Name: Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats (Video) | JoVE Journal
Uploaded: 2018-05-24T12:30:00.000+00:00
Duration: 9 min 23 s
Description: Learn auditory brainstem response and whole-cell patch clamp recording in postnatal rat pups to study outer hair cell development. This method enables precise evaluation of individual cochlear hair cell functionality during hearing onset.

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This work was supported by grants from the 973 Program (2014CB943002) and the National Natural Science Foundation of China (11534013, 31500841).

All experimental protocols involving animal subjects were approved by the Animal Ethics Committee of the Southern Medical University.
1. Prepare Solutions for Experiments
<ol>
	<li>Prepare the anesthetic (see Table of Materials): 1.5% pentobarbital sodium dissolved in ddH2O.</li>
	<li>Prepare the dissection solution (see Table of Materials): Dissolve one bag of Leiboviz&#39;s L-15 powder in 1 L of ddH2O. Adjust pH to 7.3 with 1 M NaOH. Adjust osmolarity to 300-330 mOsm by using an osmometer and 10 mM HEPES before use.</li>
	<li>Dissolve 2 mg/mL collagenase IV (see Table of Materials) in dissection solution prepared in step 1.2.</li>
	<li>Prepare the immunostaining solutions (see Table of Materials): Dissolve the 4% paraformaldehyde in phosphate buffered saline (PBS). 
	Caution: Paraformaldehyde is toxic; wear appropriate protection.</li>
	<li>Dilute the 0.3% permeability agent in PBS. Dilute the 10% normal goat serum in PBS as the blocking buffer. Dilute the prestin antibody 1:200 in blocking buffer. Dilute the Alexa488-conjugated antibody 1:600 in blocking buffer. Dilute the Phalloidin-Tetramethylrhodamine B isothiocyanate 1:200 in PBS.</li>
	<li>Prepare the extracellular solution (see Table of Materials): Prepare the Leiboviz&#39;s L-15 medium as described above (step 1.2).</li>
	<li>Prepare the intracellular solution (see Table of Materials).</li>
</ol>
2. ABR Recording
NOTE: ABR recording has been previously described in detail in our previous study9.
<ol>
	<li>Anesthetize Sprague Dawley (SD) rats (1-14 day old pups and 2-month old adults, both sexes) with a single injection of 1.5% sodium pentobarbital (22 mg/kg for pups and 30 mg/kg for adults, intraperitoneal (i.p.)).</li>
	<li>Place the anesthetized animal in a polyethylene-foam mold to immobilize the animal&#39;s body. Place the animal on an anti-vibration table in a sound-attenuating room. Keep the animal&#39;s body temperature at 37.5 &#176;C with a heating pad.</li>
	<li>Wipe the head area with 70% (vol/vol) ethanol. By using a fine scissors, make a 1-2 mm incision ventrolateral to the external pinna to place the reference electrode or ground. A subdermal needle electrode (recording electrode) is located over the skull vertex (Figure 1A).</li>
	<li>Using the function generator, generate calibrated tone bursts (1 ms rise/fall, 3 ms plateau) of various frequencies (2, 4, 8, 16, 24, and 32 kHz) and intensities (decreased from 85 to 10 dB SPL in 5 dB step). Vary the frequency and amplitude manually or using a computer. Deliver sound stimuli through an electrostatic speaker located 10 cm away toward the head of animal.</li>
	<li>Filter (100-1,000 Hz), amplify and average (256 times) the sound elicited potential using a multi-function processor to get the ABRs. The ABR traces is monitored online and stored for offline analysis. It takes about 40 min to complete the ABR recording from one animal. 
	NOTE: Usually, three to seven peaks (wave I to wave VII) with latencies less than 15 ms can be identified (Figure 1B). The ABR threshold is defined as the minimum sound level at which wave I and II could be detected.</li>
	<li>Euthanize before the animals awake from anesthesia (with an intraperitoneal injection of 0.3 mL 1.5% sodium pentobarbital).</li>
</ol>
3. The Organ of Corti (OC) Dissection
<ol>
	<li>Anesthetize rat pups. For rats less than 6 days old (&#60;P6), keep the animal in a 100 mm culture dish, and cover the dish with ice for 10 min. For rats &#62;P6, anesthetize the animal with a single injection of 1.5% sodium pentobarbital (22 mg/kg, i.p.). Decapitate the rat pups after anesthesia.</li>
	<li>Sterilize the head by spraying with 70% (vol/vol) ethanol.</li>
	<li>Open the skull along the sagittal midline with scissors; remove the brain to expose the inner ear.</li>
	<li>Transfer the inner ear into a 35-mm Petri dish filled with 3 mL of ice-cold L-15 (Figure 2A).</li>
	<li>Under the dissection microscope, use fine forceps to remove the bony capsule of the cochlea (Figure 2B).</li>
	<li>Unwrap the OC and associated stria vascularis (SV) from the modiolus.</li>
	<li>By holding the basal portion of the SV with forceps, separate the OC from the SV completely by unwinding slowly from base-to-apex (Figure 2C).</li>
	<li>Cut the OC evenly into three pieces by using fine scissors (Figure 2D).</li>
</ol>
4. Immunofluorescence Staining
<ol>
	<li>By using a 200 &#181;L pipette tip, transfer the segments of OC (step 3.8) onto a glass slide, and fix with 100 &#181;L of 4% paraformaldehyde for at least 4 h at 4 &#176;C. 
	Caution: Paraformaldehyde is toxic; wear appropriate protection.</li>
	<li>Wash the tissue by displacing the paraformaldehyde with 100 &#181;L of fresh PBS. Wash the tissue three times for 10 min.</li>
	<li>Incubate the tissue with 0.3% permeability agent in PBS for 30 min at room temperature.</li>
	<li>Discard the permeability agent in PBS and wash the tissue three times with PBS.</li>
	<li>Block with 10% normal goat serum in PBS for 1 h at room temperature.</li>
	<li>Incubate with anti-prestin-C-terminus antibody (1:200) in blocking solution for 2 h at room temperature or at 4 &#176;C overnight.</li>
	<li>Wash three times with PBS for 5 min.</li>
	<li>Incubate with Alexa488-conjugated secondary antibodies (1:600) in blocking solution for 1 h at room temperature in the dark.</li>
	<li>Wash three times with PBS for 5 min in the dark.</li>
	<li>Incubate with rhodamine-phalloidin (1:200) for 10 min at room temperature in the dark.</li>
	<li>Wash three times with PBS for 5 min in the dark.</li>
	<li>Mount on a glass slide with mounting medium (containing DAPI). Image with confocal microscopy by using 405 nm, 488 nm, and 594 nm lasers. Set the laser power at 0.1-0.5 mW and the scan speed at 0.5 frame/s.</li>
</ol>
5. Patch clamp Recording of Isolated OHCs
<ol>
	<li>Use the pipette puller and micro-forger to make patch pipettes with a tip diameter 2-3 &#181;m. Back-fill the pipettes with intracellular solution. Usually, the initial resistance of patch pipette was 2.5-3.5 M&#937; in bath solution.</li>
	<li>By using a 200 &#181;L pipette tip, transfer a piece of the OC (from step 3.8) into a 35 mm Petri dish. Digest the tissue with 100 &#181;L of enzymatic digestion medium (collagenase IV, see Table of Materials) for 5 min at room temperature.</li>
	<li>Displace the enzymatic digestion medium with 100 &#181;L of L-15. Cut the tissue into small pieces using a micro scalpel to isolate the hair cells.</li>
	<li>After gentle pipetting, transfer the cells into a homemade small plastic chamber (diameter ~ 1.5 cm) filled with enzyme-free bath solution (~ 1.5 mL, see Table of Materials).</li>
	<li>Place the chamber on the stage of an inverted microscope. Find the healthy-appearing solitary OHCs.</li>
	<li>Load the patch pipette into the head stage of a 700B amplifier. Move the patch pipette carefully by a micromanipulator and position it around the bottom of the outer hair cell (Figure 3A).</li>
	<li>Whole-cell patch clamp the OHC by sealing the lateral wall of the cell body. Apply light suction until the cell membrane is ruptured. Set the holding potential at -70 mV. Cells with access resistance ranged from 10 to 17 M&#937; and membrane resistance ranged from 100 to 500 M&#937;, and are considered a successful whole-cell configuration.</li>
	<li>Using computer control, apply hyperpolarizing and depolarizing voltage (250 ms duration and ranging from &#8722;140 to +94 mV in 13 mV steps) to the cell to elicit whole-cell currents.</li>
	<li>Amplify the whole-cell currents, filter (corner frequency of 5 kHz) by a 700B amplifier. Convert the data by a 1440A converter and store for offline analysis.</li>
	<li>Measure the membrane capacitance of OHCs using a two sine-wave voltage stimulus protocol controlled by a patch clamp software. Set the stimulate voltage range from -140 to 110 mV. Store the data for offline analysis.</li>
</ol>

<ol><li>He, D. Z., Zheng, J., Kalinec, F., Sacchi Kakehata, S. S. antos-, J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tuning+in+to+the+amazing+outer+hair+cell%3A+membrane+wizardry+with+a+twist+and+shout%5BTitle%5D)+AND+%22J+Membr+Biol%22%5BJournal%5D)">Tuning in to the amazing outer hair cell: membrane wizardry with a twist and shout.</a> J Membr Biol. 209, 119-134 (2006).</li>
<li>Dallos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cochlear+amplification%2C+outer+hair+cells+and+prestin%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Cochlear amplification, outer hair cells and prestin.</a> Curr Opin Neurobiol. 18, 370-376 (2008).</li>
<li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prestin+is+the+motor+protein+of+cochlear+outer+hair+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Prestin is the motor protein of cochlear outer hair cells.</a> Nature. 405, 149-155 (2000).</li>
<li>Abe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+expression+of+the+outer+hair+cell+motor+prestin+in+the+mouse%5BTitle%5D)+AND+%22J+Membr+Biol%22%5BJournal%5D)">Developmental expression of the outer hair cell motor prestin in the mouse.</a> J Membr Biol. 215, 49-56 (2007).</li>
<li>Waguespack, J., Salles, F. T., Kachar, B., Ricci, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stepwise+morphological+and+functional+maturation+of+mechanotransduction+in+rat+outer+hair+cells%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Stepwise morphological and functional maturation of mechanotransduction in rat outer hair cells.</a> J Neurosci. 27, 13890-13902 (2007).</li>
<li>Zhang, L. I., Bao, S., Merzenich, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+and+specific+influences+of+early+acoustic+environments+on+primary+auditory+cortex%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Persistent and specific influences of early acoustic environments on primary auditory cortex.</a> Nat Neurosci. 4, 1123(2001).</li>
<li>De, V. -S. E., Chang, E. F., Bao, S., Merzenich, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+period+window+for+spectral+tuning+defined+in+the+primary+auditory+cortex+%28A1%29+in+the+rat%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Critical period window for spectral tuning defined in the primary auditory cortex (A1) in the rat.</a> J Neurosci. 27, 180-189 (2007).</li>
<li>He, D. Z., Evans, B. N., Dallos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+appearance+and+development+of+electromotility+in+neonatal+gerbil+outer+hair+cells%5BTitle%5D)+AND+%22Hearing+Res%22%5BJournal%5D)">First appearance and development of electromotility in neonatal gerbil outer hair cells.</a> Hearing Res. 78, 77-90 (1994).</li>
<li>Hang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synchronized+Progression+of+Prestin+Expression+and+Auditory+Brainstem+Response+during+Postnatal+Development+in+Rats%5BTitle%5D)+AND+%22Neural+Plast%22%5BJournal%5D)">Synchronized Progression of Prestin Expression and Auditory Brainstem Response during Postnatal Development in Rats.</a> Neural Plast. , 4545826(2016).</li>
<li>Ehret, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+absolute+auditory+thresholds+in+the+house+mouse+%28Mus+musculus%29%5BTitle%5D)+AND+%22J+Am+Audiol+Soc%22%5BJournal%5D)">Development of absolute auditory thresholds in the house mouse (Mus musculus).</a> J Am Audiol Soc. 1, 179-184 (1976).</li>
<li>Møller, A. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aM%C3%B8ller%2C+AR+%22Hearing%3Aanatomy%2C+physiology%2C+and+disorders+of+the+auditory+system%22">Hearing:anatomy, physiology, and disorders of the auditory system</a>. , Academic Press. Dallas. (2006).</li>
<li>He, D. Z., Zheng, J., Edge, R., Dallos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+cochlear+inner+hair+cells%5BTitle%5D)+AND+%22Hearing+Res%22%5BJournal%5D)">Isolation of cochlear inner hair cells.</a> Hearing Res. 145, 156-160 (2000).</li>
<li>Tang, J., Pecka, J. L., Tan, X., Beisel, K. W., He, D. Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+Pendrin+Protein%2C+an+Anion+Transporter+and+Molecular+Motor%5BTitle%5D)+AND+%22J+Biological+Chem%22%5BJournal%5D)">Engineered Pendrin Protein, an Anion Transporter and Molecular Motor.</a> J Biological Chem. 286, 31014-31021 (2011).</li>
<li>Fu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Effects+of+Urethane+on+Rat+Outer+Hair+Cells%5BTitle%5D)+AND+%22Neural+Plast%22%5BJournal%5D)">The Effects of Urethane on Rat Outer Hair Cells.</a> Neural Plast. 2016, 1-11 (2016).</li>
</ol>

The authors have nothing to disclose.

In rats younger than day 11, no action potential in response to sound stimulation could be observed in auditory cortex6,7. Therefore, postnatal day 11 is described as &#34;hearing onset&#34;10. The development of hearing function before hearing onset was not well studied yet. Using the similar method for adult ABR recording, we demonstrate that ABRs could be elicited by pure tone burst from rat pups younger than P11 (F...

Two distinct functions of cochlear sensory hair cells are essential for mammalian hearing: mechanoelectric transduction (MET) and electromotility1,2. By MET channels located in the hair bundle, IHCs (IHCs) and OHCs convert sound vibration into membrane potential changes, as well as the electrical signals of innervated spiral ganglion neurons. OHCs change their cell length with the membrane potential and amplify the vibration of low-level sound. This activity termed electromotility is derived by the motor protein prestin located in the lateral wall of OHCs3.
In many species including rodents, the hearing function is immature in early postnatal epoch4,5. No action potential in response to sound signals could be detected in the auditory cortex before the hearing onset6,7. Development of morphology and function of the cochlea has been widely studied in mouse, gerbil, and rat4,5,8. The mechanotransduction and electromotility of hair cells are also developed in the early life epoch5.
In order to evaluate the hearing sensitivity of rats at different postnatal ages, we have developed a method for auditory brainstem response (ABR) recording in rat pups. Whole-cell patch clamp is an ideal technology to investigate the OHCs electrophysiologically. However, compared with the patch clamp performed in neurons and other epithelial cells, the low rate of whole-cell sealing limited the investigation of electromotility of isolated OHCs.
Here we describe a procedure to investigate the OHCs morphologically and electrophysiologically in acutely dissociated cochlea from postnatal rats. This method can be modified to study the molecular mechanisms that regulate inner hair cell development and function.

<table><tbody><tr><td>&lt;strong&gt;Anesthetic&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pentobarbital sodium</td><td>Sigma</td><td>P3761</td><td>1.5% in water</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Dissection solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Leiboviz&#039;s L-15 Medium</td><td>Life Technologies</td><td>41300-039</td><td>1 pack in 1 L water</td></tr><tr><td>Collagenase IV</td><td>Sigma</td><td>C5138</td><td>2 mg/mL in L-15</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>10 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunostaining solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>PH 7.3</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>4% in PBS</td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>ZS-0694</td><td>0.3% in PBS</td></tr><tr><td>Normal goat serum</td><td>Thermo Fisher Scientific</td><td>10000C</td><td>10% in PBS</td></tr><tr><td>prestin antibody</td><td>Santa Cruz</td><td>SC-22694</td><td>dil 1:200</td></tr><tr><td>Alexa Fluor 488-conjugated antibody</td><td>Thermo Fisher Scientific</td><td>A-11055</td><td>dil 1:600</td></tr><tr><td>Phalloidin-Tetramethylrhodamine B isothiocyanate</td><td>Sigma</td><td>P1951</td><td>dil 1:200</td></tr><tr><td>DAPI</td><td>Solarbio</td><td>C0060</td><td>dil 1:20</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Extracellular solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Leiboviz&#039;s L-15 Medium</td><td>Life Technologies</td><td>41300-039</td><td>1 pack in 1 L water</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>10 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Intracellular solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CsCl</td><td>Sigma</td><td>7647-17-8</td><td>140 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>7791-18-6</td><td>2 mM</td></tr><tr><td>EGTA</td><td>Sigma</td><td>67-42-5</td><td>10 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>10 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Osmometer</td><td>Gonotec</td><td>OSMOMAT 3000 basic</td><td></td></tr><tr><td>Forcep</td><td>WPI</td><td>14095</td><td>Tweezers dumont</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>MODLE-P97</td><td></td></tr><tr><td>Micro Forge</td><td>Narishigen</td><td>MF-830</td><td></td></tr><tr><td>Mini Operating System</td><td>Sutter Instrument</td><td>MP-285</td><td></td></tr><tr><td>MultiClamp</td><td>Axon</td><td>700B</td><td></td></tr><tr><td>Low-Noise Data Acquisition System</td><td>Axon</td><td>1440A</td><td></td></tr><tr><td>ES1 speaker</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>TDT system 3</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SigGenRP software</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>BioSigRP software</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>jClamp</td><td>Scientific Solutions</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Animal&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SD rat</td><td>Experimental Animal Center of Southern Medical University</td><td></td><td></td></tr></tbody></table>

auditory brainstem response and outer hair cell whole-cell patch clamp recording in postnatal rats

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).

ABR can be elicited from anesthetized rat pups older than postnatal day 7 (P7) using pure tone bursts (Figure 1A). As shown in Figure 1B, the ABR waveforms obtained from rat pups showed only three to four distinct waves with small amplitude. Usually, up to seven peaks were observed in the ABR waveforms of adult animals (Figure 1B).
For the ...

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 ...

auditory-brainstem-response-outer-hair-cell-whole-cell-patch-clamp

Research

JoVE Journal

Neuroscience

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

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.

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

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

This video demonstrates a method for recording auditory brainstem responses (ABRs) from rat pups. It outlines the steps for electrode placement, sound stimulus application, and signal processing analysis in the brainstem to assess neural responses.

Recording Auditory Brainstem Responses in a Rat Pup

This video demonstrates the procedure to implant a cochlear device in a deafened mouse and apply electrical stimuli to record auditory brainstem responses. This procedure enables to understand the basic mechanisms of electrical hearing and can contribute to advancements in cochlear implant technology and auditory neuroscience.

Recording Electrically-Evoked Auditory Brainstem Responses in a Deafened Mouse

This video demonstrates the patch-clamp technique for measuring ionic currents in inner ear sensory neuron cell bodies. It details the preparation and use of a polished patch-clamp pipette filled with an internal solution containing amphotericin B, which perforates the membrane to allow ion movement while preserving intracellular conditions. Voltage pulses are applied to trigger and record ion channel activity, providing insights into their properties and behavior.

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

This video demonstrates the examination of sensory neuron excitability in the dorsal root ganglia using the patch clamp technique to measure the rheobase and membrane potential, analyzing the neurons' responses to incremental current injections.

Whole-Cell Patch Clamp Recordings on Intact Rat Dorsal Root Ganglia

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

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.

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

Learn auditory brainstem response and whole-cell patch clamp recording in postnatal rat pups to study outer hair cell development. This method enables precise evaluation of individual cochlear hair cell functionality during hearing onset.

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

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