This protocol combines a clinically relevant methodology with a well-established animal research model. It will help answer questions about auditory development and refinement and functional changes in hearing following genetic manipulation. This technique is noninvasive, so it requires no surgery and could be combined with additional experiments.
Also, it only takes around one hour to perform. This method could be used on any small bird species. So comparative physiology is relatively easy.
The chicken ABR can also evaluate the effect on functional hearing following genetic manipulation. Begin by acquiring fertilized white leghorn chicken eggs. Next, incubate the egg at 38 degrees Celsius and humidity of 50%for 21 days before the desired testing date.
Once the egg is hatched, weigh the animal by gently placing it in a large weighing boat. After anesthesia injection, place the animal back in the incubator. Then check if the neck is limp and pinch the toe of the animal using forceps.
As a next step, determine the sex of the chicken using its wing feathers. Later, apply depilatory cream with a cotton tip applicator to the head and neck area, especially near the ear opening for the bird. Now use 70%isopropyl alcohol wipes to wipe off feathers, any remaining depilatory cream and the skin on the head and neck.
Also, sterilize the subdermal electrodes and rectal probe using a 70%isopropyl alcohol wipe. Place the animal in a sound isolation and electrically shielded chamber, ensuring that the environment has minimal electrical and acoustical noise for the best recordings. Use a heating pad to maintain animal body temperature.
Now insert the rectal probe and fix the animal's head in place or rest the beak against an object to avoid unwanted movement. Next ensure the temperature control chamber maintains the animal's temperature between 37 and 41 degrees Celsius. Use three stainless steel, silver chloride needle electrodes as a reference, active, and common ground electrodes.
Place each electrode subdermally two to three millimeters into the head, but not deep enough to penetrate the skull, and then poke the electrode out of the skin, exposing the tip. For single channel recording, place the active electrode above the skull at the midline as far caudal as the ear canal. Place the reference electrode behind the ear where the stimulus will be delivered, and place the ground electrode behind the contralateral ear in the neck.
Now check the electrode impedance. Ensure that the overall electrode impedance does not exceed five kilo ohms. Also, maintain the interelectrode impedance below three kilos ohms.
For ABR recording, depending on acquisition hardware and software, be sure to calibrate the correct sound levels across stimulus frequencies used. Now move the sound transducer apparatus toward the active ear of the animal and place it in a shallow depth of two millimeters in the ear canal. Finally, check on the animal during testing.
If the results look abnormal or absent, reposition the sound transducer in the ear canal. First, open the software to acquire ABR recordings. Set the artifact rejection upper and lower limits to approximately 25 microvolts, such that animal, movement, or noise during a sweep will exclude that sweep from the analysis.
Collect at least 1024 sweeps to obtain a grand averaged response which can be done in two recordings of 512 sweeps each. This ensures that the response is stimulus evoked and repeatable. In amplifier settings of the software, set the gain to 100, 000, the low pass filter to 3, 000 Hertz, and the high pass filter to 100 Hertz.
Set the stimulus presentation rate between 10 and 20 stimuli per second, and the time duration of the click stimulus to 100 microseconds. Next, set the sampling rate to 40 kilohertz, 25 microseconds for the best resolution data and set the stimulus polarization to alternating. If recording 512 sweeps, combine two separate tests to create a 1, 024 sweep average, and continue recording at lower and lower intensities until the evoked potential can no longer be identified.
Lower the stimulus intensity by steps of five decibel sound pressure level to find the lowest stimulus intensity that elicits an evoked response. Define ABR threshold as the lowest stimulus intensity that elicits a detectable evoked response. After euthanizing and decapitating the animal and the experiment by cleaning the heating pad, rectal probe, and silver chloride electrodes with 70%isopropyl alcohol wipes, make sure all acquired traces have been saved.
This figure represents the click ABR. Wave I peak latency increased by approximately 0.3 milliseconds for each 20 decibel decrease in stimulus intensity. Wave I also presented the largest peak amplitude and the lowest peak latency variability of all wave form peaks.
The graph shows the tone burst evoked ABR. The best response was seen at 1, 000 Hertz. The figure demonstrates that if body temperature is not maintained, latency intensity functions of the ABR are highly variable and often inaccurate.
The figure shows that ABRs recorded from hatchlings less than three hours old, labeled as P1, have peak latencies significantly prolonged and peak amplitudes reduced compared to older hatchlings, labeled as P2.The figure compares 75 decibels sound pressure level click traces in the same animal with different reference electrode placements. Wave II peak amplitude for the mastoid placement occurred one millisecond after the wave II peak for the neck placement. This time difference likely reflects the sites of ABR neural generation relative to the electrode placement.
The responses between the two ears were similar with minor changes in peak amplitudes likely due to earphone positioning. The latency of the left and right ear being equivalent supports the equally healthy function of both ears and brain stem hemispheres in the hatchling chicken. Correct sound transducer placement can distinguish between good and no results.
The chicken ABR should be robust with a good signal-to-noise ratio. All the questions addressed by ABR studies in other avian species can be applied to the chicken. Also, molecular physiology research in embryonic chicken can incorporate this in vivo methodology.
