Name: data acquisition and analysis in brainstem evoked response audiometry in mice
Uploaded: 2019-05-10T14:00:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice at JoVE.com

The authors would like to thank Dr. Christina Kolb (German Center for Neurodegenerative Diseases [DZNE]) and Dr. Robert Stark (DZNE) for their assistance in the animal breeding and animal health care. This work was financially supported by the Federal Institute for Drugs and Medical Devices (Bundesinstitut f&#252;r Arzneimittel und Medizinprodukte, BfArM, Bonn, Germany).

The authors have nothing to disclose.

This protocol provides a detailed and integrative description of how to record auditory evoked brainstem responses in mice. It puts specific focus on animal pretreatment, anesthesia, and potential methodological confounding factors. The latter include, among others, gender, mouse line, age, and housing conditions. It should be noted that all these factors can have an impact on sensorineural hearing loss and fundamental aspects of auditory information processing. Thus, appropriate stratification of auditory profiling studies is mandatory.
The instrumentation of AEP recordings has tremendously evolved in the last 50&#8211;60 years, and nowadays, commercial ABR recording systems are available which have enhanced and simplified the application of the technique but have also introduced new pitfalls. Some of these aspects are discussed here. First, the user should get used to the ABR system, that is, the instrumentation composed of desktop or laptop computer, the preamplifier, the amplifier, the electrode input box, and potential transducers (e.g., loudspeakers, insert earphones, supra-aural headphones, and bone oscillators). Notably, recording conditions are of central importance. Due to their high susceptibility, ABR recordings need to be shielded to protect them from contamination with external electrical noise and to guarantee an adequate signal-to-noise ratio.
Another important aspect is the instrumentation itself (e.g., the stimulus generator, transducers, and triggers). The most commonly used types of stimuli in mice are 100 &#181;s clicks and short-duration tone bursts with a modulated amplitude and/or frequency properties. Transducers can present a variety of acoustic stimuli either to one ear or to both ears. Here we have presented ABR results using a single loudspeaker rostral to the experimental animal. However, other approaches are also possible, including tubal-style insert earphones either in one ear or both ears. Supra-aural headphones as used in humans are not feasible in mice. As literature illustrates, different approaches can be successful, and they should be adapted depending on the experimental needs. Special attention has to be paid to the accurateness of the trigger which is essential for signal averaging as this digital pulse determines when each individual stimulus is presented. For proper recordings, the trigger and stimulus onset must be synchronous, representing time point zero. Commercially available ABR recording systems normally include self-contained triggers when the individual stimuli are presented. In many systems, there are external inputs which allow a connection from an external stimulus generator and an associated trigger. In both cases, it turned out to be valuable to control the stimulus and trigger characteristics using an external oscilloscope. Special attention also must be paid to the acquisition parameters (e.g., differential amplification, filtering, analog versus digital filters, filter designs, and parameters of signal averaging). Notably, parameters presented in the protocol presented here fit the experimental requirements of the exemplary results depicted above. However, adaptations, for instance in the sampling rate, number of stimuli applied for averaging, and their application frequency, might be necessary depending on the experimental settings.
Finally, some brief comments should be made on electrode impedance, electrode types, and electrode placement. The electrodes act like antennas, picking up voltage changes from below the skin. Subcutaneous electrode placement is mandatory as the mere application of electrodes on the skin or scalp is not suitable because of the resistance of the outer skin layer (i.e., the stratum corneum). Whereas in humans the electrical conductivity is normally improved by abrading dead skin cells and the application of an electrolyte gel or paste, this is usually not done and suitable in mice where subdermal electrodes are used. The interface of electrode and skin forms the electrode impedance which includes the electrical properties of the conductor in total. The conductor properties include the material properties of the electrode and the surface area of the contacting electrode, properties of the tissue including the debris (oil, dirt, sweat, etc.), and the electrolyte solution. The electrode material includes silver, gold, platinum, lead, tin, and stainless steel with low impedance and low electrode potentials. Care has to be taken that the electrode material is inert under recording conditions. With silver, this is achieved by using so-called complex electrodes (i.e., silver-silver chloride [Ag-AgCl] electrodes). In this case, the electrical double layer allows for a free exchange of ions which further reduces impedance. It is often recommended that electrode impedance should not exceed 5 k&#8486; and that the impedance of the individual electrodes (at least three) is comparable. It is also recommended that the interelectrode impedance should be below 2 k&#8486;. The recording electrode represents a long metal wire with an insulating coating. The wire electrode is connected via a plug to the recording equipment, in most cases the preamplifier/amplifier. In mice, the other end of the electrode wire is usually buildup of a needle electrode that might be left straight or&#8212;better&#8212;arcuated. Other electrode types, such as disk- or cup-shaped ones, no matter whether they are for reuse or pregelled disposable, are restricted to use in humans and not applicable to mice.

A central aspect of brain physiology is its capability to process environmental information resulting in different intrinsic or extrinsic output, such as learning, memory, emotional reactions, or motoric responses. Various experimental and diagnostic approaches can be used to characterize the electrophysiological responsiveness of individual neuronal cell types or clusters/ensembles of neurons within a stimulus-related neuronal circuitry. These electrophysiological techniques cover different spatiotemporal dimensions on the micro-, meso- and macroscale1. The microscale level includes voltage and current clamp approaches in different patch-clamp modes using, for instance, cultured or acutely dissociated neurons1. These in vitro techniques allow for the characterization of individual current entities and their pharmacological modulation2,3. An essential drawback, however, is the lack of systemic information as regards micro- and macrocircuitry information integration and processing. This impairment is partially overcome by in vitro techniques of the mesoscale, such as multielectrode arrays which allow for simultaneous extracellular multielectrode recordings not only in cultured neurons but also in acute brain slices4,5. Whereas microcircuitries can be preserved in the brain slices to a specific extent (e.g., in the hippocampus), long-range interconnections are typically lost6. Ultimately, to study the functional interconnections within neuronal circuitries, systemic in vivo electrophysiological techniques on the macroscale are the method of choice7. These approaches include, among other things, surface (epidural) and deep (intracerebral) EEG recordings which are carried out in both humans and animal models1. EEG signals are predominantly based on the synchronized synaptic input on pyramidal neurons in different cortical layers that can be inhibitory or excitatory in principal, despite the general predominance of excitatory input8. Upon synchronization, excitatory postsynaptic potential-based shifts in extracellular electrical fields are summed to form a signal of sufficient strength to be recorded on the scalp using surface electrodes. Notably, a detectable scalp recording from an individual electrode requires the activity of ten thousand of pyramidal neurons and a complex armamentarium of technical devices and processing tools, including an amplifier, filtering processes (low-pass filter, high-pass filter, notch filter), and electrodes with specific conductor properties.
In most experimental animal species (i.e., mice and rats), the human-based scalp EEG approach is technically not applicable, as the signal generated by the underlying cortex is too weak due to the limited number of synchronized pyramidal neurons9,10,11. In rodents, surface (scalp) electrodes or subdermal electrodes are thus severely contaminated by electrocardiogram and predominately electromyogram artifacts that make high-quality EEG recordings impossible9,11,12. When using unanesthetized freely moving mice and rats, it is therefore mandatory to directly record either from the cortex via epidural electrodes or from the deep, intracerebral structures to ensure the direct physical connection of the sensing tip of the lead/implanted electrode to the signal-generating neuronal cell clusters. These EEG approaches can be carried out either in a restraining tethered system setup or using the nonrestraining implantable EEG radio telemetry approach9,10,11. Both techniques have their pros and cons and can be a valuable approach in the qualitative and quantitative characterization of seizure susceptibility/seizure activity, circadian rhythmicity, sleep architecture, oscillatory activity, and synchronization, including time-frequency analysis, source analysis, etc.9,10,13,14,15,16,17.
Whereas tethered systems and radio telemetry allow for EEG recordings under restraining/semirestraining or nonrestraining conditions, respectively, related experimental conditions do not match the requirements for ABR recordings. The latter demand for defined acoustic stimuli which are presented repetitively over time with defined positions of a loudspeaker and experimental animal and controlled sound pressure levels (SPLs). This can be achieved either by head fixation under restraining conditions or following anesthesia18,19. To reduce the experimental stress, animals are normally anesthetized during ABR experimentation, but it should be considered that anesthesia can interfere with ABRs19,20.
As a general characteristic, the EEG is built up of different frequencies in a voltage range of 50-100 &#181;V. Background frequencies and amplitudes strongly depend on the physiological state of the experimental animal. In the awake state, beta (&#946;) and gamma (&#947;) frequencies with lower amplitude predominate. When animals become drowsy or fall asleep, alpha (&#945;), theta (&#952;), and delta (&#948;) frequencies arise, exhibiting increased EEG amplitude21. Once a sensory channel (e.g., the acoustic pathway) is stimulated, information propagation is mediated via neuronal activity through the peripheral and central nervous system. Such sensory (e.g., acoustic) stimulation triggers so-called EPs or evoked responses. Notably, event-related potentials (ERPs) are much lower in amplitude than the EEG (i.e., a few microvolts only). Thus, any individual ERP based on a single stimulus would be lost against the higher-amplitude EEG background. Therefore, a recording of an ERP requires the repetitive application of identical stimuli (e.g., clicks in ABR recordings) and subsequent averaging to eliminate any EEG background activity and artifacts. If ABR recordings are done in anesthetized animals, it is easy to use subdermal electrodes here.
Principally, AEPs include short-latency EPs, which are normally related to ABRs or BERA, and further, later-onset potentials such as midlatency EPs (midlatency responses [MLR]) and long-latency EPs22. Importantly, disturbance in the information processing of the auditory information is often a central feature of neuropsychiatric diseases (demyelinating diseases, schizophrenia, etc.) and associated with AEP alterations23,24,25. Whereas behavioral investigations are only capable of revealing functional impairment, AEP studies allow for precise spatiotemporal analysis of auditory dysfunction related to specific neuroanatomical structures26.
ABRs as early, short-latency acoustically EPs are normally detected upon moderate to high-intense click application, and there may occur up to seven ABR peaks (WI-WVII). The most important waves (WI-WV) are related to the following neuroanatomical structures: WI to the auditory nerve (distal portion, within the inner ear); WII to the cochlear nucleus (proximal portion of the auditory nerve, brainstem termination); WIII to the superior olivary complex (SOC); WIV to the lateral lemniscus (LL); WV to the termination of the lateral lemniscus (LL) within the inferior colliculus (IC) on the contralateral side27 (Supplementary Figure 1). It should be noted that WII-WV are likely to have more than one anatomical structure of the ascending auditory pathway contributing to them. Notably, the exact correlation of peaks and underlying structures of the auditory tract is still not fully clarified.
In audiology, ABRs can be used as a screening and diagnostic tool and for surgical monitoring28,29. It is most important for the identification of dysacusis, hypacusis, and anacusis (e.g., in age-related hearing loss, noise-induced hearing loss, metabolic and congenital hearing loss, and asymmetric hearing loss and hearing deficits due to deformities or malformations, injuries, and neoplasms)28. ABRs are also relevant as a screening test for hyperactive, intellectually impaired children or for other children who would not be able to respond to conventional audiometry (e.g., in neurological/psychiatric diseases such as ADHD, MS, autism etc.29,30) and in the development and surgical fitting of cochlear implants28. Finally, ABRs can provide valuable insight into the potential ototoxic side-effects of neuropsychopharmaceuticals, such as antiepileptics31,32.
The value of the translation of neurophysiological knowledge obtained from pharmacological or transgenic mouse models to humans has been demonstrated in numerous settings, particularly on the level of ERPs in auditory paradigms in mice and rats33,34,35. New insight into altered early AEPs and associated changes in auditory information processing in mice and rats can thus be translated to humans and is of central importance in the characterization and endophenotyping of auditory, neurological, and neuropsychiatric diseases in the future. Here we provide a detailed description of how ABRs can be successfully recorded and analyzed in mice for basic scientific, toxicological, and pharmacological purposes.

<table><tbody><tr><td>AEP/OAE Software for RZ6 (BioSigRZ software)</td><td>Tucker-Davis Technologies (TDT)</td><td>BioSigRZ</td><td></td></tr><tr><td>Binocular surgical magnification microscope</td><td>Zeiss Stemi 2000</td><td>0000001003877, 4355400000000, 0000001063306, 4170530000000, 4170959255000, 4551820000000, 4170959040000, 4170959050000</td><td></td></tr><tr><td>Cages (Macrolon)</td><td>Techniplast</td><td>1264C, 1290D</td><td></td></tr><tr><td>Carprox vet, 50mg/ml</td><td>Virbac Tierarzneimittel GmbH</td><td>PZN 11149509</td><td></td></tr><tr><td>Cold light source</td><td>Schott KL2500 LCD</td><td>9.705 202</td><td></td></tr><tr><td>Cotton tip applicators (sterile)</td><td>Carl Roth</td><td>EH12.1</td><td></td></tr><tr><td>Custom made meshed metal Faraday cage (stainless steel, 2 mm thickness, 1 cm mesh size)</td><td>custom made</td><td>custom made</td><td></td></tr><tr><td>5% Dexpanthenole (Bepanthen eye and nose creme)</td><td>Bayer Vital GmbH</td><td>PZN: 01578681</td><td></td></tr><tr><td>Disposable Subdermal stainless steel Needle&lt;br/&gt; electrodes, 27GA, 12mm</td><td>Rochester Electro-Medical, Inc.</td><td>S03366-18</td><td></td></tr><tr><td>Surgical drape sheets (sterile)</td><td>Hartmann</td><td>PZN 0366787</td><td></td></tr><tr><td>Ethanol, 70%</td><td>Carl Roth</td><td>9065.5</td><td></td></tr><tr><td>1/4&#039;&#039; Free Field Measure Calibration Mic Kit</td><td>Tucker-Davis Technologies (TDT)</td><td>PCB-378C0</td><td></td></tr><tr><td>Gloves (sterile)</td><td>Unigloves</td><td>1570</td><td></td></tr><tr><td>Graefe Forceps-curved, serrated</td><td>FST</td><td>11052-10</td><td></td></tr><tr><td>GraphPad Prism 6 Software, V6.07</td><td>GraphPad Prism Software, Inc.</td><td>https://www.graphpad.com/</td><td></td></tr><tr><td>Heat-based surgical instrument sterilizer</td><td>FST</td><td>18000-50</td><td></td></tr><tr><td>Homeothermic&lt;br/&gt; heating blanked</td><td>ThermoLux</td><td>461265 / -67</td><td></td></tr><tr><td>Ketanest S (Ketamine), 25mg/ml</td><td>Pfizer</td><td>PZN 08707288</td><td></td></tr><tr><td>Ringer&amp;rsquo;s solution (sterile)</td><td>B.Braun</td><td>PZN 01471434</td><td></td></tr><tr><td>Matlab software</td><td>MathWorks, Inc.</td><td>https://de.mathworks.com/products/matlab.html</td><td></td></tr><tr><td>Medusa 4-Channel Low Imped. Headstage</td><td>Tucker-Davis Technologies (TDT)</td><td>RA4LI</td><td></td></tr><tr><td>Medusa 4-Channel Pre-Amp/Digitizer</td><td>Tucker-Davis Technologies (TDT)</td><td>RA4PA</td><td></td></tr><tr><td>Microphone</td><td>PCB Pieztronics</td><td>378C01</td><td></td></tr><tr><td>Multi Field Speaker- Stereo</td><td>Tucker-Davis Technologies (TDT)</td><td>MF1-S</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>DPO3012</td><td></td></tr><tr><td>Optical PC1 express card for Optibit Interface)</td><td>Tucker-Davis Systems (TDT)</td><td>PO5e</td><td></td></tr><tr><td>Askina Braucel pads (cellulose absorbet pads)</td><td>B.Braun</td><td>PZN 8473637</td><td></td></tr><tr><td>Preamplifier</td><td>PCB Pieztronics</td><td>480C02</td><td></td></tr><tr><td>RZ6 Multi I/O Processor system (BioSigRZ)</td><td>Tucker-Davis Technologies (TDT)</td><td>RZ6-A-PI</td><td></td></tr><tr><td>0.9% saline (NaCl, sterile)</td><td>B.Braun</td><td>PZN:8609255</td><td></td></tr><tr><td>SigGenRZ software</td><td>Tucker-Davis Technologies (TDT)</td><td>https://www.tdt.com/</td><td></td></tr><tr><td>Software R (version 3.2.1) + Reshape 2 (Version 1.4.1) + ggplot 2 (version 1.0.1) + datatable (version 1.9.4), + gdata (version 2.13.3), + pastecs (version 1.3.18), + waveslim (version 1.7.5), + MassSpecWavelet (version 1.30.0)</td><td>The R Foundation, R Core Team 2015</td><td>Open Source Software (freely distributable)</td><td></td></tr><tr><td>Sound attenuating cubicle</td><td>Med Associates Inc.</td><td>ENV-018V</td><td></td></tr><tr><td>Standard Pattern Forceps, 12cm and 14.5 cm length</td><td>FST</td><td>11000-12, 11000-14</td><td></td></tr><tr><td>Leukosilk tape</td><td>BSN medical GmbH &amp;amp; Co. KG</td><td>PZN 00397109</td><td></td></tr><tr><td>Tissue Forceps- 1x2 Teeth 12 cm</td><td>FST</td><td>11021-12</td><td></td></tr><tr><td>Uniprotect ventilated cabinet</td><td>Bioscape</td><td>THF3378</td><td></td></tr><tr><td>Ventilated cabinet</td><td>Tecniplast</td><td>9AV125P</td><td></td></tr><tr><td>Xylazine (Rompun), 2%</td><td>Bayer Vital GmbH</td><td>PZN 1320422</td><td></td></tr></tbody></table>

data acquisition and analysis in brainstem evoked response audiometry in mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Click- and tone burst-evoked ABR recordings can be used to evaluate hearing threshold differences, amplitude growth function, and latency comparison. Click-evoked ABRs in the SPL increasing mode are depicted in Figure 1 for controls and two exemplary mutant mouse lines which are deficient for the Cav3.2 T-type voltage-gated Ca2+ channel (i.e., Cav3.2+/- and Cav3.2 null mutants [Cav3.2-/-]). As outlined above, a gender-specific investigation is generally recommended, due to sex-specific differences in auditory parameters in humans54,55 and mice56,57. ABRs to free-field click (0.1 ms) and tone burst (1&#8211;42 kHz in 6 kHz steps, 4.5 ms in total with a 1.5 ms ramp time) acoustic stimuli were recorded as described in the protocol. Note that the vertex-positive potentials are plotted as upward deflections as depicted in representative click-evoked recordings for female Cav3.2+/+ (Figure 1A), Cav3.2+/- (Figure 1B), and Cav3.2-/- mice (Figure 1C). In this setting, representative ABRs in females suggested an increased click-evoked ABR hearing threshold and altered amplitude growth function in female Cav3.2-/- mice compared to Cav3.2+/+ and Cav3.2+/- animals. The same tendency was observed for males which suggested an increased click-evoked ABR thresholds and reduced amplitudes in Cav2.3-/- compared to controls and heterozygous Cav3.2+/- mice. Exemplary tone burst-evoked ABRs are depicted in Figure 2 for female Cav3.2+/+, Cav3.2+/-, and Cav3.3-/- mice (all animals were 20 weeks of age).
As a first step in analyzing general hearing performance, click-evoked ABRs for different SPLs (0&#8211;90 dB) were investigated using the automated ABR threshold detection system described in section 5 of the protocol (Figure 3). The analyzed animals were age matched as aging can have a dramatic impact on sensorineural hearing loss58,59. Next, potential alterations in ABR threshold levels evoked by different tone burst frequencies (1&#8211;42 kHz, Figure 4) were analyzed. In the exemplary mouse lines, Cav2.3+/- and Cav3.2-/- exhibited increased click- and tone burst-related hearing thresholds compared to controls (all animals were 20 weeks of age).
Using the wavelet-based approach outlined above, click-evoked ABR amplitude growth function and ABR waveform latency analysis were carried out (Figure 5 and Figure 6, respectively). The latter allows insight into the possible spatiotemporal influence of the gene of interest on auditory information processing within the inner ear and brainstem.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59200/59200fig1.jpg" /> 
Figure 1: Click-evoked ABRs in controls and mutant mice (Cav3.2+/-, Cav3.2-/-). Representative ABRs obtained from (A) Cav3.2+/+, (B) Cav3.2+/-, and (C) Cav3.2-/- female mice upon click stimulation in the increasing SPL mode (from 0&#8211;90 dB with 5 dB SPL steps). For averaging, each stimulus entity was applied 300 times at 20 Hz. The acoustic stimulus onset is indicated by a vertical red line. This figure is modified from Lundt et al.60. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59200/59200fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59200/59200fig2.jpg" /> 
Figure 2: Tone burst-evoked ABRs in controls and mutant mice (Cav3.2+/-, Cav3.2-/-). Representative ABRs from (A) Cav3.2+/+, (B) Cav3.2+/-, and (C) Cav3.2-/- female mice following tone bursts of 1&#8211;42 kHz (6 kHz steps) at an SPL of 80 dB. For averaging, each stimulus entity was presented 300 times at 20 Hz. The acoustic stimulus onset is indicated by a vertical red line. This figure is modified from Lundt et al.60. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59200/59200fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59200/59200fig3.jpg" /> 
Figure 3: Click-evoked ABR-based hearing thresholds in controls and mutant mice (Cav3.2+/-, Cav3.2-/-). Click-evoked audiometric hearing threshold of (A) female and (B) male Cav3.2+/+ (female: n = 12; male: n = 13), Cav3.2+/- (female: n = 10; male: n = 9), and Cav3.2-/- mice (female: n = 10; male: n = 9). Data are plotted as mean &#177; SEM. Statistical significances were determined using &#945;-level = 0.05 and p-values defined as *p &#60; 0.05; **p &#60; 0.01; ***p &#60; 0.001; ****p &#60; 0.0001. This figure is modified from Lundt et al.60. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59200/59200fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59200/59200fig4v2.jpg" /> 
Figure 4: Tone burst-evoked ABR-based hearing thresholds in controls and mutant mice (Cav3.2+/-, Cav3.2-/-). 1&#8211;42 kHz (6 kHz steps) tone burst-evoked ABR-based audiometric hearing thresholds for Cav3.2+/+ (female: n = 12; male: n = 12; &#9650;), Cav3.2+/- (female: n = 10; male: n = 8; &#9632;), and Cav3.2-/- animals (female: n = 10; male: n = 9; &#9675;). Data are plotted as mean &#177; SEM. Statistical significances were determined using &#945;-level = 0.05 and p-values defined as *p &#60; 0.05; **p &#60; 0.01; ***p &#60; 0.001; ****p &#60; 0.0001. This figure is modified from Lundt et al.60. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59200/59200fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59200/59200fig5.jpg" /> 
Figure 5: Amplitude growth function on click-based ABR recordings in controls and mutant mice (Cav3.2+/-, Cav3.2-/-). WI&#8211;WIV amplitude (in microvolts) plotted against an increasing SPL (in decibels) for click-evoked ABR wave analysis in Cav3.2+/+ (female: n = 12; male: n = 11; black line representing the approximated control curve including the 95% confidence interval in grey), Cav3.2+/- (female: n = 8; male: n = 7; &#9632;), and Cav3.2-/- animals (female: n = 7; male: n = 9; &#9675;). Both Cav3.2-/- female and male mice exhibit significantly delayed increase in the amplitude growth across the increasing SPLs for (A and B) WI, (C and D) WII, and (G and H) WIV compared to Cav3.2+/+ and Cav3.2+/- mice. (E and F) For WIII, only Cav3.2-/- male mice displayed a significant delay in amplitude growth across the increasing SPL compared to female Cav3.2-/- animals. Data are presented as mean &#177; SEM. Statistical significances were determined using &#945;-level = 0.05 and p-values defined as *p &#60; 0.05; **p &#60; 0.01; ***p &#60; 0.001; ****p &#60; 0.0001. This figure is modified from Lundt et al.60. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59200/59200fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59200/59200fig6.jpg" /> 
Figure 6: Latency analysis upon click-evoked ABR recordings in controls and mutant mice (Cav3.2+/-, Cav3.2-/-). Latencies (in milliseconds) for each ABR wave (WI&#8211;WIV) at 65 dB SPL are depicted for Cav3.2+/+ (female: n = 12; male: n = 11), Cav3.2+/- (female: n = 8; male: n = 7), and Cav3.2-/- mice (female: n = 8; male: n = 9).&#160;Note that latency analysis can also be performed at specific sensation levels. Data are depicted as mean &#177; SEM. Statistical significances were determined using &#945;-level = 0.05 and p-values defined as *p &#60; 0.05; **p &#60; 0.01; ***p &#60; 0.001; ****p &#60; 0.0001. This figure is modified from Lundt et al.60. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59200/59200fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: ABR architecture and electrode positioning. (A) Representative ABR recording at 65 dB SPL. The initial baseline (TW1, 5 ms) was followed by the test stimulus (click or tone burst) and TW2 (10 ms) containing the early brainstem-evoked potentials. TW2 was followed by another baseline (TW3, 10 ms). The baseline periods were used to calculate the SD of the baseline noise. Whenever an individual ABR wave (WI&#8211;WIV) amplitude exceeded the SD of the baseline noise in fourfold, the hearing threshold was reached. For wave amplitude and latency comparison, a &#8220;Mexican hat&#8221;-based wavelet approach was carried out to automatically detect negative peaks (blue-yellow striped lines) and positive peaks (red-grey striped lines). Green crosses indicate the absolute maximum ABR wave amplitudes and do not display approximated values based on the wavelet approach. (B) For the ABR recordings, subdermal stainless-steel electrodes with a hook-shaped tip were used. The reference electrode was placed at the left hip, the positive (+) electrode was positioned at the vertex (axial of the pinnae), and the negative (-) electrode was inserted ventrolateral of the right pinna depending on whether a monaural or binaural recording was carried out. This figure is modified from Lundt et al.60. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59200/Suppl_Fig_1_final_MWe_30112018.pdf">Please click here to download this file.</a>

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Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

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Neuroscience

11.6K Views.  Federal Institute for Drugs and Medical Devices. Dit protocol biedt gedetailleerde informatie over het plannen, uitvoeren en analyseren van klik- en toonstormreacties bij muizen. Het belangrijkste voordeel van deze techniek is dat het zorgt voor complexe en snelle auditieve profilering van farmacologische en gemuteerde muismodellen. De nieuwe inzichten in alt-er-ed early APP en bijbehorende verandering in de auditieve verwerking bij muizen en ratten kunnen worden vertaald naar de mens.Daarom is deze methode van centraal belang in de ...

Video: Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Semi-Automated Analysis of Peak Amplitude and Latency for Auditory Brainstem Response Waveforms Using R

Many reports in the last 15 years have assessed changes in the auditory brainstem response (ABR) waveform after insults such as noise exposure. Common changes include reductions in the peak 1 amplitude and the relative latencies of the later peaks, as well as increased central gain, which is reflected by a relative increase in the amplitudes of the later peaks compared to the amplitude of peak 1. Many experimenters identify the peaks and troughs visually to assess their relative heights and latencies, which is a laborious process when the waveforms are collected in 5 dB increments throughout the hearing range for each frequency and condition. This paper describes free routines that may be executed in the open-source platform R with the RStudio interface to semi-automate the measurements of the peaks and troughs of auditory brainstem response (ABR) waveforms. The routines identify the amplitudes and latencies of peaks and troughs, display these on a generated waveform for inspection, collate and annotate the results into a spreadsheet for statistical analysis, and generate averaged waveforms for figures. In cases when the automated process misidentifies the ABR waveform, there is an additional tool to assist in correction. The goal is to reduce the time and effort needed to analyze the ABR waveform so that more researchers will include these analyses in the future.

This article describes the semi-automated measurement of the amplitudes and latencies of the first five peaks and troughs in the auditory brainstem response waveform. An additional routine compiles and annotates the data into a spreadsheet for experimenter analysis. These free computer routines are executed using the open-source statistical package R.

Many reports in the last 15 years have assessed changes in the auditory brainstem response (ABR) waveform after insults such as noise exposure. Common ...

Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly understand the mechanism of NIHL, and subsequently optimize the corresponding treatment strategies. This study aims to create an improved protocol for developing a mouse model of NIHL. Male C57BL/6J mice were used in this study. Unanesthetized mice were exposed to loud noises (1 and 6 kHz, presented simultaneously at 115-125 dB SPL-A) continuously for 6 h per day for 5 consecutive days. Auditory function was assessed 1 day and 1 week after noise exposure, using auditory brainstem response (ABR). After the ABR measurement, the mice were sacrificed, and their organs of Corti were collected for immunofluorescence staining. From the auditory brainstem response (ABR) measurements, significant hearing loss was observed 1 day after noise exposure. After 1 week, the hearing thresholds of the experimental mice decreased to ~80 dB SPL, which was still a significantly higher level than the control mice (~40 dB SPL). From the results of immunofluorescence imaging, outer hair cells (OHCs) were shown to be damaged. In summary, we created a model of NIHL using male C57BL/6J mice. A new and simple device for generating and delivering pure-tone noise was developed and then employed. Quantitative measurements of hearing thresholds and morphological confirmation of OHC damage both demonstrated that the applied noise successfully induced an expected hearing loss.

Here, we present a protocol for a mouse model of noise-induced hearing loss (NIHL). To induce NIHL, we developed a new and simple device using corrugated plastic, a rat trap cage, and a speaker. Auditory brainstem response and immunofluorescence imaging were employed to assess the hearing function and outer hair cell damage, respectively.

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly ...

Evaluation of Ribbon Synapses at Specific Frequency Regions of Mouse Cochlea

Source: Yu, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59189">Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea.</a> J. Vis. Exp. (2019).
This video demonstrates a method for evaluating ribbon synapses in specific regions of the mouse cochlea. It utilizes immunostaining and confocal imaging techniques to identify functional synapses across different frequency regions in each cochlear turn.

All procedures involving animal handling have been performed in accordance with the institute&#39;s IRB guidelines.
1. Animal Selection

	For all ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

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.

EoE Sub Articles

Recording Electrically-Evoked Auditory Brainstem Responses in a Deafened Mouse

To insert the cochlear implant electrode array, place the retractor tool back into the incision to re-access the bulla, and insert the electrode array into the scala tympani, at a depth such that the fourth platinum ring of the array is located just inside the round window.
The insertion of the electrode array can also be tricky, so take your time to find a good insertion angle using one forcep tool, the lead wire, and another to insert the array.
Coil the lead wire inside the bulla, and glue the wire to the tissue above the bulla.
After carefully removing the retractor, enclosing the insertion with tissue glue, use a scalpel to make a 0.5-millimeter incision in the neck, perpendicular to the line between where the active and reference aVR electrodes will be. Place the ground ball in the insertion, and connect the electrode array board to the Animal Stimulator Platform.
To perform an electric aVR, place the electrodes as previously demonstrated, and open the Animal Stimulator Platform Software. Then, define the electric pulse stimulation paradigm, and present the electric pulse trains recording the evoked electric eABR responses continuously via the TDT headstage pre-amplifier and auditory processor.

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

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