자금 조달 :이 작품은 재단 Bertarelli 보조금 (DJL), MED-EL 부여 (DJL), 보건 보조금 DC01089의 국립 연구소 (MCB)에 의해 지원되었다.

참고 : 모든 실험 절차는 보건 서비스 동물 애호 관리에 대한 정책 및 실험 동물의 사용을 포함하여 국가 동물 관리 지침을 따르 매사추세츠 눈의 동물 관리 및 사용위원회와 귀 의무실과 하버드 의과 대학,에 따라 수행된다 ILAR 가이드 및 동물 복지 법. 실험 절차는 왼쪽 DCN의 세부 노출 아래에 나열된. 생존 수술을 수행하는 동안 멸균 악기를 사용합니다. 1 차 개두술 등쪽 달팽이 핵 노출 <ol><li> 마취 <ol><li> 복강 내 투여를 통해 자일 라진 (xylazine) 20 ㎎ / ㎏과 케타민 100 ㎎ / ㎏ 18-24 g의 무게 8~12주, 세 마우스를, 마취. 모니터링 심박수, 호흡 수,뿐만 아니라 발가락 핀치 철수 반사에 의한 적절한 마취를 확인합니다. 눈에 장소 수의사 연고 마취 동안 건조를 방지합니다. 일단 적절하게 마취, 면도 머리 위에 놓인 t그는 수술 부위에 접근이 수월를 제공하기 위해 두피. </li></ol></li><li> 외과 위치 <ol><li> 주둥이 클램프로 고정 작은 동물 정위 홀더에 안전하게 마우스를 놓습니다. </li><li> 주둥이 클램프가 완전히 마우스의 머리를 고정하는 적절한 호흡하지만 꽉 충분히 허용 할 정도로 느슨한 있는지 확인하십시오. 마우스 머리가 느슨한 경우, 동물 절차의 개두술 부분에서 헤드 홀더에서 떨어질 가능성이있다. </li><li> 심장 박동 모니터링 할 수 표준 방식에서 청성 뇌간 이​​식 전극을 배치합니다. 호흡 비율은 시각화하여 모니터링해야합니다. 정상적인 심장 박동과 호흡이 마우스의 연령에 따라 달라질 수 있습니다. </li><li> homeothermic 가열 담요에 배치를 통해 온도계는 배치되고 euthermia이 보장된다. </li></ol></li><li> 피부 절개하고 두개골의 interparietal과 뒤통수 뼈의 노출 <ol><li> microsc에서OPE, 피부를 통해 수직 절개가 직접 날개 사이의 중간 선에서 시작하고, 후두부의 꼬리 부분까지 연장합니다. 정중선 피부 절개를 따라 횡 방향으로 피부를 변위. 두개골의 왼쪽 후방 측면을 덮고있는 근육을 시각화. </li><li> 메스 홍채 가위와 각각의 뼈에 근육 첨부 파일의 이단에 의해 근육 왼쪽 정수리, interparietal 위에 놓인, 두개골의 후두 뼈를 제거합니다. 컷 근육의 가장자리를 따라 출혈의 작은 정도를 관찰한다. 10 ~ 15 초 동안 면봉으로 부드럽게 압력이 최소화. </li></ol></li><li> 개두술을 덮는 인공 와우 핵 <ol><li> 시상 및 람다 봉합 라인 (그림 1, 왼쪽 패널)을 포함하여 관련 봉합 라인을 확인합니다. </li><li> rongeurs를 사용하여 람다 봉합 라인에 중간 선 왼쪽 interparietal 뼈를 통해 개두술, ~ 2mm의 꼬리를합니다. 이 지역은 DCN 위에 놓인다. </li><li> 따라와g의 개두술은 소뇌 위에 놓여 두라의 얇은 층을 관찰합니다. 메스 블레이드를 사용하여 경질을 제거합니다. (그림 1, 가운데 패널). 참고 : 경질의 제거는 약간의 출혈이 발생할 수 있습니다. 경질을 제거 메스 블레이드를 사용하는이 프로세스는 뇌간 표면 및 DCN 모호한 식별 할 풀, 소뇌 흡입 동안 혈액 손실의 양을 감소시키기위한 것이다. 마우스의 총 혈액량은 15 %보다&gt; ~ 1.5 ml를 초과하지 않아야합니다. 출혈이 금액을 초과하면, 마우스는 유체 보충과 함께 제공되어야한다. </li><li> 면 팁 어플리케이터 소뇌를 오버레이 응고 된 혈액을 제거합니다. 선택적으로, 부드럽게 혈액을 혈액을 멀리 취소 소뇌에 치과 지점을 사용하여 식염수 물방울. </li></ol></li><li> 소뇌 포부 <ol><li> DCN이 힘을 될 때까지 5 프랑스어 흡입을 사용하면, DCN 위에 놓인 왼쪽 소뇌의 측면 - 대부분의 부분을 대기음연간 (그림 1, 오른쪽 패널). 왼쪽 소뇌의 약 1/4 ~ 1/3를 제거합니다. DCN에 인접한 주요 랜드 마크는 우수한 반규관의 팽대부입니다. 참고 : 소뇌의 포부는 프로토콜의 핵심 단계이다. 이 출혈의 원인이됩니다이 같은 흡입의 다중 패스와 함께 하나의 시도에서 발생하지 않을 경우 소뇌의 감독 흡입은 가장 성공적으로 수행된다. 흡입을 돕기 위해, 소뇌의 표면에 약간 원위 것이다 CN의 예상 깊이 현미경의 초점면을 설정한다. CN에 초점면을 설정하면 시각화를 개선하고 선명한 이미지를 보장합니다. </li><li> DCN에 따라 흡입, 더 출혈, 뇌척수액 (CSF) 빌드 업, 그리고 소뇌 변위가 예상된다. 혈액 응고를 방지하기 위해 신속 개두술에 멸균 생리 식염수를 주입 시키 0.5 CC. 치과 점과 흡입과 부드러운 dabbing의 조합은 할 수있다DCN의 직접 시각화를위한 경로를 멀리 취소하는 데 사용됩니다. 직접 DCN의 표면에 접촉하지 마십시오. </li></ol></li></ol> 바이러스 매개 유전자 전달 및 외과 복구를위한 2. 압력 미세 주입 <ol><li> DCN은 상부의 혈액과 뇌척수액의 무료이며 명확하게 볼 수 있습니다 후 2 분 동안 10 μL 해밀턴 주사기를 사용하여 DCN에 압력 microinjections을합니다. </li><li> 팁은 더 이상 DCN의 표면 아래에 보일 때까지 미세 조작기로 바늘을 도입하지 않는다. </li><li> 최적의 성능을 위해, 외상을 최소화하고 얇은 DCN 내 주입 볼륨을, 지역화, 45 °로, 얕은 경사와 등을 33 또는 34 게이지 바늘 (34 게이지 바늘 기밀 주사기를 사용)을 사용하는 얕은 뇌간 구조 (&lt;300 μm의 두께). 참고 : 다른 미세 주입 장비가 활용 기초 할 수있다. 이상적으로, 주사 기기는 약간의 벤 있도록 유연해야D는 경우 직접 D​​CN을 대상으로 할 필요가 있었다. 마우스 인해 호흡에 절차 전반에 걸쳐 약간 이동하면서 또한, 악기는 운동의 소량을 견딜 수 있도록 충분히 견고해야한다. </li></ol> 3. 수술 복구 <ol><li> 주사 직후 피부를 다시 근사 및 마우스 표준 복구 절차 당 복구 할 수있다. 남은 소뇌와 흉터 조직은 소뇌의 흡인에 의한 공동으로 채울 것입니다. 충분한 의식이 흉골 드러 누움을 유지하기 위해 회복 될 때까지 지속적으로 동물을 모니터링 할 수 있습니다. 심박수, 호흡 수,뿐만 아니라 공급하는 능력을 모니터한다. 참고 : 완전히 회복 될 때까지 아니 동물 수술을 한 다른 동물 케이지에 반환됩니다. </li><li> 마우스는 수술 후 원에서 도보로 시작하는 경우가 가능성이 힘든 시간 공급을하는 것처럼 (&lt;사례 5 %), 피부 절개의 폐쇄 후 일반적으로 2-4 시간은, 바로, 마우스를 희생. 티그의 부작용으로 인해 소뇌 흡인하기 쉽다. 동물은 수술 후 통증에 대해 모니터링되어야하고, 적절한 마약 제도적 표준을 기반으로 동물의 편안함을 보장하기 위해 부여해야합니다. 항생제는 필요 감염의 경우 증거 할 수 있습니다. </li></ol> 4. 차 개두술 달팽이 핵 노출 <ol><li> 치유와 바이러스 매개 유전자 전달 배양 2-4 주 후, 이전에 주입 마우스 단계를 반복 1.1-1.5 최적의 배양 시간이 전송 유전자의 종류에 따라 다릅니다 다시 마취. </li><li> 핀셋, 메스 및 rongeurs의 조합에 의해 개두술 사이트를 통해 흉터 조직을 제거합니다. </li><li> 개두술을 시각화 한 후, DCN을 덮는 소뇌 및 흉터 조직을 나머지의 조합을 식별한다. 위에있는 소뇌 / (이전 소뇌의 열망과 유사) 흉터 조직을 흡인하기 위해 5 프랑스어 흡입을 사용합니다. </li><li> 흡인 후, 추가 출혈, 뇌척수액 B 기대uild 업 및 소뇌의 나머지 비트. 빠르게 혈액 응고를 방지하기 위해 식염수에 개두술을 소개한다. DCN의 직접 시각화에 대한 경로를 취소 부드러운 치과 점 dabbing 및 흡입의 조합을 사용합니다. </li><li> DCN의 표면을 관찰한다. DCN 이제 액세스 가능한 optogenetics 기반 생리학 실험을 수행합니다. optogenetics 기반 실험을 구동하기 위해, 광섬유 (도 2)와, 예를 들면 광 자극을 소개. </li></ol> 5. 조직학 <ol><li> 실험의 결론에 따라, 마취제의 과다 복용으로 마우스를 안락사. 4 % 파라 포름 알데히드이어서 생리 식염수로 마우스를 관류. </li><li> 2 시간의 두개골 및 사후 수정에서 뇌간의 압축을 풉니 다. 24 ~ 48 시간 동안 30 % 자당의 뇌간을 Cryoprotect. 제 60 ㎛의 섹션을 사용하여 표준 저온 유지 장치를 이용 뇌간. </li></ol>

<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. Res. Otolaryngol. Abstr. (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>

저자가 공개하는 게 없다.

이 논문은 중앙 청각 시스템의 조작 생쥐 모델에서 DCN의 직접 시각화 기술을 설명합니다. 직접 시각화의 설명 방식은 정위 적 방법을 주요 대안에 비해 상당한 장점을 제공합니다. 정위 방법은 직접 시각화 여유가없는 반면, 주로, DCN의 직접 시각화, 뇌간의 사이트를 즉시 확인 할 수 있습니다. 바이러스 - 매개 유전자 전달의 경우에서와 같이, 인큐베이션 기간을 필요로 장기간 실험에서, 주입 대?...

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.

<table><tbody><tr><td>Stereotaxic holder</td><td>Stoelting</td><td>51500</td><td></td></tr><tr><td>Homeothermic blanket</td><td>Harvard</td><td>507214</td><td></td></tr><tr><td>Scalpel blade #11</td><td>Fine Surgical Tools</td><td>10011-00</td><td></td></tr><tr><td>Iris scissor</td><td>Fine Surgical Tools</td><td>14084-08</td><td></td></tr><tr><td>5 French suction</td><td>Symmetry Surgical</td><td>2777914</td><td></td></tr><tr><td>Dental Points</td><td>Henry Schein</td><td>100-8170</td><td></td></tr><tr><td>Bone rongeur</td><td>Fine Surgical Tools</td><td>16020-14</td><td></td></tr><tr><td>10 &amp;micro;l Hamilton syringe</td><td>Hamilton&amp;nbsp;</td><td>7633-01</td><td></td></tr><tr><td>34 gauge, needle</td><td>Hamilton&amp;nbsp;</td><td>207434</td><td></td></tr><tr><td>Rongeurs</td><td>Fine Surgical Tools</td><td>16021-14</td><td></td></tr></tbody></table>

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

체포 또는 청력 손실을 반대하는 바이러스 매개 유전자 전달의 사용에 대한 조사는 크게 말초 청각 시스템에 이관되었다. 연구는 거의 중앙 청각 시스템에 유전자 전달을 조사 하였다. 청각 경로의 2 차 뉴런을 포함 뇌간의 등쪽 달팽이관 핵 (DCN)를, 유전자 전달을위한 잠재적 인 위치이다. 이 프로토콜에서 후두와 접근법을 통해 쥐의 DCN의 직접적이고 최대 노출에 대한 기술은 증명된다. 이 방법은 하나 급성 또는 생존 수술을 할 수 있습니다. DCN의 직접적인 시각화에 이어, 실험 호스트는 청색 레이저에 연결된 광 파이버에 의한 인공 핵 및 후속 자극으로 opsins의 주입을 포함 할 수있다. 이러한 전기 자극과 신경 인젝터 트레이싱과 같은 다른 신경 생리학 실험도 가능하다. visualiza의 수준기 및 자극 지속 기간은 달성 실험 광범위한 적용이 접근법 만든다.

부분 소뇌 흡입은 코 클리어 핵에 대한 액세스를 보여줍니다 두개골을 덮는 피부와 근육이 같은 관상과 람다 봉합 선으로, 두개골 표면 랜드 마크를 제거한 후, 개두술의 대략적인 현지화를 보여줍니다. rongeurs와 개두술에 따라, 소뇌가 보여집니다. 소뇌의 작은 부분을주의 깊게 흡입 후 주입 할 수있는 CN, (그림 1)의 시각화를 보여줍니다. <p class="jove_con...

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.

청각 통로의 Optogenetic 자극에 대한 쥐의 등쪽 달팽이 핵의 직접 시각화

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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9.2K 조회수.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

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

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

성인 및 신생아 쥐의 내 귀에 로컬 약물 전달에 대 한 외과 접근으로 Canalostomy

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

연구

유전학

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic 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 in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

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

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

만성에 대한 성인 태어나 강한 뉴런의 선택 바이러스성 전달 생체내에 Optogenetic의 자극

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

신경과학

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

차 문화와 Corti의 Murine 오르간의 플라스미드 Electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

생물학

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

설치류 중추 신경계의 생체 Optogenetic 자극

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

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

에 가이드<em&gt; 생체</emOptogenetically 확인 된 대뇌 피질의 억제의 interneurons에서&gt; 단일 단위 기록

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

청각 신경의 자극 Optogenetic

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

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

신생아 쥐의 달팽이관 Explant 기법으로<em&gt; 체외에서</em&gt; 청력 연구의 스크리닝 도구

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

생체 내 신경 회로 연결성 모방을 위한 체외 쐐기 슬라이스 준비

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

청각 뉴런을 시각화하기 위한 쐐기 뇌 슬라이스 준비하기

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain ...

청각 통로의 Optogenetic 자극에 대한 쥐의 등쪽 달팽이 핵의 직접 시각화

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