Name: data acquisition and analysis in brainstem evoked response audiometry in mice
Uploaded: 2019-05-10T14:00:00
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).

All animal procedures were performed according to the guidelines of the German Council on Animal Care and all protocols were approved by the local institutional and national committee on animal care (Landesamt f&#252;r Natur, Umwelt, und Verbraucherschutz, State Office of North Rhine-Westphalia, Department of Nature, Environment and Consumerism [LANUV NRW], Germany). The authors further certify that all animal experimentation was carried out in accordance with the National Institutes of Health Guide for the Care and Use ...

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 stud...

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>
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			<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>
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		<tr>
			<td>Cotton tip applicators (sterile)</td>
			<td>Carl Roth</td>
			<td>EH12.1</td>
			<td></td>
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			<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>
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			<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 
			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&#39;&#39; 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>
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			<td>Homeothermic 
			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&#8217;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 &#38; 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-/-</...

Watch this Scientific Journal Video about Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice at JoVE.com

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 ...

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.

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