Name: direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway
Uploaded: 2015-01-20T15:00:00
Description: Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Finansman: Bu çalışma Vakfı Bertarelli hibe (DJL), bir MED-EL hibe (DJL) ve Sağlık Hibeler DC01089 bir National Institutes (MCB) tarafından desteklenmiştir.

NOT: Bütün deneysel prosedürler Halk Sağlığı Servisi İnsanca Bakım Politikası ve laboratuvar Hayvanların Kullanımı, dahil olmak üzere ulusal hayvan bakımı yönergeleri izleyin Massachusetts Göz Hayvan Bakımı ve Kullanımı Komitesi ve Kulak Revir ve Harvard Tıp Okulu, uygun yapılmaktadır Ilar Kılavuzu ve Hayvan Refahı Yasası. Deneysel işlemler sol DCN detay maruz aşağıda listelenen. Sağkalım ameliyat yaparken steril aletler kullanın. 1. İlköğretim Kraniyotomi ve Dorsal Cochlear N...

<ol>
	<li>Lalwani, A., Mhatre, A. <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+therapy.">Cochlear gene therapy.</a> Ear Hear. 24 (4), 342-348 (2003).</li><li>Lalwani, A., Jero, J., Mhatre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+issues+in+cochlear+gene+transfer.">Current issues in cochlear gene transfer.</a> Audiol Neurootol. 7 (3), 146-151 (2002).</li><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>Koh, S., Pettis, R., Mhatre, A., Lalwani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+microinjection+and+its+effects+upon+auditory+function+in+the+guinea+pig.">Cochlear microinjection and its effects upon auditory function in the guinea pig.</a> Eur Arch Otorhinolaryngol. 257 (9), 469-472 (2000).</li><li>Lalwani, A., Walsh, B., Reilly, P., Muzyczka, N., Mhatre, A. <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>Wareing, M., Lalwani, A. <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+therapy:+current+perspectives.">Cochlear gene therapy: current perspectives.</a> Int J Pediatr Otorhinolaryngol. 5 (49), 27-30 (1999).</li><li>Han, 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=Transgene+expression+in+the+guinea+pig+cochlea+mediated+by+a+lentivirus-derived+gene+transfer+vector.">Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.</a> Hum Gene Ther. 10 (11), 1867-1873 (1999).</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>Hernandez, V. H., 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=Optogenetic+stimulation+of+the+auditory+pathway.">Optogenetic stimulation of the auditory pathway.</a> J Clin Invest. 124 (3), 1114-1129 (2014).</li><li>Adamantidis, 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=Optogenetic+interrogation+of+dopaminergic+modulation+of+the+multiple+phases+of+reward-seeking+behavior.">Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior.</a> J Neurosci. 30 (31), 10829-10835 (2011).</li><li>Kim, 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=Optogenetic+mimicry+of+the+transient+activation+of+dopamine+neurons+by+natural+reward+is+sufficient+for+operant+reinforcement.">Optogenetic mimicry of the transient activation of dopamine neurons by natural reward is sufficient for operant reinforcement.</a> PloS One. 7 (4), e33612 (2012).</li><li>Britt, J., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogations+of+the+neural+circuits+underlying+addiction.">Optogenetic interrogations of the neural circuits underlying addiction.</a> Curr Opin Neurobiol. 23 (4), 539-545 (2013).</li><li>Abbott, S., Coates, M., Stornetta, R., Guyenet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+c1+and+retrotrapezoid+nucleus+neurons+causes+sleep+state-dependent+cardiorespiratory+stimulation+and+arousal+in+rats.">Optogenetic stimulation of c1 and retrotrapezoid nucleus neurons causes sleep state-dependent cardiorespiratory stimulation and arousal in rats.</a> Hypertension. 61 (4), 835-841 (2013).</li><li>Carter, 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=Tuning+arousal+with+optogenetic+modulation+of+locus+coeruleus+neurons.">Tuning arousal with optogenetic modulation of locus coeruleus neurons.</a> Nat Neurosci. 13 (12), 1526-1533 (2010).</li><li>Darrow, 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=Optogenetic+control+of+central+auditory+neurons.">Optogenetic control of central auditory neurons.</a> Assoc. 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(695), (2012).</li><li>Shimano, 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=Assessment+of+the+AAV-mediated+expression+of+channelrhodopsin-2+and+halorhodopsin+in+brainstem+neurons+mediating+auditory+signaling.">Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling.</a> Brain Res. 1511, 138-152 (2013).</li><li>Doucet, J., Ryugo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+from+the+ventral+cochlear+nucleus+to+the+dorsal+cochlear+nucleus+in+rats.">Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats.</a> J Comp Neurol. 385, 245-264 (1997).</li><li>Brown, M., Drottar, M., Benson, T., Darrow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissural+axons+of+the+mouse+cochlear+nucleus.">Commissural axons of the mouse cochlear nucleus.</a> J Comp Neurol. 521, 1683-1696 (2013).</li><li>Verma, R., 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> Hear Res. 310, 69-75 (2014).</li><li>Taberner, A. M., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+single+auditory+nerve+fibers+in+the+mouse.">Response properties of single auditory nerve fibers in the mouse.</a> J Neurophysiol. 93 (1), 557-569 (2005).</li><li>Rolls, 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=Optogenetic+disruption+of+sleep+continuity+impairs+memory+consolidation.">Optogenetic disruption of sleep continuity impairs memory consolidation.</a> Proc Natl Acad Sci. 108 (32), 13305-13310 (2011).</li><li>Huff, M., Miller, R., Deisseroth, K., Moorman, D., LaLumiere, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraining+optogenetic+manipulations+of+basolateral+amygdala+activity+modulate+consolidation+of+inhibitory+avoidance+memory+in+rats.">Posttraining optogenetic manipulations of basolateral amygdala activity modulate consolidation of inhibitory avoidance memory in rats.</a> Proc Natl Acad Sci. 110 (9), 3597-3602 (2013).</li><li>Stortkuhl, K., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Smell+of+Blue+Light:+A+New+Approach+toward+Understanding+an+Olfactory+Neuronal+Network.">The Smell of Blue Light: A New Approach toward Understanding an Olfactory Neuronal Network.</a> Front Neurosci. 5 (72), (2011).</li><li>Hira, R., 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=Transcranial+optogenetic+stimulation+for+functional+mapping+of+the+motor+cortex.">Transcranial optogenetic stimulation for functional mapping of the motor cortex.</a> J Neurosci Methods. 179 (2), 258-263 (2009).</li><li>Ayling, O., Harrison, T., Boyd, J., Foroshkov, A., Murphy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+light-based+mapping+of+motor+cortex+by+photoactivation+of+channelrhodopsin-2+transgenic+mice.">Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.</a> Nat Methods. 6 (3), 219-224 (2009).</li><li>Boyden, E., Zhang, F., Bamberg, E., Nagel, G., 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=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat. Neurosci. 8, 1263-1268 (2005).</li></ol>

Bu kağıt, merkezi işitme sisteminin manipülasyonu için fare modelinde DCN doğrudan görselleştirme tekniği anlatılmaktadır. Doğrudan görselleştirme özetlenen yaklaşım stereotaksik yaklaşımlar ana alternatif üzerinde önemli avantajlar sağlar. Stereotaksik yaklaşımlar doğrudan görselleştirme göze değil oysa Öncelikle, DCN doğrudan görselleştirme, beyin sapı sitenin hemen onay veriyor. Virüs-aracılı gen transferi durumunda olduğu gibi, inkübasyon sürelerini uzun süreli gerektirecek d...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

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direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

Tutuklama veya işitme kaybı ters virüs aracılı gen transferi kullanımı soruşturma büyük ölçüde çevresel işitme sisteminin küme olmuştur. Birkaç çalışma, merkezi işitme sistemine gen transferi inceledik. işitsel yolun ikinci dereceden nöronları içeren beyin sapı dorsal koklear nükleus (DCN), gen transferi için potansiyel bir sitedir. Bu protokol, posterior fossa yaklaşımı ile kemirgen DCN doğrudan ve maksimal maruz kalma bir tekniktir gösterilmiştir. Bu yaklaşım, akut ya da hayatta kalma cerrahi sağlar. DCN doğrudan görselleştirme sonra, deney bir ana bilgisayar, bir mavi ışık lazer bağlanmış bir fiber optik ile koklear nükleus ve daha sonra stimülasyon içine opsins püskürtülmesi de dahil olmak üzere uygulanabilir. Böyle elektrik stimülasyonu ve nöral enjektör iz gibi diğer nörofizyoloji deneyler de mümkün bulunmaktadır. Visualiza düzeyiyon ve uyarılma süresi elde deneyler geniş bu yaklaşım uygulanamaz hale.

Kısmi Serebellar Aspirasyon Koklear Nucleus Erişim gösterir Kafatası örten deri ve kas gibi koronal ve lamda sütür hatları gibi, kafatası yüzey işaretlerini, kaldırıldıktan sonra, Kraniotomi yaklaşık lokalizasyonu göstermektedir. Rongeurs ile kraniyotomi ardından, beyincik görüntülenmiştir. Serebellumun küçük bir kısmı dikkatlice aspire enjekte edilebilir CN, (Şekil 1) görselleştirme gösterir. Mavi Iş?...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

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

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

İşitsel Yolu ve optogenetic Stimülasyon için Murin Dorsal Koklear Çekirdek Doğrudan Görselleştirme

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

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