We thank the grants from the Ministry of Science and Technology (MOST) of the Taiwan Government (MOST 110-2314-B-715-005, MOST 111-2314-B-715-009-MY3), and intramural research grants from Mackay Medical College (MMC-RD-110-1B-P030, MMC-RD-111-CF-G002-03).

Animal experiments in this study were approved by the Animal Care Committee of Mackay Medical College. Eight-week-old Male C57BL/6J mice were purchased from the National Laboratory Animal Center (New Taipei City, Taiwan). All mice were bred and housed in accordance with the standard animal protocol.
1. Induction of NIHL in mice
<ol>
	<li>Prepare the cage for the experimental mice
	<ol>
		<li>To do so, use a rat trap cage with dimensions of 14 cm &#215; 17 cm &#215; 24 cm. Cut four pieces of corrugated plastic boards into appropriate sizes, making them fit into the cage (13 cm &#215; 23 cm and 13 cm &#215; 16 cm).</li>
		<li>To prevent the mice from getting their feet cut by the mesh grid, place two of the pieces at the bottom and on the back side, respectively. Place the other two pieces perpendicularly interlocked with each other, to divide the space within the cage into quarters.</li>
	</ol>
	</li>
	<li>Carry out this study in a soundproof box. Place a speaker 8.5 cm in front of the cage, and place both the speaker and the cage in a soundproof box.</li>
	<li>Open the CLIO application software</li>
	<li>Move the cursor to the speaker icon, then click on TwoSin. Enter and change the value of Freq 1 to 1000 Hz, Freq 2 to 6000 Hz, and click on OK to start playing the sound.</li>
	<li>Under the Leq tab, change dBV to dBSPL. After setting the time on the software interface, click the green triangle button to play the sound.</li>
	<li>Place a microphone in front of the speaker at a distance of 8.5 cm to calibrate the noise level (see Supplementary File 1). Adjust the noise level to 125 dB SPL-A and continuously monitor for no less than 3 min to ensure that the noise level is sufficiently stable. Use a generator, an analyzer, and an amplifier to create and control the noise. (Figure 1) 
	NOTE: Do this step in a soundproof box to avoid damage to the operator&#39;s hearing.</li>
	<li>Place four male C57BL/6J mice into the cage (one for each quarter) for noise exposure. Randomly assign the mice in each quarter during the noise exposure. Place a microphone onto the top of the cage to monitor the noise level during the noise exposure (Figure 2). 
	NOTE: Do this step in a soundproof box to avoid damage to the operator&#39;s hearing.</li>
	<li>Expose the mice to the noise at frequencies of 1 kHz and 6 kHz continuously for 6 h per day, for 5 consecutive days.</li>
	<li>Measure the hearing thresholds of the mice 1 day after the noise exposure (i.e., on the 6th day) by measuring the ABR. Repeat these ABR measurements again 1 week after the noise exposure (i.e., on the 13th day). After the ABR measurements, sacrifice all the involved mice and harvest their cochleae (Figure 3).</li>
</ol>
2. Auditory brainstem response (ABR)-based assessment of hearing threshold
<ol>
	<li>Use a commercial ABR testing system specifically designed for small animals10.</li>
	<li>Intraperitoneally inject a mixture of tiletamine and zolazepam (40 mg/kg) and xylazine (9.3 mg/kg) into the mice for general anesthesia. 
	NOTE: ABR assessment takes ~2 h. Make sure to provide thermal support via a heating pad and apply eye ointment to prevent dryness while the mouse is under anesthesia.</li>
	<li>Measure the ABR in the mouse under general anesthesia. Place subdermal needle electrodes (12 mm) at the vertex, behind the pinna of the left ear, and back near the tail to measure the hearing threshold.</li>
	<li>Present acoustic stimuli using a speaker placed 1 cm from the animal&#39;s left ear.</li>
	<li>Use an oscilloscope to control the acoustic stimuli. Choose Sine Wave for the stimuli and 10 k for the window scale. Turn the Frequency knob to obtain the desired frequency of the acoustic stimuli.</li>
	<li>Adjust the stimulus intensity by turning the AMLP knob on the function generator. Obtain the desired stimulus intensity by turning the AMLP knob to a suitable voltage, calculated from calibration.</li>
	<li>Collect the ABR measurements under a series of stimulus intensities, from 10 dB SPL to 100 dB SPL, with a 10 dB step size. 
	NOTE: Measure the hearing threshold in a soundproof box. The minimum stimulus intensity level that could give rise to a discernable Wave V in the collected signal was assumed to be the ABR threshold10,11 (Figure 4). Post-ABR assessment, monitor the animal until it recovers from anesthesia (~1 h).</li>
</ol>
3. Microscopic examination
<ol>
	<li>Fixing the harvested tissue
	<ol>
		<li>After the ABR measurements, sacrifice the mouse by intraperitoneally injecting a mixture of tiletamine and zolazepam (100 mg/kg) and xylazine (23.25 mg/kg) for the microscopic examination.</li>
		<li>Harvest the cochleae from the mouse and immediately immerse them in 10% formaldehyde (FA) for fixation (two cochlea/mL) for at least 8 h at 4 &#176;C.</li>
		<li>Following fixation, replace the FA solution with a 10% ethylenediaminetetraacetic acid (EDTA) solution for decalcification for 3-4 days at 4 &#176;C. Then, check each cochlea with tweezers to confirm that the cochleae are sufficiently softened.</li>
		<li>Place the cochleae in a Petri dish filled with phosphate-buffered saline (PBS), and cut the spiral-shaped organ of Corti (OC) (each decalcified cochlea) into three sections-the basal turn, middle turn, and apical turn-for tissue staining.</li>
		<li>Under a dissecting microscope (magnification: 8x-35x), remove the bony structures (scala vestibuli, scala tympani, and modiolus) of the decalcified cochleae and obtain the soft tissue, including the OC (thickness: about 40 &#181;m).</li>
	</ol>
	</li>
	<li>Cochlea immunofluorescence staining
	<ol>
		<li>Preparation of the blocking buffer: Prepare 2% bovine serum albumin (BSA) and 0.2% Triton X-100 solutions in PBS.</li>
		<li>Transfer the tissue to be stained from the Petri dish to a microcentrifuge tube, and immerse the tissue in 0.1 mL of the blocking buffer for 2 h at room temperature (RT).</li>
		<li>Preparation of the Myo7A primary antibody buffer: Dilute the Myo7A primary antibody in the blocking buffer at a volume ratio of 1:200.</li>
		<li>Treat the tissue in the microcentrifuge tube with 100 &#181;L of Myo7A primary antibody buffer for 2 h at RT.</li>
		<li>Rinse the tissue three times in PBS for 5 min each.</li>
		<li>Preparation of the Myo7A secondary antibody (Myo7A-Rb-488) and phalloidin antibody (phalloidin-594) buffer: Dilute Myo7A secondary antibody (1:400) and phalloidin antibody (1:200) in the blocking buffer.</li>
		<li>Treat the tissue with 100 &#181;L of Myo7A secondary antibody and phalloidin antibody buffer for 2 h at RT.</li>
		<li>After antibody + phalloidin antibody treatment, rinse the tissue in PBS three times, for 5 min each.</li>
		<li>Transfer the rinsed tissue from the microcentrifuge tube to a Petri dish filled with PBS using a 1 mL plastic transfer pipette with a cut tip.</li>
		<li>To prepare for the microscopic examination, unfold the tissue, place it onto a glass slide, and add 15 &#181;L of 4&#39;,6-diamidino-2-phenylindole (DAPI) fluoromount medium onto the tissue to fully cover it. Place a glass coverslip gently over the tissue. Leave the slide overnight at RT before sealing it with nail polish.</li>
	</ol>
	</li>
	<li>Image acquisition
	<ol>
		<li>Acquire 2D images using an upright fluorescence microscope and image acquisition software.</li>
		<li>Before performing the microscopic examination, center the tissue in the field of view and adjust the sensitivity to ISO 100. Adjust the exposure time initially by clicking on the Automatic Mode button of the software, and then fine-tune manually by clicking on the Adjust button to optimize the signal-to-noise ratio.</li>
		<li>Adjust the wavelength of the incident light to excite the fluorophores by rotating the fluorescence cube. Pseudo colors were used to mark the emission from different fluorescent labels (blue light: DAPI; green light: Myo7A; red light: phalloidin).</li>
		<li>By scanning the tissue sample, generate and collect the imaging data as .tif and .jpg image files.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>World Report on Hearing. World Health Organization Available from: <a target="_blank" href="https://www.who.int/publications/i/item/9789240020481">https://www.who.int/publications/i/item/9789240020481</a> (2021)</li><li>Fernandez, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+cochlear+synaptopathy+with+and+without+sensory+cell+loss.">Noise-induced cochlear synaptopathy with and without sensory cell loss.</a> Neuroscience. 427, 43-57 (2020).</li><li>Kujawa, S. G., Liberman, M. 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L., Matthews, I. R., Li, J., Sherr, E. H., Chan, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+stress+response+inhibition+provides+sex-dependent+protection+against+noise-induced+cochlear+synaptopathy.">Integrated stress response inhibition provides sex-dependent protection against noise-induced cochlear synaptopathy.</a> Scientific Reports. 10 (1), 18063 (2020).</li><li>Amanipour, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+hearing+loss+in+mice:+Effects+of+high+and+low+levels+of+noise+trauma+in+CBA+mice.">Noise-induced hearing loss in mice: Effects of high and low levels of noise trauma in CBA mice.</a> Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2018, 1210-1213 (2018).</li><li>Edderkaoui, B., Sargsyan, L., Hetrick, A., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+duffy+antigen+receptor+for+chemokines+ameliorated+cochlear+damage+from+noise+exposure.">Deficiency of duffy antigen receptor for chemokines ameliorated cochlear damage from noise exposure.</a> Frontiers in Molecular Neuroscience. 11, 173 (2018).</li><li>Hultcrantz, M., Simonoska, R., Stenberg, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+hearing:+a+summary+of+recent+investigations.">Estrogen and hearing: a summary of recent investigations.</a> Acta Oto-laryngologica. 126 (1), 10-14 (2006).</li><li>Tsai, S. C. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intravenous+administration+of+skin-derived+mesenchymal+stem+cells+ameliorates+hearing+loss+and+preserves+cochlear+hair+cells+in+cisplatin-injected+mice:+SMSCs+ameliorate+hearing+loss+and+preserve+outer+hair+cells+in+mice.">The intravenous administration of skin-derived mesenchymal stem cells ameliorates hearing loss and preserves cochlear hair cells in cisplatin-injected mice: SMSCs ameliorate hearing loss and preserve outer hair cells in mice.</a> Hearing Research. 413, 108254 (2022).</li><li>Choi, M. Y., Yeo, S. W., Park, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+restoration+in+a+deaf+animal+model+with+intravenous+transplantation+of+mesenchymal+stem+cells+derived+from+human+umbilical+cord+blood.">Hearing restoration in a deaf animal model with intravenous transplantation of mesenchymal stem cells derived from human umbilical cord blood.</a> Biochemical and Biophysical Research Communications. 427 (3), 629-636 (2012).</li><li>Yu, S. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+evaluation+of+ribbon+synapses+at+specific+frequency+regions+of+the+mouse+cochlea.">Morphological and functional evaluation of ribbon synapses at specific frequency regions of the mouse cochlea.</a> Journal of Visualized Experiments. (147), e59189 (2019).</li></ol>

No conflict of interest to disclose.

NIHL can be divided into two types: temporary NIHL, which shows a temporal shift of the hearing threshold, and permanent NIHL, which is featured by a permanent hearing-threshold shift. The hearing loss that we observed on the 6th day (1 day after the noise exposure) is believed to be a combination of these two types. In this case, the hearing threshold would show a gradual recovery over time owing to the temporal component of hearing loss. In our preliminary experimental studies, the results acquired with the ...

About 1.3 billion people worldwide suffer from hearing loss due to noise exposure1. In this study, we aimed to establish a clear step-by-step process for inducing and confirming noise-induced hearing loss (NIHL). NIHL results from a degeneration/destruction of the hair cells (HCs) and spiral ganglion neurons (SGNs), damage in the HC stereocilia, and/or loss of synapses between the cochlear inner HCs and SGNs. Such abnormalities may also cause tinnitus and impaired speech perception (especially in a complex acoustic environment) besides NIHL. Social, psychological, and cognitive functions may be sequentially affected by these physiological deficiencies2,3,4,5,6.
In NIHL-related preclinical studies based on mice, the most popular mouse strains are CBA/CaJ2,3,6,7 and C57BL/64,5,8. The male3,4,7 mice, furthermore, are more commonly used than the female ones, as estrogen has a protective effect on hearing. Therefore, we only used male mice in this study9. After referring to the literature, we chose 1 kHz and 6 kHz as the frequencies of the applied noise. The intensity of the applied noise was 115 dB SPL-A (surrounding the cage) to 125 dB SPL-A (in the center of the cage). After exposing the experimental mice to the noise continuously for 6 h per day, for 5 consecutive days, an optimum increase in hearing threshold indicated an optimum extent of NIHL was generated in the experimental mice. The operations for handling the animals, building the experimental setup, and inducing noise are all clearly described step-by-step in the provided protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;1/4&#34; CCP Free-field Standard Microphone Set</td>
			<td>GRAS</td>
			<td>428158</td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Amplifier Input Module, AMI100D</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Bio-amplifier, BIO100C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A9647</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Cellsens software</td>
			<td>Olympus life science</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Corrugated plastic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI fluoromount</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>SIGMA</td>
			<td>E5134</td>
			<td>Decalcification</td>
		</tr>
		<tr>
			<td>Evoked Response Amplifier, ERS100C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>APLHA</td>
			<td>F030410</td>
			<td>Fixation of cochlear</td>
		</tr>
		<tr>
			<td>High Performance Data Acquisition System, MP160</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Modular Extension Cable, MEC110C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Myo7A primary antibody</td>
			<td>Proteus</td>
			<td>25-6790</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Myo7A secondary antibody</td>
			<td>Jackson immunoresearch</td>
			<td>711-545-152</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Needle Electrode, Unipolar 12 mmTp, EL452</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>phalloidin antibody</td>
			<td>Alexa Fluor</td>
			<td>A12381</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>phosphate-buffered saline</td>
			<td>SIGMA</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat trap cage</td>
			<td></td>
			<td></td>
			<td>14 cm x 17 cm x 24cm</td>
		</tr>
		<tr>
			<td>ROMPUN- xylazine injection, solution&#160;</td>
			<td>Bayer HealthCare, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sound amplifier, MT-1000</td>
			<td>unika</td>
			<td></td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Sound generator/analyzer/miscellaneous, FW-02</td>
			<td>CLIO</td>
			<td>620300719</td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Soundproof chamber</td>
			<td>IEA Electro-Acoustic Technology</td>
			<td></td>
			<td>For noise exposure and ABR</td>
		</tr>
		<tr>
			<td>Speaker&#160;</td>
			<td>IEA Electro-Acoustic Technology</td>
			<td></td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Stimulator Module, STM100C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SIGMA</td>
			<td>T8787</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Tubephone Set, OUT101</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Upright Microscope, BX53</td>
			<td>Olympus</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A shift in ABR hearing threshold 
The hearing threshold of the mice was measured using tone-burst ABR either 1 day or 1 week after the noise exposure. A significant increase in the hearing threshold at all three tested frequencies was observed (12 kHz: 84.29 &#177; 2.77 dB SPL; 24 kHz: 91.43 &#177; 0.92 dB SPL; 32 kHz: 98.57 &#177; 1.43 dB SPL) 1 day after the noise exposure (i.e., the 6th day). Partial hearing recovery occurred 1 week after the noise exposure (i.e., the 13th d...

Watch this Scientific Journal Video about Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage at JoVE.com

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

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 ...

modified-experimental-conditions-for-noise-induced-hearing-loss-mice

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Neuroscience

1.9K Views.  MacKay Medical College. 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.

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

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

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 ...

Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for inducing CHL in rodents. The protocol demonstrates CHL by tympanic membrane puncture. Verification of CHL surgery was by otoscope examination and behavioral assessment by clap startle response, both replicable and reliable, and are simple methods to demonstrate hearing loss has occurred. The simple CHL procedure is advantageous due to its reproducibility and flexibility to different pursuits in hearing loss research. The limitations of inducing CHL by a surgical approach are associated with the learning curve to perform the surgical procedure and confidence in audiological examination. Inducing a hearing impairment by CHL allows one to readily study the neural manifestations and behavioral outcomes of hearing loss.

Here, we present a protocol to establish a replicable conductive hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle.

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for ...

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 ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, ...