This protocol provides detailed information about how to plan, carry out and analyze click and tone-burst evoked auditory brainstem responses in mice. The main advantage of this technique is that it allows for complex and fast auditory profiling of pharmacological and mutant mouse models. The new insights into alt-er-ed early APP and associated change in the auditory processing in mice and rats can be translated to humans.
Therefore, this method is of central importance in the characterization and the phenotyping of auditory, neurological and diseases. This method is most important for the identification of dysacusis, hypoacusis and anacusis. For example, in age related, noise-induced metabolic, congenital and aspi-met-acury hearing loss as well as in hearing deficits due to deformities or malformations, injuries and neoplasms.
Users new to the technique, should pay special attention to proper electro-placement and pre-experimental calibration of the system. Visual demonstration of the method is critical to illustrate anesthesia, ABR recording, ABR filtering processes and automated ABR neur-o-lass-es. Begin by turning on the pre-amplifier connected to the microphone, at least five minutes prior to the calibration, to allow for the equilibration of the system.
Turn on the oscilloscope. Then position the microphone, connected to a pre-amplifier inside the sound attenuating cubical, to mimic the experimental murine ear. Next, open the commercially available processing and acquisition softwares and program the stimulus protocols for the clicks and tone bursts.
Start with the click stimulus entity to analysis and determine click thresholds. Followed by ABR symmetry of the left and right ear. And ABR amplitudes and latencies later on.
Next, use the same software to verify tone burst stimulation protocol using Sig-Gen RZ stimulus design software. And checks stimulus settings, under Bio-Sig RZ acquisition software. Program the appropriate frequency range to be tested, depending on the scientific question, and ensure that the frequency ranges to be applied meet the technical capabilities of the loud speaker.
For averaging, set the number of sequential acoustics stimuli, either clicks or tone bursts for instance, at 300 times, with a rate of 20 per second;an averaging duration of 25 milliseconds and the amplification factor of the pre-amplifier, 20 times. Next, verify appropriate sampling rate for ABR data acquisition and then pass filter using a six-poll butter-worth filter. Activate the notch filter if necessary.
Start the tone burst calibration by selecting the calibration:CAL200K file;within the software, to activate the calibration configuration mode. And choose perimeters according to the experimental conditions. Use the processor system to execute the calibration procedure.
Make sure that the technical specifications of the microphone and loud speaker, in terms of sound pressure level or SPL limits, frequency range and distribution harmonize. Then, select and start the pre-defined click stimulation protocol. Run a single click SPL to verify that the spectrum of sound stimuli is analyzed by online fast four-ay transformation of the oscilloscope, matches the requirements.
Select and start the pre-defined tone burst stimulation protocol, within the range of one to 42 kilohertz. Confirm the frequency spectrum of the recorded acoustic test stimuli, by using an oscilloscope and online FFT. Finally, complete the tone burst calibration by loading the created calibration file to the tone burst stimulus protocol.
Begin by placing the anesthetized mouse inside a sound-attenuating cubicle, lined with acoustical foam. For the recording of monaural brain stem evoked auditory potentials, insert sub-dermal stainless steel electrodes at the vertex, axial of the pin-eye and ventral-lateral of the right or left pinna depending on the ear to be measured. On the other end, for binaural recordings, place the negative electrodes at both the right and left pinna.
Position the ground electrode at the hip of the animal. Prior to the insertion, form a hook shape at the tip of the stainless steel electrode that's sub-dermal fixation of the electrodes is guaranteed. And once inserted, properly place the rostrum of the mouse 10 centimeters opposite to the loud speaker.
Connect all electrodes to the head stage and check for their impedance. Then, perform impedance measurements of all electrodes, prior to each recording, to verify proper electrode positioning and conductivity. Record ABRs under free field conditions using a single loud speaker.
Finally, conduct ABR analysis via automated threshold detection and wave latency analysis to determine positive peaks and negative waves. As a first step in analyzing general hearing performance, click-evoked ABRs for different SPLs, between zero and 90, were investigated using the automated ABR threshold detection system. Potential alterations in ABR threshold levels evoked by different tone burst frequencies were analyzed.
In the exemplary mouse lines, Cav3.2 plus, minus and Cav3.2 minus, minus exhibited increased click and tone burst related hearing thresholds compared to controls. Lastly, click-evoked ABR amplitude growth function and ABR wave form latency analysis were carried out via wavelet based approach. The latter allows insight into the possible spacio-temporal influence of the gene of interest on auditory information processing within the inner ear and brainstem.
Proper ABR electrode placement in pinna's measurement and system calibration are essential for performing this technique. The auditory approach presented here, can also be used in combination with a telemetry system to analyze complex, mid-latency and late-auditory evoked potentials. This helps in the characterization and in the phenotyping of auditory, neurological and neuro diseases.
