound pressure to reach the Microphone. The microphone is connected to a Microphone Amplifier which feeds the ear-canal sound pressure waveform to the A-D converter.

Although the probe-tube Microphone can monitor sound pressure near the eardrum, it "sees" the world through an acoustic filter (the probe tube). A correction for this acoustic filtering has been made by coupling a calibrated second microphone to the end of the acoustic system where the animal's ear will be during recordings. This "acoustic calibration" is stored in the form of a look-up table, with the conversion factors for each input frequency between voltage at the probe tube microphone and dB SPL at the ear . Even with this probe-tube system, calibration at high frequencies (> 10 kHz) is difficult. As the wavelength of sound becomes small comparable to the dimensions of the ear canal, standing waves form, and SPL is not uniform within the canal. Thus, the SPL measured at the

probe-tube tip will be but one sample of many different pressures at different points on and near the eardrum. Only one Earphone is required for ABRs. Two Earphones are required for DPOAEs, because if a single

acoustic transducer were driven with two tones, it would produce its own distortion products. All acoustic transducers have significant non-linearities , like the inner ear, when driven over the large dynamic range necessary for auditory experiments (80 dB).

3.3 Response Amplification and Filtering

For ABR measurement, the tiny electric signals from the subdermal electrodes are fed to a Low-Impedance Amplifier, where the gain is set at 10,000X and the signals are filtered through a 0.3 - 3 kHz passband. The recording is made in differential fashion, i.e. the difference is taken between the skin at the "vertex" of the head and the skin behind the "pinna", and neither of these electrodes is grounded. Such a recording configuration will tend to minimize voltages from distant sources such as the heart, which produces a huge EKG signal which you will see on the oscilloscope.

Figure 5: Cross-section of the acoustic coupler used to present and stimuli and record ear-canal sound pressure

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For DPOAE measurements, the "response" is extracted from the ear-canal sound pressure which is amplified as described above.

4. Animal Preparation All animal handling should be done with gloves and lab coats to minimize exposure to allergens. If you have known animal allergies, please discuss with Staff. 4.1 Anesthetization and monitoring anesthesia level (done by TA)

The TA will weigh the animal and determine the anesthesia dose. We will inject into the peritoneal cavity (abdomen) a mixture of a short-acting sedative, xylazine, and a long-acting anesthetic, urethane. We will monitor anesthesia level initially by the toe-pinch reflex: i.e. foot withdrawal when the toes are vigorously pinched. When the toe-pinch reflex disappears, the animal has reached a "surgical level" of anesthesia. The animal may need more anesthetic during the lab. Once the animal is connected to the test apparatus, we will monitor depth of anesthesia by measuring heart rate (extracted from the EKG on the electrodes used to measure ABR). Note: The experimental chamber is kept at about 90 F to keep the animals' temperature from dropping.

4.2 Insertion of electrodes and placement of acoustic coupler. STEP 1: OPEN EAR CANAL FOR DIRECT VIEW OF EARDRUM (Figure 6)

• Lay animal on the wooden block with its right side up. We will measure responses from the right ear. • Looking through the operating microscope on low power, try to sight down the ear canal to the eardrum.

[Manipulate the pinna as if you were trying to stretch it away from the animal's head] • Use an alcohol-soaked Q-tip to clean the region around the ear and soak the fur. • Pick up the pinna with tweezers and make a small slit inferiorly with scissors. After the cut, you should

be able to see the eardrum by pulling apart the left and right sides of the slit. • Widen the slit to a rectangular cutout so you can see the eardrum without any pinna manipulation • If there is bleeding, stop it by applying pressure with a cotton applicator.

Figure 6: After the animal is deeply anesthetized, use small scissors to open the portion of the pinna (external ear) which hides the entrance to the ear canal. After the surgical exposure, you should be able to directly see the eardrum and ossicles.

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STEP 2: INSERT SUBDERMAL ELECTRODES TO MEASURE ABR • Three needle electrodes are inserted: green- (ground) in the back near the tail, yellow- in the scalp

between the ears and blue- just behind the pinna near the slit. • Each needle should be inserted through skin only (you don’t want to record muscle activity!). Pick up

some loose skin with tweezers and insert the needle so that the tip comes out the other side • If necessary, manipulate the pinna electrode so that it helps hold the pinna slit open, and tape the wire in

position. • DO NOT PROCEED TO THE NEXT STEP UNLESS YOU CAN SEE THE EARDRUM AND THERE

IS NO BLOOD AROUND THE SLIT STEP 3: POSITION ACOUSTIC COUPLER

• Tilt the viewing axis of the operating microscope (~30 off vertical) so you can watch the front of the acoustic coupler approach the eardrum. Using the "z-control" on the manipulator, lower the acoustic coupler such that the output/input ports are within a few mm of the eardrum.

• It's OK for the probe tube of the coupler to touch the animal, but do not create an airtight seal and don't let the tube contact any blood (it may wick up the tubes and damage the equipment)

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5. Data Acquisition and Retrieval 5.1 The LabVIEW environment

In this lab, we use LabVIEW, a graphical programming language designed for data acquisition, analysis and display. Each LabVIEW window is the "front panel", or graphical user interface, for a different program.

5.1.1 LabVIEW application-level controls: these controls,

shown in Figure 7, appear at the top left of each LabVIEW window. NEVER PRESS THE STOP BUTTON. DATA WILL BE LOST

5.1.2 The LabVIEW Toolbox: With the cursor in an active labVIEW window, hitting the Tab key scrolls through the toolbox. You want to be in "pointer mode" where the cursor looks like a pointing hand. 5.2 The HST721 Lab Controller Interface - Main Menu

Your "main menu" for the lab appears in the HST721 Lab Controller, which has links to all the other programs you need. The HST721 Lab Controller front panel is shown in Figure 8. STEP 1: START THE LAB CONTROLLER

• In the LabVIEW window displaying the front panel shown in Figure 8, make sure the recycle arrow at the top left of the window is highlighted (See 5.1).

STEP 2: INITIALIZE EQUIPMENT AND SIGN IN

• Under STEP OPTION, click EQPT SETUP. This initializes all the computer-controlled equipment

• Click NEW GROUP. Enter your group number in the new front panel that appears. Hit "ENTER" and the new window disappears. This sign-in insures that all data files you create have your group's ID .

• When you return to the HST721 Lab Controller window, your group number should appear in the red-rimmed box labeled "Group ID#"

• DO EACH OF THESE STEPS ONLY ONCE STEP 3: DATA ACQUISITION RUNS

• There are two pairs of programs (a pair for ABR, a pair for DP) listed under RUN OPTIONS. Pressing any of these blue buttons brings up the front panel of the appropriate program.

• Each pair includes an ACQUISITION program to acquire new data and a PLOT program to display previously acquired data.

• Each execution of an acquisition program is called a "Run". After each Run (described below), the software increments the appropriate Run # and displays it in the red-rimmed box in the HST721 Lab Controller.

• You will run each acquisition program repeatedly during this lab exercise.

Run Recycle Stop

Figure 7: LabVIEW aaplication controls visible at upper left corner of the window.

Figure 8: The front panel of the HST721 Lab Controller

3

Group ID #

ABR ACQUISITION ABR DATA PLOT

EQPT SETUP

QUIT

NEW GROUP

RUN OPTIONS

DP ACQUISITION

0

DP RUN #DP DATA PLOT

SETUP OPTIONS(do each only once)

0

ABR RUN #

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5.3 Acquiring DPOAE Data The front panel of the HST721 DP Controller is shown in Figure 9 and 10. Figure 9 shows the controls (ways you tell the software what to do); Figure 10 shows the indicators (ways the software shows you what it has done). 5.3.1 DPOAE Acquisition Controls

The controls for each acquisition program are at the left of the window and are numbered in the order you perform them. To change the value of a numerical control, either 1) place the cursor over the box, double click and enter a new value, or 2) press on the up or down scroll arrow at the left of each control.

1) Set Stimuli. There are five numerical controls that set parameters of the DPOAE Run:

• f2 frequency (kHz) - [defaults = 4, 8, 16, 32]. Note: f1 will automatically be set

• start dB - the lowest SPL to measure [default = 20] • stop dB - the highest SPL to measure [default = 80] • dB Step - dB increment between start and stop levels [default =

5] • #Waves per spectrum - number of segments of ear-canal sound

pressure to average at each f2 level [default = 25] 2) Start. Press the Start button when all "Set Stimuli" controls have been set. 3) Watch Data Acquisition. The program automatically presents f1 and f2 at the SPLs you requested. Data display is described below. Press ABORT button to stop acquisition, purge data and return to Main Menu without incrementing Run # . 4) Add Notes (Optional). You can store away text with the data file. such as notes about manipulations you made before the run. 5) Save. Four beeps signal that the Run is finished - all SPLs have been presented. Press the Save button to write data to a spreadsheet file. Run # will automatically increment [6. Note FilePath for data retrieval. Note the filepath and filename of the datafile just created.] See below for information on data storage format 7. Return to Main Menu. Press Return and the DP Controller will disappear returning you to the Main Menu

4 . 0

f2 Frequency

1 5

Start dB

8 0

Stop dB

5

dB Step

2 5

#Waves per Spectrum

1. Set Stimuli

2. Start START

3. Watch Data Acquisition

ABORT

4. Add Notes (Optional)

5. Save SAVE DATA

6. Note FilePath

7. Return to Main Menu RETURN

Type stuff here if you want

Figure 9: Controls from the LabVIEW program used to acquire DPOAEs

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5.3.2 DPOAE Acquisition Displays

Figure 10 shows the graphs and indicators from the DPOAE acquisition program 1) Response Waveform graph: Segment of the averaged time waveform of the ear-canal sound pressure as recorded by the probe-tube microphone. It displays a new waveform at every SPL presented. The vertical axis is the amplified voltage from the probe-tube microphone, as

seen at the input to the A-D converter (Fig. 4); the horizontal axis is time in msec. We show only a fraction of the waveform so you see the periodicity 2. Response Spectrum graph: The fast fourier transform (FFT), i.e. spectrum, of the averaged probe-tube microphone response. The vertical axis is voltage from the probe-tube microphone in dB re 1 Volt (e.g. -20 dB = 0.1 V; -40 dB = 0.01 V, etc.); the horizontal axis is frequency in Hz. To convert dB volts from the probe-tube microphone into dB SPL in the ear, the program uses the look-up table derived from the acoustic calibration (Section 3.2). 3. DPOAE Amplitude vs Level graph: Amplitudes, in dB SPL, of the spectral components at f2, f1 and 2f1-f2 (vertical axis) vs. commanded SPL of the primaries (horizontal axis). Black symbols are levels of f2 and f1. The measured level of f2 (filled symbols) should equal the commanded level, and f1 (unfilled symbols) should be 10 dB higher. The blue symbols show levels of 2f1-f2 (circles) and the surrounding noise floor (X's). [See Section 2.2]. 4. ID#, Run# and Filepath indicators: ID # displays your Group # and the Run # displays the current DPOAE Run #. The filename created for this run will be DP-[Group#]-[Run#], e.g. "DP-3-1" for Group 3, Run 1. Note that Run #s for DPOAE acquisition increment independently of Run #s for ABR acquisition. The filepath for the folder in which you will find this file is: "C:\Data\HST721- DATA\HST721-[Group#]", e.g. "C:\Data\HST721- DATA\HST721-3", if you are in Group 3. 5. Miscellaneous Indicators: These top two indicators display the exact frequencies of f1 and 2f1-f2. These values will be stored in the data file. The bottom two show the maximum SPL the acoustic system can deliver at f2 and f1.

Figure 10: Graphs and Indicators from the LabVIEW program used to acquire DPOAEs

0

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Frequency (Hz)

500001 0 0 0 10000

Response Spectrum

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ID #

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DPOAE Amplitude vs Level

f 1

105 .46103 .25 MAX SPL

f 1 f 2

1 .2513

-1.2199-1.0000

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C:\Data\HST721- DATA\HST721-2\DP-2-1

2 f 1 - f 2

3

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f 1f 22f1 - f2Noise Flr

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5.4 Acquiring ABR Data The front panel of the HST721ABR Controller is shown in Figure 11 and 12. Figure 11 shows the controls (ways you tell the software what to do); Figure 12 shows the indicators (ways the software shows you what it has done). 5.4.1 ABR Acquisition Controls

Controls are at the left of the window and are numbered in the order you perform them. To change the value of a numerical control, either 1) place the cursor over the box, double click and enter a new value, or 2) press on the up or down scroll arrow at the left of each control. 1) Set Stimuli. There are six numerical controls that set the parameters of the ABR Run:

• tone pip frequency (kHz): [defaults = 4, 8, 16 and 32 kHz] • start dB - the lowest SPL to present [default = 20] • stop dB - the highest SPL to present [default = 80] • dB Step - dB increment between start and stop levels [default

= 5] • #Averages per level - number of alternate-polarity, tone-pip

pairs to average at each sound pressure level [default = 512 pairs, i.e. 1024 tone pips]

• Rep Rate - the number of tone pips presented per second [default = 35 /sec]

2) Start. Press the Start button when all "Set Stimuli" controls have been set. 3) Watch Data Acquisition / ABORT / Reject Threshold. The program automatically presents tone pips at the SPLs you requested. Data display is described below.

• During acquisition, press ABORT button to stop Run, purge data and return to Main Menu without incrementing Run # .

• During acquisition, start with Reject Threshold at 15 µVolts. Increase if a large fraction of responses are being rejected (See Acquisition Display #2 below)

4) Add Notes (Optional). You can store away text with the data file. such as notes about manipulations you made before the run. 5) Save. Four beeps signal that the Run is finished - all SPLs have been presented. Press the Save button to write data to a spreadsheet file. Run # will automatically be incremented

[6. Note FilePath for data retrieval. Note the filepath and filename of the datafile just created.] See below for information on data storage format 7. Return to Main Menu. Press Return and the ABR Controller will disappear returning you to the Main Menu

START

ABORT

1 0START (dB)

8 0STOP (dB)

5 1 2# AVERAGES per LEVEL

8 . 0 0FREQUENCY

5dB STEP

Tone Pip Stimuli

SAVE DATA

3 5

REP RATE (pips/sec)

1 5

REJECT THRESH (µV)

2. Start

1. Set Stimuli

5. Save

3. Watch Data Acquisition

4. Add Notes (Optional)

6. Note FilePath:

RETURN7. Return to Main

Figure 11: The controls from the LabVIEW program used to acquire ABRs

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5.4.2 ABR Acquisition Displays Figure 12 shows the graphs and indicators from the ABR acquisition program 1) Stimulus Waveform graph: the waveform (voltage on the vertical axis, time in msec on the horizontal axis) being sent to the earphone. 2) Current Average graph plus 3 Indicators: accumulating ABR average response waveform, i.e. the amplified voltage difference between the vertex and pinna electrodes. The response amplitude has been corrected for amplifier gain, to read in microvolts at the electrode.

The three indicators show a) SPL: the current SPL of the tone pips being measured, b) # Aves: the number of non-rejected averages thus far accumulated, and c) # Rejects: the number of response waveforms "rejected" because their peak to peak voltage was higher than the "reject threshold" [see Acquisition Control #3].

3) Amplitude vs Level graph: peak-to-peak amplitude of the averaged ABR (in microvolts; vertical axis) obtained at each tone pip SPL (horizontal axis). The noise floor, i.e. the peak-to-peak amplitude at sub-threshold levels, should be between 1.0 and 1.5 microvolts.

4) Stacked Waveforms graph and SPL list: Stack of ABR waveforms for each sound level: to see waves at low SPLs, each waveform is normalized so peak-to-peak display is ~ 1 inch of vertical space To estimate threshold, pick the lowest SPL at which any of the major waves is still present. The SPL List indicator reminds you of all the SPLs so far presented. 5) ID#, Run# and Filepath indicators: The ID # displays your Group # and the Run # displays the current ABR Run #. The filename created for this run will be ABR-[Group#]-[Run#], e.g. "ABR-3-1" for Group 3, Run 1. Run #s for ABR acquisition increment independently of Run #s for DPOAE acquisition. The filepath for the folder in which you will find this file is: "C:\Data\HST721- DATA\HST721-[Group#]", e.g. "C:\Data\HST721- DATA\HST721-3", if you are in Group 3.

Figure 12: Graphs and indicators from the LabVIEW program used to acquire ABRs

3ID #

1 5

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1 0

1 0 00 2 0 4 0 6 0 8 0

Amplitude vs Level

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512 # Averages

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Stimulus Waveform

Level (dB SPL

C:\Data\HST721- DATA\HST721-3\ABR-3-1

2

35

Stacked Waveforms - All Levels

SPLList

1

4

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5.5 Retrieving and Decoding your data Filenames and Locations: The file names are of the form "ABR-[Group#]-Run#]" and "DP-[Group#]-[Run#]". They are located in a folder called "HST721-[Group#]" within "HST721- DATA" within "Data" on Drive C. File format: ABR and DP data are saved as text files. Each file starts with 5-6 "header lines" (Figs. 13 and 14). Each header line starts with a colon and contains self-explanatory information, including the run type, the time of day, and the values of all stimulus parameters. After the header lines, come the data lines, in a "tab-delimited" spreadsheet form suitable for importation into a variety of plotting and analysis programs including Matlab, Excel, Kaleidagraph, etc. :RUN-0 ABR I-O 10/24/2003 4:47 PM : EAR: R FREQ: 16.00 # AVES: 400 REP RATE (/sec): 80 SAMPLING (µsec): 10 :NOTES- :CHAMBER-4 :SPLs TESTED-20;30;40;50;60;70;80; :ANALYZED DATA :SPL "NEURAL" P-P "CM" P-P 20.000 1.563 1.296 30.000 1.587 1.529 40.000 1.514 1.017 50.000 1.562 1.566 60.000 1.820 1.490 70.000 2.368 2.671 80.000 3.563 4.549 :RAW WAVEFORMS -0.084572 -0.060116 -0.104443 0.059216 0.167206 -0.009075 -0.132732 -0.046043 -0.051418 -0.105358 0.054485 0.158127 -0.014187 -0.088329 -0.042152 -0.030743 -0.097805 0.046932 0.125854 -0.006329 -0.072536 etc. Figure 13: Text showing data storage formats for an ABR Run Data Storage Conventions: Data stored are of two types: "Analyzed Values" and "Raw Waveforms". Each type is identified by one header line immediately above it.

• Analyzed Values: o Analyzed values for the DP Runs (Fig. 13) consist of the numerical values of the data plotted in

panel 3 of Figure 10: a spreadsheet in which each row is different commanded f2 SPL and the 5 columns contain the commanded f2 level, and the measured SPLs for f2, f1, 2f1-f2 and the noise floor near 2f1-f2 (as identified in the associated header line).

o Analyzed values for the ABR Runs (Fig. 14) consist of the numerical values of the data plotted in panel 3 of Figure 12: a spreadsheet in which each row is a different tone pip SPL and the 2 columns contain 1) the SPL and 2) the peak-to-peak voltage of the ABR wave at that level.

• Raw Waveforms:

o At the end of each DP File (Fig. 13) are the average time waveforms of the probe-tube microphone voltage at each SPL. Each column shows the response at a different f2 level.

o At the end of each ABR File (Fig 14) are the average time waveforms of the ABR response. Each column shows the response at a different tone pip SPL.

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:RUN-1 DP I-O 3:54 PM :Level Step (dB):5 Start dB: 20 Stop dB: 50 # Buffers Averaged: 25 Sampling Interval:4 Ear:Right :NOTES- :CHAMBER-4 :ANALYZED DATA :Command dB Measured f1(dB) Measured f2(dB) 2f1-f2(dB) 2f1-f2Nse(dB) 20.00 30.01 19.97 -24.01 -25.33 25.00 35.05 24.98 -26.75 -23.71 30.00 40.04 30.01 -25.80 -25.42 35.00 45.04 35.01 -25.51 -24.86 40.00 50.04 40.04 -22.93 -25.06 45.00 55.11 45.05 -20.88 -24.54 50.00 60.10 50.05 -23.63 -24.94 :RAW WAVEFORMS 0.002390 0.003187 0.004161 0.006183 0.009836 0.016199 0.027711 0.002397 0.003308 0.004397 0.006392 0.010246 0.016801 0.028828 0.002385 0.003362 0.004509 0.006510 0.010535 0.017273 0.029696 etc. Figure 14: Text showing data storage formats for a DP Run

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6. The Experiment

6.1 Baseline Measurements

Each group should start by obtaining baseline measurements of DPOAE and ABR "thresholds" as a function of stimulus frequency.

To determine "threshold" for each response type (ABR and DPOAE) obtain a response-vs.-level function for each stimulus frequency: 4, 8, 16 and 32 kHz. Response levels should be varied from 20 to 80 dB SPL in 5 dB steps. Your data will all be saved, so you can define "threshold" criteria and assess "threshold" offline (after the lab).

Time requirements:

Each ABR response (one tone-pip frequency, one SPL) requires ~30 seconds. Thus an entire response-vs-level function (13 sound levels) requires 6.5 minutes, and acquiring data to define "threshold" at four frequencies will take ~30 minutes Each DPOAE response (one primary tone pair, one SPL pair) requires ~5 seconds. Thus an entire response-vs. level function requires ~1 minute and acquiring data to define "threshold" at four frequencies will take ~ 5 minutes.

Questions to ponder: Thresholds estimated by both ABR and DPOAE will likely show a frequency dependence - where does

this dependence originate? Are "thresholds" measured by one technique lower at all test frequencies than by the other? Discuss

possible origins of any systematic differences you see. Do threshold differences between the two techniques show any dependence on stimulus frequency?

Discuss possible origins of any frequency dependence you see?

6.2 Experimental Questions

The method(s) you use to investigate your experimental question(s) are up to you. Feel free to pose and

investigate questions other than those on the list, if you think they can be addressed with the data acquisition interfaces provided. The references at the end might help you to develop some ideas.

6.2.1 Effects of tympanic membrane perforation on middle ear transmission.

Children are often fitted with "tympanostomy tubes" to treat chronic middle ear infections. These tubes are placed in the eardrum to vent the middle ear space; acousticallly they act like perforations. You can use the DPOAE and/or ABR "threshold" measures as a type of biological measurement device to assess the effects on sound transmission of perforating the tympanic membrane. Using either ABRs or DPOAEs, you can gauge the effects of perforation size or perforation placement on sound transmission (in dB), and you can determine the frequency dependence of any sound transmission changes observed.

Would you expect such a manipulation of the middle ear to affect DPOAEs and ABRs equally? Is one measure better suited for this task than the other? Can you explain the magnitude and frequency dependence of your results based on what you have learned about acoustics and middle ear mechanics in your other courses? Can you suggest an ideal placement site for tympanostomy tubes based on your results?

[Note: You can attempt to simulate "healing" of these perforations by patching with a small piece of wet lens paper]

6,2,2. Measurement Artifact and System Distortion: How do you know your responses are animal generated?

If you were the first person to notice distortion products in ear canal, you might well have assumed they were generated by your equipment, since it is impossible to build sound sources that do not distort if driven to high SPLs. Try to determine quantitatively 1) how much of the distortion you measure is an otoacoustic

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emission and how much is generated by the equipment used to generate and measure the sound and 2) determine whether the answer depends on stimulus frequency as well as stimulus level. One key question to think about is how any system distortion limits your ability to measure a threshold shift with DPOAEs.

There are two general approaches to this problem: 1) re-measure baseline responses after coupling the acoustic system to a passive air cavity, such as syringe barrel, instead of a living mouse's ear or 2) measure baseline responses before vs. after the animal is dead. (The TA can help you overdose the animal with anesthetic - how will you know it is dead?). Do you get the same answer with approach 1 and approach 2? Might it take some time after death before the dead ear responds like the passive cavity? Might it matter how big the air space in the passive cavity is?

6.2.3. Reproducibility of "threshold" measures

It is unlikely that the response you get will be identical to those obtained by other lab groups. Estimated "thresholds" may differ by many dB - Why? Is it because your animals' hearing acuity is really different, or is it because of uncontrolled variability in the data? It is always important to know how reproducible your results are and to understand the sources of variability and their relative magnitude.

Variability can arise in the animal or in the measurement technique. Animal-based variability can arise because 1) the responses are probabilistic in nature and will never be exactly the same twice; or 2) there are true underlying changes in animal state, such as body temperature, blood pressure etc., which affect the response magnitude. Measurement-based variability can arise because of differences in placement and alignment of the acoustic coupler, or of the subdermal electrodes to name just two.

Try to assess the magnitude of one or more of these sources of variability by making repeated measures of some subset of the "baseline" responses with or without manipulation of some variable[s] (e.g. coupler or electrode placement). You may find that responses at some frequencies are more variable than those at others. If so, discuss possible reasons why.

[Note: you could vary animal temperature by placing it on an ice block. We can insert a rectal temperature probe.]

7. References Melcher, J.R. et al (1996) Generators of the brainstem auditory evoked potential in cat. I. An experimental approach to their identification. Hear Res., Apr 93(1-2):1-27 Horner, K.C. et al (1985) Distortion product otoacoustic emissions in hearing-impaired mutant mice J Acoust Soc Am. Nov; 78 (5):1603-11 Parham, K., et al (2001) Noninvasive assessment of Auditory Function in Mice: Auditory Brainstem Response and Distortion Product Otoacoustic Emissions in Handbook of Mouse Auditory Research (Willot, J.F., ed.), CRC Press LLC