Name: optogenetic stimulation of the auditory nerve
Uploaded: 2014-10-08T15:00:00
Description: Watch this Scientific Journal Video about Optogenetic Stimulation of the Auditory Nerve at JoVE.com

Dette arbejde blev støttet af det tyske Forbundsministeriet for Uddannelse og Forskning (Bernstein Fokus for Neurotechnology yde 01GQ0810, T. Moser, og MED-EL Tyskland); den tyske Research Foundation gennem Center for Nanoscale mikroskopi og Molekylær Fysiologi af hjernen (FZT 103 T. Moser) og gennem SFB889, til N. Strenzke og T. Moser).

Alle eksperimenter præsenteres i dette arbejde blev udført med de etiske standarder, der er defineret af den tyske lov om beskyttelse af forsøgsdyr. The University of Göttingen bestyrelse for dyrevelfærd og dyrevelfærd kontor i staten Niedersachsen godkendt forsøgene. 1. Fremstilling af μLED-stimulator <ol><li> For μLEDs, først forberede μLED-stimulator. Brug blå lysdioder med 200 af 200 um aktive overflade (μLED se Materialer tabel). </li><li> Solder ledningerne til μLED. De...

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A., Litvak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place-pitch+discrimination+of+single-+versus+dual-electrode+stimuli+by+cochlear+implant+users+(L).">Place-pitch discrimination of single- versus dual-electrode stimuli by cochlear implant users (L).</a> The Journal of the Acoustical Society of America. 118 (2), 623-626 (2005).</li><li>Srinivasan, A. G., Shannon, R. V., Landsberger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+virtual+channel+discrimination+in+a+multi-channel+context.">Improving virtual channel discrimination in a multi-channel context.</a> Hearing research. 286 (1-2), 19-29 (2012).</li><li>Zeng, F. 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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=Auditory+responses+to+electric+and+infrared+neural+stimulation+of+the+rat+cochlear+nucleus.">Auditory responses to electric and infrared neural stimulation of the rat cochlear nucleus.</a> Hearing research. 310, 69-75 (2014).</li><li>Fenno, L., Yizhar, O., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+application+of+optogenetics.">The development and application of optogenetics.</a> Annual review of neuroscience. 34, 389-412 (2011).</li><li>Hegemann, P., Nagel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+channelrhodopsins+to+optogenetics.">From channelrhodopsins to optogenetics.</a> EMBO molecular medicine. 5 (2), 173-176 (2013).</li><li>Packer, A. M., Roska, B., H&#228;usser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+neurons+and+photons+for+optogenetics.">Targeting neurons and photons for optogenetics.</a> Nature neuroscience. 16 (7), 805-815 (2013).</li><li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (24), 13940-13945 (2003).</li><li>Nagel, G., Szellas, T., Kateriya, S., Adeishvili, N., Hegemann, P., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsins:+directly+light-gated+cation+channels.">Channelrhodopsins: directly light-gated cation channels.</a> Biochemical Society transactions. 33 (Pt 4), 863-866 (2005).</li><li>Nagel, G., Brauner, M., Liewald, J. F., Adeishvili, N., Bamberg, E., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Current biology: CB. 15 (24), 2279-2284 (2005).</li><li>Boyden, E. 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De beskrevne forsøg viser den optogenetic stimulering af SGNs, og kan i princippet også anvendes til at stimulere indre og / eller ydre hårceller, hvis ekspressionen af ​​opsiner. Disse eksperimenter kræver meget tålmodighed og omsorg. Som nævnt før, er de mest kritiske trin er en god cochleostomy / runde vindues indsættelse samt en passende position og orientering af lyskilden. Der er begrænsninger med optogenetic stimulering ved brug CHR2. I vores tilfælde oABR amplitude stig...

Ifølge World Health Organization, 360 millioner mennesker verden over lider af høretab. I døve forsøgspersoner direkte elektrisk stimulation af SGNs af kreditinstitutter muliggøre åben taleforståelse i de fleste af dem 1,2,4,5. Selvom kreditinstitutter er blevet implanteret i mere end 200.000 mennesker og derfor er den mest succesfulde neuroprosthesis, lyd kodning drevet af de nuværende cochlear implantater er begrænset. CIs er baseret på elektrisk stimulation af en række elektroder, hvor hver aktiverer en tonotopic region hørenerven således omgår den dysfunktionelle sanseorgan Corti i cochlea. Normal hørelse lyttere kan skelne mere end 2.000 frekvenser, men nutidens kreditinstitutter kun bruge op til 12-22 frekvenskanaler 4. Dette skyldes udbredt strøm fra hver stimulerende elektrode 7,9, aktivere et stort antal SGNs der repræsenterer mange forskellige lydfrekvenser 8,15. Dettebegrænsning kan forbedres ved hjælp af multipolær stimulation, men på bekostning af højere strømforbrug 16,17. Deres produktion dynamikområde for en sund intensitet er også begrænset, typisk under 6-20 dB 4,18. Af disse grunde, forbedre hyppighed og intensitet opløsning er vigtige mål for at øge CI ydeevne til at lindre talegenkendelse i støjende omgivelser, prosodi forståelse og musik perception. En anden mulighed for at stimulere hørenerven er optisk stimulering. Lys kan være praktisk fokuseret på at målrette en lille SGN befolkning, lover bedre fysisk indespærring, stigende frekvens opløsning og også udvide dynamikområde, hvilket resulterer i en bedre intensitet opløsning. Faktisk har cochlear stimulering med infrarødt lys vist glimrende frekvens opløsning i dyremodeller 10,11,19. En ulempe ved denne form for stimulering er, at det kræver højere energi end elektrisk stimulering <sup&gt; 10,11. Desuden har bekymringer om metodens evne til direkte at stimulere auditive neuroner blevet rejst 12,20. Som et alternativ til infrarød stimulering, vi ansætter optogenetics at gøre SGNs lysfølsomt. Optogenetics er en ny tilgang, der kombinerer genetiske og optiske teknikker til ikke-invasivt og specifikt eksportkontrol på celler med høj tidsmæssig præcision (anmeldelser 21- 23). Den aktuelt mest anvendte modalitet beskæftiger ekspressionen af mikrobielle channelrhodopsin 2 (CHR2) genet af Chlamydomonas reinhardtii og varianter deraf, der koder for en let-gatede kationkanal 24. CHR2 er en 7-transmembran-helix protein, som, når transduceres ind i neuroner og aktiveres af blåt lys, fungerer som ikke-selektive kationkanal, således depolariserende cellerne 24-27. CHR2 er blevet godt karakteriseret 24,28- 31 og mange varianter er blevet udviklet til at ændre action spektret, gating og permeabilitet egenskaber 32,33. Målet med vores arbejde er at etablere cochlear optogenetics til aktivering af det auditive vej. Vi bemærker, at optogenetic strategi for at stimulere hørenerven kræver genetisk manipulation af spiral ganglion til ekspressionen af ​​channelrhodopsin. Arbejde med mus og rotter tillader brugen af tilgængelige transgene dyr 13,34,35, som giver udtryk for channelrhodopsin med lidt variation langs tonotopic akse og på tværs af dyr 36. Kombination af betingede alleler 37 med passende Cre-linjer giver mulighed for celle-specifikke ekspression. Genoverførsel til spiral ganglion af andre dyr kræver brug af virus, såsom adenoassocieret virus, der er en standard tilgang optogenetics 38 og at vi viste sig at fungere godt i mus 36. Genmanipulation og ekspressionen af ​​transgener, der koder for fremmede proteiner, bear risici for bivirkninger såsom IMMUne svar og / eller spredning, kompromitteret tilstand eller endda død af genetisk manipulerede celler. Med henblik på denne demonstration vi bruger transgene mus, der udtrykker CHR2 i spiral ganglion neuroner under Thy-1 promotoren 13 til optisk stimulere den auditive vej. Vi bemærker, at andre channelrhodopsin varianter kan anvendes til samme formål som vi påvist under anvendelse af virus-medieret overførsel af varianten griberen 14 i SGNs 39. Mens cochlear optogenetics kræver genetisk manipulation, det giver molekylær tuning til optimeret SGN stimulering og løfter forbedret frekvens og intensitet opløsning i forhold til elektrisk stimulation. Optogenetic stimulering af den auditive vej er yderst relevant for hørelse forskning. For eksempel, det lover fremskridt i undersøgelser af aktiviteten-afhængige forfinelse af tonotopy under udvikling, i analysen af ​​kravet om spektral integration i lyd localization og af omfanget af samspillet mellem frekvens-specifikke afferente fremskrivninger i det centrale auditive system.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr>
			<td>Urethane</td>
			<td>Sigma Aldrich</td>
			<td>U2500-100G</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Xylazine HCl</td>
			<td>RXV</td>
			<td></td>
			<td>Sedative and analgesic</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Reckitt Benckiser</td>
			<td></td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>It is used to hold hard tissue, e.g. bone or materials. Never use them to hold soft delicated tissue&#160;</td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Only to be used to hold soft tissue</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td>To open the skin and help with the muscle dissection</td>
		</tr>
		<tr>
			<td>Lempert Rongeurs&#160;</td>
			<td>Fine Science Tools</td>
			<td>16004-16</td>
			<td>They are very useful to easily remove the bone from the bulla</td>
		</tr>
		<tr>
			<td>473-nm laser&#160;</td>
			<td>Changchun New Industries</td>
			<td>MLL-III473</td>
			<td>100 mW solid state 473 nm laser</td>
		</tr>
		<tr>
			<td>Laser driver&#160;</td>
			<td>Changchun New Industries</td>
			<td>DPSSL MLL 100 mW</td>
			<td>TTL operated laser driver</td>
		</tr>
		<tr>
			<td>250 &#181;m optical fiber</td>
			<td>Any comercial ; e.g. Thorlabs</td>
			<td>M42L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Acousto-optical modulator</td>
			<td>Crystal Technology, Inc.</td>
			<td>PCAOM VIS</td>
			<td>Control the amount of light coupled into the fiber from the laser</td>
		</tr>
		<tr>
			<td>Controller for Acousto-optical modulator</td>
			<td>Crystal Technology, Inc.</td>
			<td>160T1-8SAR-24-0.8</td>
			<td>Control the acousto-optic modulator</td>
		</tr>
		<tr>
			<td>Solo2 laser power &#38; energy meter</td>
			<td>Gentec-EO</td>
			<td></td>
			<td>Used to measure light intensity of the LED and the fiber coupled laser</td>
		</tr>
		<tr>
			<td>Blue &#181;LED</td>
			<td>Cree</td>
			<td>C470UT200</td>
			<td>It is necessary to build several &#956;LED devices because easily get damaged or the isolation is not good enough</td>
		</tr>
		<tr>
			<td>TDT System&#160;</td>
			<td>Tucker-Davis Technologies</td>
			<td>RZ6-A-P1</td>
			<td>It can be used any system for stimulus generation&#160; presentation and data acquisition</td>
		</tr>
		<tr>
			<td>Single-shank, 16-channel silicon probe</td>
			<td>Neuronexus</td>
			<td>a1x16-5mm-100-177-CM16LP&#160;</td>
			<td>These are fragile devises, must be handled carefully and cleaned after use</td>
		</tr>
		<tr>
			<td>Omnidrill</td>
			<td>World Precision Instruments</td>
			<td>503598</td>
			<td>Perform craniotomy for IC recordings and reference screw implantation</td>
		</tr>
		<tr>
			<td>Micro Drill Steel Burrs</td>
			<td>any commercial; e.g. Fine Science Tools</td>
			<td>19007-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Self tapping bone screw</td>
			<td>any commercial; e.g. Fine Science Tools</td>
			<td>19010-10</td>
			<td>Reference screw</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>any commercial; e.g. Luigs+NeumannInVivo Unit Junior 4 axis</td>
			<td></td>
			<td>Positioning of recording probe</td>
		</tr>
	</tbody>
</table>

optogenetic stimulation of the auditory nerve

Direkte elektrisk stimulation af spiral ganglion neuroner (SGNs) ved cochlear implantater (CIS) giver mulighed for åben taleforståelse i de fleste implanterede døve fag 1- 6. Ikke desto mindre, lyd kodning med aktuelle CIs har dårlig frekvens og intensitet opløsning på grund af en bred strøm spredes fra hver elektrode kontakt aktiverer et stort antal SGNs langs tonotopic akse cochlea 7- 9. Foreslås Optisk stimulation som et alternativ til elektrisk stimulering, der lover rumligt mere begrænset aktivering af SGNs og dermed højere frekvens opløsning af kodning. I de senere år har direkte infrarød belysning af cochlea blevet anvendt til at fremkalde reaktioner i hørenerven 10. Metoden kræver dog højere energier end elektrisk stimulation 10,11 og usikkerhed om den underliggende mekanisme 12. Her beskriver vi en metode baseret på optogenetics at stimulere SGNsmed lav intensitet blåt lys, ved hjælp af transgene mus med neuronal udtryk for channelrhodopsin 2 (CHR2) 13 eller virus-medieret ekspression af CHR2-varianten Catch 14. Vi anvendte mikro-lysemitterende dioder (μLEDs) og fiber-koblede lasere til at stimulere CHR2-udtrykkende SGNs gennem en lille kunstig åbning (cochleostomy) eller runde vindue. Vi analyseret svarene ved hovedbunden optagelser af lys-fremkaldte potentialer (optogenetic auditive hjernestamme respons: oABR) eller ved mikroelektrode optagelser fra den auditive vej og sammenlignede dem med akustisk og elektrisk stimulation.

<html> En optimal cochleostomy er kritisk og øger sandsynligheden for et vellykket eksperiment. Dette betyder, at vinduet er regelmæssige, små, og der er ingen skade af de interne cochlear strukturer. For eksempel blødning indikerer skade af stria vascularis. Et godt eksempel er vist i figur 1B. Brug CHR2-transgene mus, er CHR2 udtrykt i SGNs i cochlea (figur 1C). Blåt lys belysning, enten ved μLED eller laser, fremkalder store oABR, so...

Watch this Scientific Journal Video about Optogenetic Stimulation of the Auditory Nerve at JoVE.com

Optogenetic Stimulation of the Auditory Nerve

Optogenetic Stimulering af hørenerven

Cochlear implants (CIS) gør det muligt at høre ved direkte elektrisk stimulation af hørenerven. , Dårlig hyppighed og intensitet opløsning begrænser imidlertid kvaliteten af ​​at høre med kreditinstitutter. Her beskriver vi optogenetic stimulering af hørenerven i mus som en alternativ strategi for auditiv forskning og udvikling af fremtidige kreditinstitutter.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Research

JoVE Journal

Neuroscience

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selektiv Viral transduktion af Voksen-fødte olfaktoriske neuroner for Kronisk In vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Fiberoptisk implantation for kronisk Optogenetic Stimulation af hjernevæv

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Stimulering af flugt Opførsel i<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Selektiv Tracing af Auditive fibre i de Avian embryoer Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo optogenetic Stimulering af Rodent centralnervesystemet

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Direkte visualisering af den murine Dorsal Cochlear Nucleus for optogenetic Stimulering af Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Vurdering af neuromuskulære funktion Brug Perkutan elektrisk nervestimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...