recording auditory brainstem responses in a rat pup

Recording Auditory Brainstem Responses in a Rat Pup

To begin this procedure, place the anesthetized animal in a polyethylene foam mold to immobilize the animal&#39;s body. Then, place the animal on an anti-vibration table in a sound-attenuating room. Keep the animal&#39;s body temperature at 37.5 degrees Celsius with a heating pad.
Next, wipe the head area with 70% ethanol. Make a 1 to 2-millimeter incision ventrolaterally to the external pinna, to place the reference electrode or ground electrode. Then, place a subdermal recording electrode over the skull vertex.
Using the function generator, generate calibrated tone bursts of various frequencies and intensities. Deliver the sound stimuli through an electrostatic speaker located 10 centimeters away from the head of the animal.
To obtain the auditory brainstem responses, the sound elicited potentials are filtered, amplified, and averaged using a multifunction processor. The 40-minute ABR recording is monitored online and stored for offline analysis.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Auditory Brainstem Response (ABR) Recording
<ol>
	<li>Anesthetize Sprague Dawley (SD) rats (1-14 day old pups and 2-month-old adults, both sexes) with a single injection of 1.5% sodium pentobarbital (22 mg/kg for pups and 30 mg/kg for adults, intraperitoneal (i.p.).</li>
	<li>Place the anesthetized animal in a polyethylene-foam mold to immobilize the animal&#39;s body. Place the animal on an anti-vibration table in a sound-attenuating room. Keep the animal&#39;s body temperature at 37.5 &#176;C with a heating pad.</li>
	<li>Wipe the head area with 70% (vol/vol) ethanol. By using a fine scissors, make a 1-2 mm incision ventrolateral to the external pinna to place the reference electrode or ground. A subdermal needle electrode (recording electrode) is located over the skull vertex (Figure 1A).</li>
	<li>Using the function generator, generate calibrated tone bursts (1 ms rise/fall, 3 ms plateau) of various frequencies (2, 4, 8, 16, 24, and 32 kHz) and intensities (decreased from 85 to 10 dB SPL in 5 dB step). Vary the frequency and amplitude manually or using a computer. Deliver sound stimuli through an electrostatic speaker located 10 cm away toward the head of the animal.</li>
	<li>Filter (100-1,000 Hz), amplify, and average (256 times) the sound elicited potential using a multi-function processor to get the ABRs. The ABR traces are monitored online and stored for offline analysis. It takes about 40 min to complete the ABR recording from one animal. 
	NOTE: Usually, three to seven peaks (wave I to wave VII) with latencies less than 15 ms can be identified (Figure 1B). The ABR threshold is defined as the minimum sound level at which wave I and II could be detected.</li>
	<li>Euthanize before the animals awake from anesthesia (with an intraperitoneal injection of 0.3 mL 1.5% sodium pentobarbital).</li>
</ol>

Source: Chang, A. et al., <a href="https://app.jove.com/v/56678">Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats.</a> J. Vis. Exp. (2018).
This video demonstrates a method for recording auditory brainstem responses (ABRs) from rat pups. It outlines the steps for electrode placement, sound stimulus application, and signal processing analysis in the brainstem to assess neural responses.

<img alt="Figure 1" src="/files/ftp_upload/22835/22835_Figure1.jpg" /> 
Figure 1. Auditory brainstem response recording. (A) The recording electrode was inserted into the scalp between two ears. Ground and reference electrodes were placed under the left and right pinna, respectively. An open field speaker was placed 10 cm away from the nasal tip of the rat. (B) Two representative ABR waveforms in response to 16 kHz, 80 dB SPL tone bu...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immobilize an anesthetized rat pup in a foam mold.
Position the pup on an anti-vibration table within a soundproof room to minimize vibration and noise-related artifacts. Place a heating pad underneath to maintain body temperature.
Sterilize the head. 
Make incisions near the outer ears to place the reference and ground electrodes.
Next, insert the recording electrode beneath the skin at the skull vertex.
Using a speaker connected to a sound generator, deliver sound pulses of varying frequencies and intensities.
The mechanical vibrations from the sound pulses deflect the hair cell projections in the rat&#39;s inner ear. 
This deflection opens mechanically-gated ion channels, inducing a positive ion influx and hair cell depolarization.
The depolarized hair cells release neurotransmitters, which bind to the inner ear neuronal receptors, generating action potentials.
The action potentials propagate through the auditory nerve to the brainstem.
The recording electrode detects the signal, which a processor converts into the auditory brainstem response.

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Recording Auditory Brainstem Responses in a Rat Pup

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