This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

NOTA: tutte le procedure e la gestione degli animali rispettate le linee guida etiche NIH e requisiti del protocollo approvati Care and Use Committee della University of California Animal Istituzionale, San Francisco. 1. Preparare la Animal di Chirurgia <ol><li> Eseguire interventi chirurgici in un ambiente pulito, spazio dedicato. Autoclave tutti gli strumenti chirurgici, sterilizzare con una sterilizzatrice vetro tallone prima dell&#39;intervento. NOTA: In questo protocollo, utilizzare giorno postnatale 10-12 (P10-12) topi FVB. Età diverse e ceppi di topi possono essere utilizzati per soddisfare le esigenze di uno specifico progetto. I topi di età superiore a P40 può essere impegnativo, perché l&#39;osso bulla diventa più difficile. </li><li> Anestetizzare topi con iniezione intraperitoneale di una miscela di ketamina cloridrato (100 mg / kg), xilazina cloridrato (10 mg / kg), e acepromazina (2 mg / kg). Inizia la preparazione chirurgica solo dopo che l&#39;animale non risponde più agli stimoli dolorosi, come pinch punta. Se necessary, somministrare una dose di richiamo (un quinto della dose originale) del cocktail anestetico per ripristinare il piano anestetico originale. NOTA: Fare attenzione a questo passaggio perché la maggior parte della mortalità osservata a questa età è causata da overdose di anestetico. </li><li> Coprire gli occhi dell&#39;animale con una pomata oftalmica protettiva per mantenere gli occhi umidi durante l&#39;anestesia, sopprimendo riflesso corneale dell&#39;animale. </li><li> Posizionare il mouse con il collo esteso su una piastra elettrica per tutta la procedura fino a quando il mouse è completamente sveglio, per evitare post-anestesia ipotermia. </li><li> Radere la regione post-auricolare sinistro con un clipper e disinfettare con il 70% di etanolo e iodopovidone prima manipolazione chirurgica. </li></ol> 2. La procedura chirurgica e Vector Injection <ol><li> Utilizzare un approccio post-auricolare per esporre la bolla timpanica. Incidere il tessuto sottocutaneo con piccole forbici per esporre il muscolo post-auricolare. Dopo ritrarre il tessuto adiposo in Oce lato posteriore dell&#39;incisione, separare i muscoli a destra ea sinistra perpendicolare alla incisione per esporre l&#39;osso temporale. Assicurarsi che questa incisione è abbastanza a lungo per lavorare comodamente con visualizzazione adeguata. </li><li> Perforare bolla timpanica con un ago 25 G ed espandere il foro come necessario con pinze staccando la spina dorsale per consentire l&#39;accesso al giro basale della coclea, e quindi allargare sufficientemente visualizzare l&#39;arteria stapediale e la membrana della finestra rotonda (RWM) . </li><li> Forare la RWM delicatamente al centro con una pipetta borosilicato capillare. Osservare efflusso fluido attraverso il RWM a questo punto; questo è normale. Attendere che l&#39;efflusso è stabilizzata (il fluido effluxed dalla coclea viene asciugata con carta da filtro sterile). NOTA: Fare attenzione quando si tiene e avanza l&#39;ago di vetro in modo che la perforazione RWM è il più piccolo possibile. A seconda del microscopio utilizzato, detengono le pipette con un micromanipolatore oa mano con una pipettatitolare. </li><li> Preparare le pipette per l&#39;iniezione con una pipetta estrattore lo stesso senso pipette patch clamp sono preparati. Assicurarsi che il diametro della punta è di grandi dimensioni (circa 15 micron di diametro) in modo che il virus pipettando dentro e fuori è fatto facilmente. </li><li> Elaborare 1 a 2 ml di AAV1-VGLUT3, AAV1-GFP o AAV2-GFP in una pipetta di iniezione. Dopo l&#39;efflusso è stabilizzato (da 5 a 10 min), microinject questo volume fisso nella scala timpanica attraverso lo stesso foro precedentemente realizzato in RWM. </li><li> Sigillare la nicchia RW subito dopo aver estratto la pipetta con un piccolo tampone di muscoli e fissarlo con una piccola goccia di tessuto adesivo posto sul muscolo per evitare perdite dalla finestra rotonda. NOTA: Seal questo buco molto bene dopo l&#39;ago viene rimosso con un piccolo tappo di muscoli per evitare qualsiasi perdita perilymph dalla coclea-questo passaggio è fondamentale per preservare l&#39;udito. La mancata sigillare completamente il RWM comporterà la perdita dell&#39;udito nel corso del tempo. </li><li> Dopo l&#39;iniezione, coprire ilbuco nella bolla uditiva con tessuto adiposo, restituire i muscoli post-auricolari e tessuto adiposo alla loro posizione normale e suturare la ferita a strati con un assorbibile sutura cromico 6-0 o più piccolo. </li><li> Disinfettare la ferita con povidone-iodio. </li></ol> 3. postoperatoria Cura <ol><li> Mettere i topi in una gabbia calda e non lasciarli incustoditi fino a quando non sono completamente recuperati poi spostarli nuovamente con la madre. Si consiglia di rimuovere il maschio dalla gabbia prima di mettere i cuccioli di nuovo con la madre. </li><li> Somministrare per via sottocutanea Carprofen (2 mg / kg) per l&#39;analgesia dopo l&#39;intervento e ogni 24 ore in seguito per 3 giorni, per gestire l&#39;infiammazione e il dolore. Monitorare gli animali ogni giorno per i segni di disagio, perdita di peso anormale, dolore, o infezione. Generalmente dal 3 ° giorno dopo l&#39;intervento chirurgico tutti i topi vanno agiscono normalmente. Se i sintomi di disagio o di malattia appaiono in un mouse dopo il 3 ° giorno, in considerazione eutanasia tegli mouse come da linee guida istituzionali. </li></ol> 4. Valutazione della funzione cocleare dopo la consegna Viral Utilizzando uditiva del tronco encefalico (ABR) Risposta Recordings NOTA: La risposta del tronco encefalico uditivo (ABR) è un Potenziali evocati uditivi estratto da attività elettrica in corso nel cervello e registrato attraverso elettrodi posti sotto il cuoio capelluto. L&#39;animale viene stimolato con il suono. La registrazione risultante consiste di cinque onde che riflettono l&#39;attività elettrica dei punti successivi nel percorso uditivo nei primi 10 msec dopo l&#39;insorgenza di uno stimolo uditivo. <ol><li> Anestetizzare topi mediante iniezione intraperitoneale di una miscela di ketamina cloridrato e xilazina cloridrato come descritto nella sezione 1.2 del presente protocollo, salvo non acepromazina. </li><li> Posizionare il mouse su una piastra elettrica per tutto il test dell&#39;udito fino a quando il mouse è completamente sveglio, per evitare post-anestesia ipotermia. NOTA: Usai seguenti stimoli sonori in questo esperimento: clicca (5 msec durata; 31 Hz tasso presentazione). </li><li> Inserire elettrodi sottocutanei aghi al vertice, sotto la pinna dell&#39;orecchio sinistro (riferimento), e sotto l&#39;orecchio controlaterale (massa) e iniziare ABRs registrazione come precedentemente descritto in una camera insonorizzata 27,28. Record ABR forme d&#39;onda a intervalli di 5 dB del livello di pressione sonora giù dal massimo di ampiezza. </li><li> Determinare le soglie ABR dopo l&#39;intervento già a quattro giorni dopo la consegna virale NOTA: soglia è definita come il livello di stimolo minimo al quale risposta picchi di onde I-V erano chiaramente e ripetutamente presente un controllo visivo. </li><li> Misura onda Io per analizzare l&#39;attività del nervo cocleare. Il livello di stimolo più basso che produce una forma d&#39;onda rilevabile ABR è definito come soglia. </li></ol> 5. Cochlear Transgene Protein Expression Usando immunofluorescenza <ol><li> Anestetizzare topi da un overdose di intrapiniezione eritoneal di una miscela di ketamina cloridrato e xilazina cloridrato come descritto nella sezione 1.2 del presente protocollo, salvo non acepromazina. </li><li> Decapitare il capo solo dopo che l&#39;animale non risponde più agli stimoli dolorosi, come il pinch punta. </li><li> Sezionare il coclee fuori e di processo per tutto il montaggio immunofluorescenza come descritto 26. Un protocollo dettagliato di cocleare tutto-mount immunofluorescenza utilizzando anti-VGLUT3 e anticorpi anti-miosina 7a è descritto in Akil et al., 2013 29. </li><li> Per l&#39;etichettatura GFP, incubare le cocleari tutto-monti notte a 4 ° C con un coniglio anti-GFP anticorpi a 1: 250 di diluizione. </li><li> Risciacquare la coclee due volte per 10 min con PBS e poi incubare per 2 ore a IgG anti-coniglio di capra coniugata Cy2 diluito 1: 4000 in PBS. </li><li> Sciacquare la coclee con PBS due volte per 10 minuti e incubare con DAPI per 15 min. </li><li> Montare la coclee su vetrini e osservare under un microscopio con immunofluorescenza confocale. </li></ol>

<ol>
	<li>Jero, 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=Cochlear+gene+delivery+through+an+intact+round+window+membrane+in+mouse.">Cochlear gene delivery through an intact round window membrane in mouse.</a> Hum. Gene Ther. 12 (5), 539-548 (2001).</li><li>Nam, H. 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=Structure+of+adeno-associated+virus+serotype+8,+a+gene+therapy+vector.">Structure of adeno-associated virus serotype 8, a gene therapy vector.</a> J. Virol. 81 (22), 12260-12271 (2007).</li><li>Ryan, A. F., Mullen, L. M., Doherty, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+targeting+for+cochlear+gene+therapy.">Cellular targeting for cochlear gene therapy.</a> Adv Otorhinolaryngol. 66, 99-115 (2009).</li><li>Xia, L., Yin, S., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+ear+gene+trasfection+in+neonatal+mice+using+adeno-associate+viral+vwctor:+a+comparison+of+two+approaches.">Inner ear gene trasfection in neonatal mice using adeno-associate viral vwctor: a comparison of two approaches.</a> PLoS One. 7 (8), e43218 (2012).</li><li>Husseman, J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+in+the+inner+ear+using+adenovirus+vectors.">Gene therapy in the inner ear using adenovirus vectors.</a> AdvOtorhinolaryngol. 66, 37-51 (2009).</li><li>Ballana, E., 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=Efficient+and+specific+transduction+of+cochlear+supporting+cells+by+adeno-associated+virus+serotype+5.">Efficient and specific transduction of cochlear supporting cells by adeno-associated virus serotype 5.</a> Neurosci. Lett. 442 (2), 134-139 (2008).</li><li>Praetorius, 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=Adenoviral+vectors+for+improved+gene+delivery+to+the+inner+ear.">Adenoviral vectors for improved gene delivery to the inner ear.</a> Hear. Re. 248 (1-2), 31-38 (2009).</li><li>Kay, M. A., Glorioso, C. G., Naldini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+therapy:+the+art+of+turning+infectious+agents+into+vehicles+of+therapeutics.">Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics.</a> Nature Medicine. 7 (1), 33-40 (2001).</li><li>Kesser, B. W., Lalwani, A. K., Ryan, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+and+Stem+Cell+Transplantation:+Strategies+for+Hearing+Restoration.">Gene Therapy and Stem Cell Transplantation: Strategies for Hearing Restoration.</a> Adv Otorhinolaryngol. 66, 64-86 (2009).</li><li>Cooper, L. B., 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=AAV-mediated+delivery+of+the+caspase+inhibitor+XIAP+protects+against+cisplatin+ototoxicity.">AAV-mediated delivery of the caspase inhibitor XIAP protects against cisplatin ototoxicity.</a> Otol. Neurotol. 27 (4), 484-490 (2006).</li><li>Gratton, M. A., Salvi, R. J., Kamen, B. A., Saunders, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+cisplatin+and+noise+on+the+peripheral+auditory+system.">Interaction of cisplatin and noise on the peripheral auditory system.</a> Hear. Res. 50 (1-2), 211-223 (1990).</li><li>Lalwani, A. K., Walsh, B. J., Reilly, P. G., Muzyczka, N., Mhatre, A. N. <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+in+vivo+gene+therapy+for+hearing+disorders:+introduction+of+adeno-associated+virus+into+the+cochlea+of+the+guinea+pig.">Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.</a> Gene Ther. 3 (7), 588-592 (1996).</li><li>Kesser, B. W., Hashisaki, G. T., Holt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Transfer+in+Human+Vestibular+Epithelia+and+the+Prospects+for+Inner+Ear+Gene+Therapy.">Gene Transfer in Human Vestibular Epithelia and the Prospects for Inner Ear Gene Therapy.</a> Laryngoscope. 118 (5), 821-831 (2008).</li><li>Izumikawa, 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=Auditory+hair+cell+replacement+and+hearing+improvement+by+Atoh1+gene+therapy+in+deaf+mammals.">Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals.</a> Nat. Med. 11 (3), 271-276 (2005).</li><li>Praetorius, 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=Adenovector-mediated+hair+cell+regeneration+is+affected.">Adenovector-mediated hair cell regeneration is affected.</a> Acta Otolaryngol. 130 (2), 215-222 (2009).</li><li>Friedman, L. M., Dror, A. A., Avraham, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+to+study+inner+ear+development+and+hereditary+hearing+loss.">Mouse models to study inner ear development and hereditary hearing loss.</a> Int. J. Dev. Biol. 51 (6-7), 609-631 (2007).</li><li>Chang, E. H., Van Camp, G., Smith, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+connexins+in+human+disease.">The role of connexins in human disease.</a> Ear Hear. 24 (4), 314-323 (2003).</li><li>Cohen-Salmon, 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=Targeted+ablation+of+connexin26+in+the+inner+ear+epithelial+gap+junction+network+causes+hearing+impairment+and+cell+death.">Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death.</a> Curr. Biol. 12 (13), 1106-1111 (2002).</li><li>Nickel, R., Forge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+and+connexins+in+the+inner+ear:+their+roles+in+homeostasis+and+deafness.">Gap junctions and connexins in the inner ear: their roles in homeostasis and deafness.</a> Curr. Opin. Otolaryngol. Head Neck Surg. 16 (5), 452-457 (2008).</li><li>Lv, P., Wei, D., Yamoah, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv7-type+channel+currents+in+spiral+ganglion+neurons:+involvement+in+sensorineural+hearing+loss.">Kv7-type channel currents in spiral ganglion neurons: involvement in sensorineural hearing loss.</a> J. Biol. Chem. 285 (45), 34699-34707 (2010).</li><li>Leibovici, M., Safieddine, S., Petit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+human+hereditary+deafness.">Mouse models for human hereditary deafness.</a> Curr. Top. Dev. Biol. 84, 385-429 (2008).</li><li>Dror, A. A., Avraham, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+loss:+mechanisms+revealed+by+genetics+and+cell+biology.">Hearing loss: mechanisms revealed by genetics and cell biology.</a> Annu. Rev. Genet. 43, 411-437 (2009).</li><li>Richardson, G. P., de Monvel, J. B., Petit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+genetics+of+deafness+illuminates+auditory+physiology.">How the genetics of deafness illuminates auditory physiology.</a> Annu. Rev. Physiol. 73, 311-334 (2011).</li><li>Jero, J., Tseng, C. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+approach+appropriate+for+targeted+cochlear+gene+therapy+in+the+mouse.">A surgical approach appropriate for targeted cochlear gene therapy in the mouse.</a> Hearing Research. 151 (1-2), 106-114 (2001).</li><li>Seal, R. P., 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=Sensorineural+deafness+and+seizures+in+mice+lacking+vesicular+glutamate+transporter+3.">Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3.</a> Neuron. 57 (2), 263-275 (2008).</li><li>Akil, O., 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=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75 (2), 283-293 (2012).</li><li>Akil, O., 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=Progressive+deafness+and+altered+cochlear+innervation+in+knock-out+mice+lacking+prosaposin.">Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin.</a> J. Neurosci. 26 (5), 13076-13088 (2006).</li><li>Fremeau, R. 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=Vesicular+glutamate+transporters+1+and+2+target+to+functionally+distinct+synaptic+release+sites.">Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites.</a> Science. 304 (5678), 1815-1819 (2004).</li><li>Akil, O., Lustig, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Cochlear+Whole+Mount+Immunofluorescence.">Mouse Cochlear Whole Mount Immunofluorescence.</a> Bio-protocol. , (2013).</li><li>Kho, S. T., Pettis, R. M., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlea+microinjection+and+its+effects+upon+auditory+function+in+guinea+pig.">Cochlea microinjection and its effects upon auditory function in guinea pig.</a> Eur Arch Otorhinolaryngol. 257 (9), 469-472 (2000).</li><li>Iizuka, 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=Noninvasive+in+vivo+delivery+of+transgene+via+adeno-associated+virus+into+supporting+cells+of+the+neonatal+mouse+cochlea.">Noninvasive in vivo delivery of transgene via adeno-associated virus into supporting cells of the neonatal mouse cochlea.</a> Hum. Gene Ther. 19 (4), 384-390 (2008).</li><li>Kilpatrick, L. 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=Adeno-associated+virus-mediated+gene+delivery+into+the+scala+media+of+the+normal+and+deafened+adult+mouse+ear.">Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.</a> Gene Ther. 18 (6), 569-578 (2011).</li><li>Yamasoba, T., Yagi, M., Roessler, B. J., Miller, J. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+Ear+Transgene+Expressionafter+Adenoviral+Vector+Inoculation+in+the+Endolymphatic+Sac+Hum.">Inner Ear Transgene Expressionafter Adenoviral Vector Inoculation in the Endolymphatic Sac Hum.</a> Gene Ther. 10 (5), 769-774 (1999).</li><li>Praetorius, M., Baker, K., Weich, C. M., Plinkert, P. K., Staecker, 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+preservation+after+inner+ear+gene+therapy:+the+effect+of+vector+and+surgical+approach.">Hearing preservation after inner ear gene therapy: the effect of vector and surgical approach.</a> ORL J. Otorhinolaryngol. Relat. Spec. 65 (4), 211-214 (2003).</li><li>Carvalho, G. J., Lalwani, A. 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+effect+of+cochleaostomy+and+intracochlear+infusion+on+auditory+brain+stem+response+threshold+in+the+guinea+pig.">The effect of cochleaostomy and intracochlear infusion on auditory brain stem response threshold in the guinea pig.</a> Am. J. Otol. 20 (1), 87-90 (1999).</li><li>Kawamoto, K., Oh, S. H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Functional+and+Structural+Outcome+of+Inner+Ear+Gene+Transfer+via+the+Vestibular+and+Cochlear+Fluids+in+Mice.">The Functional and Structural Outcome of Inner Ear Gene Transfer via the Vestibular and Cochlear Fluids in Mice.</a> Mol. Ther. 4 (6), 575-585 (2001).</li><li>Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N., Mhatre, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+reporter+for+gene+transfer+studies+in+the+cochlea.">Green fluorescent protein as a reporter for gene transfer studies in the cochlea.</a> Hear Res. 114 (1-2), 139-147 (1997).</li><li>Lalwani, A. 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=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. 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=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

In questo lavoro, si descrive in dettaglio una tecnica che può essere utilizzato per la terapia genica cocleare, con l&#39;obiettivo di ripristino o salvataggio normale funzione uditiva che viene compromessa da un difetto genetico. Come è tipicamente atraumatica, questo approccio è sicuro per il trasferimento di geni cocleare o altri potenziali terapie molecolari 30. Altri approcci per la terapia cocleare sono stati descritti, tra cui un approccio ventrale 24, cocleostomia 31,32 e sac...

La terapia genica è stato a lungo suggerito come un potenziale trattamento per la perdita dell&#39;udito genetica, ma il successo in questo settore è rimasta inafferrabile 1. Ad oggi, le metodologie viralmente mediati hanno prevalso grazie alla capacità teorica di indirizzare specifici tipi cellulari all&#39;interno della coclea relativamente inaccessibile. Entrambi adenovirus (AV) e il virus adeno-associato (AAV) sono stati utilizzati per la consegna del gene cocleare. AAV sono vantaggiosi nella coclea per una serie di motivi. Sono virus replica-carenti e possono efficacemente trasferire molecole transgenici a diversi tipi di cellule, tra cui i neuroni, un obiettivo importante per una serie di cause di perdita dell&#39;udito. AAV ingresso nella cellula è mediata da recettori specifici 2; pertanto, la scelta di un particolare sierotipo deve essere compatibile con i tipi cellulari da trasdotte. AAV può efficacemente trasfettare cellule ciliate 3 e incorporare nel genoma dell&#39;ospite, conseguente, espressione stabile a lungo termine del TRAproteine ​​nsgenic e il cambiamento fenotipico nella cella 4. Anche se non necessariamente vantaggioso per applicazioni a breve termine, come la rigenerazione delle cellule ciliate, espressione a lungo termine è molto importante per il salvataggio stabile di difetti genetici. Perché AAV non sono associati a qualsiasi malattia umana o infezione e non mostrano ototossicità 5,6,7, sono un candidato ideale per l&#39;uso in terapia genica per forme ereditarie di perdita dell&#39;udito 8. Trasferimento di materiale genetico esogeno nell&#39;orecchio interno dei mammiferi utilizzando vettori virali è stata studiata negli ultimi dieci anni e sta emergendo come una tecnica promettente per il trattamento di forme sia genetiche e acquisite di perdita dell&#39;udito 9. La coclea è potenzialmente un bersaglio ideale per la terapia genica per diverse ragioni: 1) la sua piccolo volume richiede una quantità limitata del virus necessaria; 2) il suo relativo isolamento da altri effetti limiti organi laterali; e 3) le sue camere piene di liquido facilitano viraleconsegna in tutto il labirinto 10, 11,12,13,14, 15. I modelli murini di sordità congenita consentono uso di molti metodi di studio per monitorare lo sviluppo dell&#39;orecchio interno in un modo replicabile sistematica. Mentre le dimensioni ridotte del topo coclee non presenta difficoltà chirurgica, il mouse serve come modello estremamente importante nello studio della sordità genetica, con diversi vantaggi sperimentali su altre specie 16. Modelli murini consentono la valutazione di una serie di caratteristiche, attraverso analisi di linkage genetico, raccolta di osservazioni dettagliate morfologiche, e la simulazione di scenari patogeni; come tali, sono buoni candidati per la terapia genica mediata viralmente. Ampi studi genetici nei topi combinate con i progressi tecnologici hanno permesso di generare topi geneticamente modificati in modo riproducibile attraverso laboratori 17,18, 19, 20,21. Furthermore, esistono numerosi modelli sia per acquisite ed ereditarie udito fenotipi di perdita nei topi, consentendo test rigorosi in questo modello animale 22, 23,24. Così, correggendo l&#39;udito con la terapia genica virale mediato in un modello di topo è un primo passo opportuno nella ricerca di una cura per le malattie umane. Abbiamo precedentemente dimostrato che i topi transgenici privi di glutammato vescicolare Transporter 3 (VGLUT3) nascono sordi a causa della mancanza di rilascio di glutammato a nastro sinapsi IHC 25. Poiché questa mutazione non porta ad una degenerazione primaria delle cellule ciliate sensorie, questi topi mutanti sono potenzialmente un ottimo modello in cui sperimentare la terapia genica per cocleare sordità congenita. Fino ad oggi, una serie di tecniche di consegna virali per la terapia genica cocleare sono stati descritti, tra andata diffusione membrana finestra, rotondo iniezione membrana finestra e distribuirla su cocleostomia. Ci sono potentivantaggi ial e svantaggi di ciascuno di questi approcci 9. Qui riportiamo un metodo chirurgico per la consegna del gene virale mediata all&#39;orecchio interno VGLUT3 KO mouse attraverso la membrana della finestra rotonda (RWM). Il metodo di iniezione RWM post-auricolare è minimamente invasiva con ottima conservazione dell&#39;udito, ed è relativamente veloce. Come precedentemente pubblicato, nel tentativo di ripristinare dell&#39;udito in questo modello di topo, un vettore che porta il gene AAV1 VGLUT3 (AAV1-VGLUT3) è stato introdotto nella coclea di questi topi sordi al giorno postnatale 12 (P! @), Con conseguente il restauro di sentire 26. Audizione in topi VGLUT3 KO è stato verificato dai uditivo risposta tronco encefalico (ABR), mentre l&#39;espressione della proteina transgene è stata verificata mediante immunofluorescenza (IF). Questa metodologia dimostra quindi che la terapia genica mediata viralmente può correggere un difetto genetico che altrimenti provoca sordità.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Name</td>
			<td style="width:177px;">Company</td>
			<td style="width:161px;">Catalog Number</td>
			<td style="width:111px;">Comments</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Ketamine</td>
			<td style="width:177px;">Butler Schein</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Xylazine</td>
			<td style="width:177px;">AnaSed</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Acepromazine</td>
			<td style="width:177px;">Provided by UCSF LARC</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Carprofen analgesia</td>
			<td style="width:177px;">Provided by UCSF LARC</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Betadine</td>
			<td style="width:177px;">Betadine Puredue Pharma</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">dexamethasone ophthalmic ointment (TobraDex)</td>
			<td style="width:177px;">Alcon</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Heating pad</td>
			<td style="width:177px;">Braintree scientific, inc.</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">25G needle</td>
			<td style="width:177px;">BD</td>
			<td align="right" style="width:161px;">305127</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Borosilicate capillary pipette</td>
			<td style="width:177px;">World precision instruments, inc.</td>
			<td style="width:161px;">1B100F-4</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Suture PDS*plus Antibacterial</td>
			<td style="width:177px;">Ethicon</td>
			<td style="width:161px;">PDP149</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Tissue glue (Vetcode)</td>
			<td style="width:177px;">Butler Schein</td>
			<td align="right" style="width:161px;">31477</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Rabbit Anti-GFP antibody</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:161px;">A11122</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope&#160;&#160;&#160;&#160;&#160;</td>
			<td>Leica</td>
			<td></td>
			<td>MZ95</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flaming/ Brown Micropipette&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sutter Instrument Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puller Model P-97&#160;&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TDT BioSig III System&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>&#160;Tucker-Davis Technologies</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

La terapia genica, utilizzato per realizzare il recupero funzionale di sordità neurosensoriale, promette di concedere una migliore comprensione dei meccanismi molecolari e genetici alla base che contribuiscono alla perdita dell&#39;udito. Introduzione di vettori nell&#39;orecchio interno deve essere fatto in modo che l&#39;agente ampiamente distribuisce tutta la coclea minimizzando lesioni alle strutture esistenti. Questo manoscritto descrive un approccio chirurgico post-auricolare che può essere utilizzato per il mouse terapia cocleare usando molecolari, farmacologico, e consegna virale per topi giorno postnatale 10 e anziani attraverso la membrana finestra rotonda (RWM). Questo approccio chirurgico consente la consegna rapida e diretta nella scala timpanica, riducendo al minimo la perdita di sangue ed evitare la mortalità degli animali. Questa tecnica comporta trascurabile o nessun danno alle strutture essenziali dell&#39;orecchio interno e medio nonché muscoli del collo mentre tutto conservando udito. Per dimostrare l&#39;efficacia di questa tecnica chirurgica, la glutam vescicolaremangiato transporter 3 knockout (VGLUT3 KO) topi verrà utilizzato come esempio di un modello murino di sordità congenita che recupera l&#39;udito dopo la consegna del VGLUT3 all&#39;orecchio interno tramite un virus adeno-associato (AAV-1).

Per verificare le caratteristiche tecniche e l&#39;utilità di un approccio post-auricolare per la terapia molecolare cocleare, AAV1-VGLUT3, AAV1-GFP e AAV2-GFP sono state consegnate in topi P10-12 all&#39;orecchio interno tramite l&#39;RWM. Questo approccio dimostra espressione del transgene successo all&#39;interno delle cellule ciliate interne (IHC) (VGLUT3 Figura 1 e Figura 2 e GFP GFP figura 3A), le cellule ciliate esterne (OHC) (GFP Figura 2) e ce...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Metodo chirurgico per Virally Mediated Gene consegna all&#39;orecchio mouse interno attraverso la finestra rotonda a membrana

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.1K Views.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

Video: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Microscopia intravitale della microcircolazione cervello di topo usando una finestra chiusa del cranio

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Ricerca

Neuroscienze

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Elettrofisiologia II: extracellulare fabbricazione di elettrodi di aspirazione

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolando LacZ cellule che esprimono da tessuti dell&#39;orecchio interno del mouse in citometria a flusso

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

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.

Gene Transfer per l&#39;orecchio del mouse Sviluppo Inner<em&gt; In Vivo</em&gt; Elettroporazione

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 ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

Un lucido e rinforzato Assottigliato cranio-Window a lungo termine Imaging del cervello di topo

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

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.

Registrazioni patch clamp in cellule interne dell&#39;orecchio Capelli Isolato da Zebrafish

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imaging pHluorin-tagged inserimento recettore alla membrana plasmatica nella Primaria neuroni mouse coltivate

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizzando metodologie combinati per definire il ruolo della membrana plasmatica di consegna Durante Axon ramificazione e neuronale morfogenesi

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Tecnica mouse Microchirurgia infusione per Targeted Sostanza consegna nel sistema nervoso centrale<i&gt; via</i&gt; Carotide interna

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Laminectomia e impianto della finestra del cavo spinale nel mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...