Name: auditory brainstem response and outer hair cell whole-cell patch clamp recording in postnatal rats
Uploaded: 2018-05-24T12:30:00
Description: Watch this Scientific Journal Video about Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats at JoVE.com

Dette arbejde blev støttet af tilskud fra den 973 Program (2014CB943002) og National Natural Science Foundation of China (11534013, 31500841).

Alle eksperimentelle protokoller der involverer dyrs emner blev godkendt af dyr etiske komité i den sydlige medicinske universitet.
1. Forbered løsninger for eksperimenter
<ol><li>Forberede narkose (Se Tabel af materialer): 1,5% pentobarbital natrium opløst i ddH2O.</li>
	<li>Forberede dissektion løsning (Se Tabel af materialer): opløses en taske af Leiboviz's L-15 pulver i 1 L i ddH2O. Juster pH til 7,3 med 1 M NaOH. Justere osmol...

<ol>
	<li>He, D. Z., Zheng, J., Kalinec, F., Sacchi Kakehata, S. S. a. n. t. o. s. -., J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+in+to+the+amazing+outer+hair+cell:+membrane+wizardry+with+a+twist+and+shout.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+amplification,+outer+hair+cells+and+prestin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prestin+is+the+motor+protein+of+cochlear+outer+hair+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+expression+of+the+outer+hair+cell+motor+prestin+in+the+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+morphological+and+functional+maturation+of+mechanotransduction+in+rat+outer+hair+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+and+specific+influences+of+early+acoustic+environments+on+primary+auditory+cortex.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+period+window+for+spectral+tuning+defined+in+the+primary+auditory+cortex+(A1)+in+the+rat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+appearance+and+development+of+electromotility+in+neonatal+gerbil+outer+hair+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronized+Progression+of+Prestin+Expression+and+Auditory+Brainstem+Response+during+Postnatal+Development+in+Rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+absolute+auditory+thresholds+in+the+house+mouse+(Mus+musculus).">Development of absolute auditory thresholds in the house mouse (Mus musculus).</a> J Am Audiol Soc. 1, 179-184 (1976).</li><li>M&#248;ller, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hearing:anatomy, physiology, and disorders of the auditory system. , (2006).</li><li>He, D. Z., Zheng, J., Edge, R., Dallos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+cochlear+inner+hair+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Pendrin+Protein,+an+Anion+Transporter+and+Molecular+Motor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Urethane+on+Rat+Outer+Hair+Cells.">The Effects of Urethane on Rat Outer Hair Cells.</a> Neural Plast. 2016, 1-11 (2016).</li></ol>

I rotter yngre end dag 11, kunne ingen aktionspotentialet i respons til lyd stimulation observeres i auditive cortex6,7. Derfor er postnatal dag 11 beskrevet som "høring debut"10. Udvikling af hørelse funktion før retsmødet debut ikke var godt undersøgt endnu. Ved hjælp af metoden svarer til voksen ABR optagelse, vi viser, at ABRs kunne være fremkaldt af ren tone brast fra rotte hvalpe yngre end P11 (figur 1</st...

To forskellige funktioner af cochlear sensoriske hårcellerne er afgørende for pattedyr høring: mechanoelectric transduktion (MET) og electromotility1,2. Af markedsøkonomisk kanaler beliggende i til håret bundt, IHCs (IHCs) og OHCs konvertere lyd vibrationer i membranen potentielle ændringer samt de elektriske signaler innerverede spiral ganglion neuroner. OHCs ændre deres celle længde med membran potentiale og forstærke vibrationer af laveste-niveau-lyd. Denne aktivitet betegnes electromotility er afledt af den motoriske protein prestin placeret i den laterale væg af OHCs3.
I mange arter inklusive gnavere, er funktionen hørelse umodne i tidlige postnatal epoke4,5. Ingen handling potentiale i respons til lyd signaler der kunne påvises i den auditive cortex før høringen debut6,7. Udvikling af morfologi og funktion af øresneglen er blevet udbredt undersøgt i mus og ørkenrotte rotte4,5,8. Mechanotransduction og electromotility af hårcellerne er også udviklet i den tidlige liv epoke5.
For at vurdere retsmødet følsomhed af rotter på forskellige postnatal alderstrin, har vi udviklet en metode til auditive hjernestammen svar (ABR) optagelse i rotte unger. Hele-celle patch klemme er en ideel teknologi til at undersøge OHCs electrophysiologically. Men sammenlignet med patch klemme udført i neuroner og andre epitelceller, den lave udnyttelsesgrad for hele-celle forsegling begrænset undersøgelsen af electromotility af isolerede OHCs.
Her beskriver vi en metode til at undersøge OHCs morfologisk og electrophysiologically i akut dissocierede øresneglen fra postnatal rotter. Denne metode kan ændres for at studere de molekylære mekanismer, der regulerer inderste hår celle udvikling og funktion.

<table>
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		<tr>
			<td>Anesthetic</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>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dissection solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leiboviz&#39;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immunostaining solutions</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Extracellular solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leiboviz&#39;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>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Intracellular solution</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>MgCl2</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Animal</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

Den ydre hår celle er en af de to typer af sensoriske hår celler i pattedyr cochlea. De ændrer deres celle længde med receptor potentielle til at forstærke svage vibrationer af low-level lydsignal. Morfologi og elektrofysiologiske ejendom af ydre hårceller (OHCs) udvikler sig i begyndelsen postnatal aldre. Modning af ydre hår celle kan bidrage til udviklingen af det auditive system. Processen for OHCs udvikling er imidlertid ikke godt undersøgt. Dette er dels fordi var vanskeligheden at måle deres funktion af en elektrofysiologiske tilgang. Med det formål at udvikle en enkel metode til at løse de ovenstående spørgsmål, beskrive her vi en trinvis protokol for at studere funktionen af OHCs i akut dissocierede øresneglen fra postnatal rotter. Med denne metode, kan vi evaluere de cochlear reaktion på ren tone stimuli og undersøge udtrykket niveau og funktion af motor protein prestin i OHCs. Denne metode kan også bruges til at undersøge de indre hårceller (IHCs).

ABR kan være fremkaldt fra bedøvede rotte pups ældre end postnatal dag 7 (P7) ved hjælp af ren tone byger (figur 1A). Som vist i figur 1B, opnået ABR waveforms fra rotte pups viste kun tre til fire forskellige bølger med lille amplitude. Normalt blev op til syv tinder observeret i ABR kurveformer af voksne dyr (figur 1B).
Til følgend...

Watch this Scientific Journal Video about Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats at JoVE.com

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats (Video) | JoVE

Denne protokol beskriver metoder til at registrere den auditive hjernestammen svar fra postnatal rotte unger. For at undersøge den funktionelle udvikling af ydre hårceller, er forsøgsmetoden af hele-celle patch klemme optagelse i isolerede ydre hårceller beskrevet trin for trin.

Auditive hjernestammen svar og ydre hår celle hele-celle Patch klemme optagelse i Postnatal rotter

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 ...

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Neuroscience

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

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