저자는 초기 embryogenesis 동안 여자는 귀에 해부학 적 구조에 대한 전문 지식을위한 이미징 기술과 박사 도리스 우와 제안과 도움을 박사 캔디스 Hsieh 감사하고 싶습니다. 이 작품은 NSF IOS-0642346, NIH T32-DC010775, NIH T32-GM008620, NIH R01-DC010796 및 DOE GAANN P200A120165에 의해 지원되었다.

1. 다음 해부 도구와 시약을 준비 <ul><li> 인공 뇌척수 (aCSF, 130 MM NaCl, 3 MM KCl, 1.2 MM KH 2 PO 4, 20 MM NaHCO 3, 3 MM HEPES, 10 MM 포도당, 2 MM CaCl 2, 1.3 MM MgSO 4) 지속적으로 95% O와 함께 주입 실온에서 2 / 5퍼센트 CO 2. 주입의 경우, 2 / 3 뚜껑에 구멍을 뚫었 구멍 500 ML 넓은 입 Nalgene 병에 입력하십시오. 탱크는 병 뚜껑에있는 구멍을 통해 aCSF을 침투 한 잔 줄기 버블에 튜브가 첨부됩니다. 액체의 1 / 3에 대한 접근하지만 림을 통해 splashing 느낄 수있을만큼 힘없는 거품의 일정한 스트림을 얻기 위해 압력을 조정합니다. 서로와 가스 흐름 난류에서 개별 샘플을 분리 할 수​​있는 Nalgene 병에 잠기 5 ML 유리 병 (최대 6). ACSF는 모든 조직 배양하기 전에 최소한 20 분 동안 산소를해야합니다. </li><li> 작은 실리콘 분해 요리 (35mm × 10작은 육각형 폴리스티렌으로 배치이 분해 핀 (47 밀리미터 아래)와 함께) Sylgard Elastomere 키트를 사용하여 mm 페트리 코팅은 보트를 무게. </li><li> 두 미세 팁 포셉 한 곡선 팁 포셉, 50 ML의 비커, 그리고 조직 폐기물에 대한 컨테이​​너입니다. </li><li> 유리 micropipette (1.2 OD / 0.9 ID) 끝에서 열어 약 20-50 μm로 분류하고있는 6.25 %의 용액에 rhodamine dextran 아민 (RDA, MW = 3,000 엔, Invitrogen)로 가득을 뽑아 0.4 % 트리톤 PBS에서 X-100 . micromanipulator에 picospritzer하고 안정화하기 위해 고급 튜브에 연결합니다. </li></ul> 2. 마이크로 해부 E8 4,5에서 뇌 바닥 유두을 나타 내기 위해 <ol><li> 곡선 포셉으로 배를 무게 내에서 실리콘 코팅 분해 음식에 직접 머리를 배치, 그냥 brainstem 아래 E8 배아를 잘라. </li><li> 관심의 측면에 귀를 약간 볼 수 있도록 분해 핀 사용, 복부 전망을 감상 할 수있는 머리를 배치, 머리가 약간 dorsally 기울여서 10-30도 멀리 CE에서 포인팅nter (그림 2A). 즉시 핀으로 머리를 보호합니다. 양국 추적을 원하는 경우, 센터는 0도에서 머리, 또는 전체 복부 전망은되도록 양쪽에 액세스 할 수 있습니다. 왼쪽에 액세스 할 수 있도록 마찬가지로, 왼쪽 양면 분사를 들어, 센터에서 -10에서 -30도까지 머리를 보호합니다. </li><li> 50 ML의 비커를 사용하여 완전히 조직을 잠수, 그릇에 산소를 aCSF의 30 ML을 전송하기 만하면됩니다. </li><li> 부드럽게 파악하고 미세 팁 집게를 사용하여 아래턱 아래로 껍질을 벗기면 후 관심의 측면에 더 낮은 눈꺼풀을 제거합니다. </li><li> 남은 미각과 식도 등 놓인의 혈관 조직을 제거합니다. 이 부드러운 조직은 벗겨 버릴 표면에 특유의 척추 동맥과 chondrocranium을 개발의 원활한 표면을 공개해야합니다. 이 시점까지, 특성 흰색 otoconial 질량이 표시됩니다. </li><li> 에 떠있는 동안 외부 meatus, 턱 관절의 연골, 그리고 중간 귀 주위 피부를주의 깊게 해부NER 귀 그대로. 부드럽게 이러한 연골 성의 구조를 떼어 포셉을 사용합니다. </li><li> 고급 집게 팁과 부드러운 한눈에 모션을 사용하여 척추 동맥과 풀링 혈액을 제거합니다. 피가보기를 흐릿하게 할 수 있으며 응고은 주입 피펫 열기를 연결 할 수 있습니다. </li><li> 인접 감각 상피 (뇌 바닥 유두) 및 음향 신경과 달팽이 덕트는 chondrocranium 6 바로 아래에 위치해 있으며, 앞쪽 근처의 말초 - 대부분의 팁 (꼭대기)와, ventrally 내이 (内耳)에서 연장 가늘고 긴 손가락과 같은 프로젝션으로 시각화 할 수 있습니다 중간 선 5. 시각화에 도움이 수있는 달팽이 덕트 (그림 2B)의 꼭대기에있는 해부 및 / 또는 otolithic calcifications (흰색)에서 약간의 피가 풀링 (빨간색)가있을 수 있습니다. 꼭대기에서 lagenar 망막 황반은, 감각 패치 vestibular 기능을 7,8 될 생각. </li></ol> 3. CN VIII 청각 섬유의 선택적 라벨링 <ol><li&gt; 달팽이 덕트 및 뇌 바닥 유두 지역에서 배아 표면 근처의 주입 피펫를 놓습니다. </li><li> 천천히 첫번째 펑크 두개골에 micropipette을 낮 춥니 다. 이 두개의 지역은 주변 지역보다 얇은 적은 힘 침투해야합니다. micropipette 이제 달팽이 덕트 내에 있어야합니다. 선택 사항 : 추적 염료의 작은 주사는 뇌 바닥 유두를 시각화에 도움이 여기를 만들 수 있습니다. </li><li> micropipette를 변경하지 않고, 단지 위반 달팽이 덕트의 깊은 경계 (그림 1)까지 낮추고 계속 진행합니다. 여기 주사를 수행하고 vestibular lagena가있는 대부분의 근위 팁을 제외 뇌 바닥 유두 (그림 2C, 2D)의 길이를 따라 여러 지역에서 반복합니다. 10-30 PSI 사이에 자리 잡고 picospritzer으로 여러 주사가 만들어집니다. 주입 염료 유체의 볼륨은 다양하고, 원하는 라벨의 범위에 따라 달라집니다. 각각의 주입은 형광 염료 라가 될약 200 beled 명소 - 400 μm의 직경 (그림 2D). </li><li> 주입이 완료되면, brainstem 위의 샘플을 가로로 쪼개다 지속적으로 95% O 5분의 2 % CO 2와 perfused되는 aCSF의 컨테이너 내에서 작은 병으로 꼬리 부분을 배치 전송 피펫을 사용하여 집게를 사용합니다. </li><li> 각 배아를 들어, 원하는 중앙 추적의 거리에 따라, 염색 전송을위한 20-30 분 수 있습니다. </li><li> 조직을 제거하고 조직 학적 sectioning를 준비합니다. 여기에 표시된 예에서, 조직은 4 % paraformaldehyde (1X PBS에서) 밤 밤, 30 %의 자당 (1X PBS에서)에 몰두하고 있습니다. </li></ol> 4. Counterstaining 및 분석 <ol><li> Counterstaining는 방향뿐만 아니라 관심의 특정 지역을 파악로 해부학 적 위치에 관찰자 도움 유용합니다. 이 준비 효과적인 counterstain 강력히 리터 neurofilament의 immunolabeling (그림 1, 3B, 3E)입니다abels는 axons과 CNS의 axonal 책자의 경계 할 수 있습니다. </li><li> 예측이 rostrocaudal 축을 따라 수백 미크론 이상을 연장 할 수 있으므로 분석이 표시된 지역에 걸쳐 수행해야합니다. 기본적인 분석의 몇 가지 예는 다음과 같습니다 타겟팅 오류 속도, 타이밍 및 / 또는 프로젝션 패턴의 발산의 변화. RDA - 표시 axons은 고해상도 (인물 3A, 3D)와 주변 장치 및 중앙 신경 시스템의 시각화 수 있습니다. </li></ol>

<ol>
	<li>Groves, A. K., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+sound+in+space:+the+regulation+of+inner+ear+patterning.">Shaping sound in space: the regulation of inner ear patterning.</a> Development. 139, 245-257 (2012).</li><li>Pflieger, J. F., Cabana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vestibular+primary+afferents+and+the+vestibulospinal+projections+in+the+developing+and+adult+opossum,+Monodelphis+domestica.">The vestibular primary afferents and the vestibulospinal projections in the developing and adult opossum, Monodelphis domestica.</a> Anatomy and Embryology. 194, 75-88 (1996).</li><li>Molea, D., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+topography+of+nucleus+magnocellularis+innervation+by+the+cochlear+ganglion.">Timing and topography of nucleus magnocellularis innervation by the cochlear ganglion.</a> The Journal of Comparative Neurology. 466, 577-591 (2003).</li><li>Bissonnette, J. P., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+atlas+of+the+gross+anatomy+of+the+developing+inner+ear+of+the+chicken.">Standard atlas of the gross anatomy of the developing inner ear of the chicken.</a> The Journal of Comparative Neurology. 368, 620-630 (1996).</li><li>Brigande, J. V., Kiernan, A. E., Gao, X., Iten, L. E., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+pattern+formation+in+the+inner+ear:+do+compartment+boundaries+play+a+role.">Molecular genetics of pattern formation in the inner ear: do compartment boundaries play a role.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 11700-11706 (1073).</li><li>Bellairs, R., Osmond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The atlas of chick development. , (2005).</li><li>Manley, G. A., Haeseler, C., Brix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+patterns+and+spontaneous+activity+of+afferent+fibres+to+the+lagenar+macula+and+apical+basilar+papilla+of+the+chick's+cochlea.">Innervation patterns and spontaneous activity of afferent fibres to the lagenar macula and apical basilar papilla of the chick's cochlea.</a> Hearing Research. 56, 211-226 (1991).</li><li>Code, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efferent+neurons+to+the+macular+lagena+in+the+embryonic+chick.">Efferent neurons to the macular lagena in the embryonic chick.</a> Hearing Research. 82, 26-30 (1995).</li><li>Maklad, A., Fritzsch, B. <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+vestibular+afferent+projections+into+the+hindbrain+and+their+central+targets.">Development of vestibular afferent projections into the hindbrain and their central targets.</a> Brain Research Bulletin. 60, 497-510 (2003).</li><li>Rubel, E. W., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+system+development:+primary+auditory+neurons+and+their+targets.">Auditory system development: primary auditory neurons and their targets.</a> Annual Review of Neuroscience. 25, 51-101 (2002).</li><li>Satoh, T., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+analysis+of+inner+ear+cells+using+genomic+tags+for+clonal+identification.">Lineage analysis of inner ear cells using genomic tags for clonal identification.</a> Methods Mol. Biol. 493, 47-63 (2009).</li><li>Bok, J., Chang, W., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+and+morphogenesis+of+the+vertebrate+inner+ear.">Patterning and morphogenesis of the vertebrate inner ear.</a> The International Journal of Developmental Biology. 51, 521-533 (2007).</li><li>Appler, J. M., Goodrich, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+the+ear+to+the+brain:+Molecular+mechanisms+of+auditory+circuit+assembly.">Connecting the ear to the brain: Molecular mechanisms of auditory circuit assembly.</a> Progress in Neurobiology. 93, 488-508 (2011).</li><li>Bulankina, A. V., Moser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuit+development+in+the+mammalian+cochlea.">Neural circuit development in the mammalian cochlea.</a> Physiology (Bethesda). 27, 100-112 (2012).</li><li>Fekete, D. M., Campero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+in+the+inner+ear.">Axon guidance in the inner ear.</a> The International Journal of Developmental Biology. 51, 549-556 (2007).</li><li>Momose-Sato, Y., Glover, J. C., Sato, K. <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+functional+synaptic+connections+in+the+auditory+system+visualized+with+optical+recording:+afferent-evoked+activity+is+present+from+early+stages.">Development of functional synaptic connections in the auditory system visualized with optical recording: afferent-evoked activity is present from early stages.</a> Journal of Neurophysiology. 96, 1949-1962 (2006).</li><li>Marrs, G. S., Spirou, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+assembly+of+auditory+circuits:+spiral+ganglion+and+brainstem.">Embryonic assembly of auditory circuits: spiral ganglion and brainstem.</a> The Journal of Physiology. 590, 2391-2408 (2012).</li><li>Milo, 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=Genomic+analysis+of+the+function+of+the+transcription+factor+gata3+during+development+of+the+mammalian+inner+ear.">Genomic analysis of the function of the transcription factor gata3 during development of the mammalian inner ear.</a> PloS One. 4, e7144 (2009).</li><li>Fritzsch, B., Eberl, D. F., Beisel, K. W. <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+bHLH+genes+in+ear+development+and+evolution:+revisiting+a+10-year-old+hypothesis.">The role of bHLH genes in ear development and evolution: revisiting a 10-year-old hypothesis.</a> Cellular and Molecular Life Sciences : CMLS. 67, 3089-3099 (2010).</li><li>Jahan, I., Kersigo, J., Pan, N., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurod1+regulates+survival+and+formation+of+connections+in+mouse+ear+and+brain.">Neurod1 regulates survival and formation of connections in mouse ear and brain.</a> Cell and Tissue Research. 341, 95-110 (2010).</li><li>Huang, E. 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=Brn3a+is+a+transcriptional+regulator+of+soma+size,+target+field+innervation+and+axon+pathfinding+of+inner+ear+sensory+neurons.">Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons.</a> Development. 128, 2421-2432 (2001).</li><li>Jones, J. M., Warchol, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Gata3+transcription+factor+in+the+acoustic+ganglion+of+the+developing+avian+inner+ear.">Expression of the Gata3 transcription factor in the acoustic ganglion of the developing avian inner ear.</a> The Journal of Comparative Neurology. 516, 507-518 (2009).</li><li>Lu, C. C., Appler, J. M., Houseman, E. A., Goodrich, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+profiling+of+spiral+ganglion+neurons+reveals+insights+into+auditory+circuit+assembly.">Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 31, 10903-10918 (2011).</li></ol>

관심의 충돌이 선포되지 않습니다.

VIIIth 신경의 초기 개발 연구로 인해 여러 별개의 신경절에서 발생하는 배아 axons을 식별하는 어려움의 일부에 제한되어 있습니다. 몇몇 연구는 초기 개발 기간 동안 청각 및 vestibular 감각 세포와 신경 세포의 운명을지도 분자 신호, 5,11,12을 탐험했지만 중앙 innervation을 규제 프로세스는 결정하지 못하고있다. 이하는 주요 핵에 투영 중심 프로세스에 대해 알려진 반면, 음향 신경절 세포 계?...

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 폴리스티렌은 요리를 체중 </td><td> 피셔 과학 </td><td> 02-202-101 </td><td></td></tr><tr><td> 페트리 접시 35 X 10mm </td><td> 피셔 과학 </td><td> 50820644 </td><td> 실리콘 해부 요리를 만들기 위해 사용 </td></tr><tr><td> Sylgard 실리콘 엘라스토머 키트 </td><td> 세계 정밀 계측기 </td><td> SYLG184 </td><td> 코트 페트리는 분해 요리를하기 위해 </td></tr><tr><td> 해부 핀 </td><td> 여러 </td><td></td><td> 해부 동안 장소에 배아를 가지고 </td></tr><tr><td> NaCL </td><td> 여러 </td><td></td><td> aCSF 제조법의 일부 </td></tr><tr><td> KCl </td><td> 여러 </td><td></td><td> aCSF 제조법의 일부 </TD&gt; </tr><tr><td> KH 2 PO 4 </td><td> 여러 </td><td></td><td> aCSF 제조법의 일부 </td></tr><tr><td> NaHCO 3 </td><td> 여러 </td><td></td><td> aCSF 제조법의 일부 </td></tr><tr><td> 포도당 </td><td> 여러 </td><td></td><td> aCSF 제조법의 일부 </td></tr><tr><td> CaCl 2 </td><td> 여러 </td><td></td><td> aCSF 제조법의 일부 </td></tr><tr><td> MgSO 4 </td><td> 여러 </td><td></td><td> aCSF 제조법의 일부 </td></tr><tr><td> aCSF에 대한 컨테이​​너입니다. 뚜껑과 반투명 넓은 입 Nalgene 병, 500 ML (16 오즈를) 제안합니다. </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td> 유리 bubling 줄기에 뚜껑의 상단에 드릴 홀 개구는 액체 침투하는 </td></tr><tr><td> 빈 5 ML의 유리관 또는 유사한 투명 유리 병 </td><td> 미국의 제약 파트너, Inc의 </td><td> 6332300105 </td><tD&gt; 서로와 버블 링 스트림에서 샘플을 별도의 유지 aCSF의 배양시 사용 </td></tr><tr><td> carbogen의 탱크 (95% O 5분의 2 % CO 2) 버블에 튜브로 연결 </td><td> 여러 </td><td></td><td> aCSF에 주입을위한 유리의 줄기 버블에 튜브하여 첨부 </td></tr><tr><td> 유리 줄기 버블 </td><td> 여러 </td><td></td><td> aCSF에 carbogen을 불러 일으킬 수있는 </td></tr><tr><td> 곡선 팁 포셉 </td><td> 세계 정밀 계측기 </td><td> 501008 </td><td> 난자에서 배아의 머리를 제거하려면 </td></tr><tr><td> 두 미세 팁 포셉 </td><td> 세계 정밀 계측기 </td><td> 501985 </td><td> 마이크로 해부에 대한 </td></tr><tr><td> 50 ML의 비커 </td><td> 여러 </td><td></td><td></td></tr><tr><td> Rhodamine Dextran 아민 (RDA) </td><td> Invitrogen </td><td> 여러 </td><td> 형광 축삭 트레이서 </td></tr><tr><td> 트리톤 X-100 </td><td> ICN의 Biomedicals </td><td></td><td></td></tr><tr><td> 인산염은 (1X PBS), 식염 버퍼 </td><td> 여러 </td><td></td><td> 표준 실험실 시약 </td></tr><tr><td> 얇은 벽 유리 모세 혈관, 1.2 OD, 0.9 ID 4 &quot;(100 ㎜) 길이 </td><td> 세계 정밀 계측기 </td><td> TW120F-4 </td><td> RDA와 함께로드합니다. 각 모세는 두 유리 micropipettes하게 </td></tr><tr><td> 니들 / 피펫 풀러 </td><td> 데이비드 Kopf 악기 </td><td> 모델 720 </td><td> 사용 설정 : 열 16.4, 솔레노이드 2.2 </td></tr><tr><td> Picospritzer </td><td> 파커 계측 </td><td> 여러 </td><td> 유리 micropipette으로 미세 튜브에 첨부 </td></tr><tr><td> Micromanipulator </td><td> Narishige </td><td> 여러 </td><td></td></tr><tr><td> 형광과 해부 현미경 </td><td> 여러 </td><td></td><td></td&gt; </tr><tr><td> 4 % Paraformaldehyde </td><td> 여러 </td><td></td><td> 표준 실험실 시약 </td></tr><tr><td> 안티 Neurofilament 항체, 선택 사항 </td><td> Millipore </td><td> AB1991 </td><td> 제조업체에서 권장하는 조직 학적 프로토콜에 따라 </td></tr><tr><td> 그라 이오 스탯과 sectioning에 대한 관련 자료 </td><td> Leica </td><td> 여러 </td><td></td></tr><tr><td> 이미지에 대한 Epifluorescent 현미경 </td><td> 다양한 Zeiss, </td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

배아 여자가 주변 및 중앙 신경 세포 투영의 연구 널리 사용되는 모델입니다. 청각 시스템에서 VIIIth 뇌신경에서 청각 axons의 선택적 라벨 중앙의 청각 회로 개발 연구를 강화합니다. 내이 (内耳)의 여러 감각 기관에 VIIIth 신경 하나에 기여하기 때문에이 방법은 도전입니다. 또한, 안정적으로 조류 VIIIth 신경 내 axons의 vestibular 그룹 비해 청각을 구분 마커 식별 할하지 못하고있다. 회로가 형성되기 전에 감각 - evoked 응답이 표시되지로 청각 및 vestibular 경로는 초기 배아에서 기능적으로 구별 할 수는 없습니다. 중앙 돌출 VIIIth 신경 axons는 일부 연구에서 추적 된 있지만, 청각 축삭 라벨은 다른 VIIIth 신경 구성 요소 2,3에서 라벨을 동반되었다. 여기, 우리는 선택적으로 labe로 음향 신경절에서 anterograde 추적하는 방법을 설명개발 VIIIth 신경에서 난 청각 axons. 첫째, 산소 인공 뇌척수에 몰두 8 일 여자는 배의 앞쪽 두부 지역의 부분 절개 한 후, 달팽이 덕트는 해부학 적 랜드 마크 식별 할 수 있습니다. 다음, 고급 당겨 유리 micropipette은 음향 신경절 세포이있는 덕트와 인접 깊은 영역으로 rhodamine dextran 아민의 작은 양을 주입하는 위치입니다. 주사 후 30 분 내에, 청각 axons은 hindbrain에 중앙 추적하고 나중에 histologic 준비에 따라 시각화 할 수 있습니다. 이 방법은 중앙 청각 회로 형성에 주변의 개발 연구를위한 유용한 도구를 제공합니다.

VIIIth 신경과 신경 자체의 해부학 적 구조의 구성 요소는 (그림 1, 3) 복잡하고 복잡합니다. 선택적으로 음향 신경절 세포에서 발생하는 섬유를 추적함으로써, VIIIth 신경뿐만 아니라 brainstem 내의 기본 청각 afferents의 세그먼트는 완전히 추적 할 수 있으며, 자신의 vestibular 대응 (그림 2, 3)에서 구별. 마찬가지로,이 기법은 음향 신경절 세포의 주변 계획 (그림 3G)을</strong...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

여기 우리는 신경과 hindbrain의 청각 축삭 섬유의 추적 선택을위한 초기 여자의 태아의 청각 신경에 형광 염료를 주입 한 다음 microdissection 기술을 설명합니다.

조류 배아 Vestibulocochlear 신경 청각 섬유의 선택적 추적

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

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.1K 조회수.  University of California, Irvine. 여기 우리는 신경과 hindbrain의 청각 축삭 섬유의 추적 선택을위한 초기 여자의 태아의 청각 신경에 형광 염료를 주입 한 다음 microdissection 기술을 설명합니다.

비디오: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

피부와 사지 본즈 Innervating 마우스 감각 신경 섬유의 조직 준비 및 Immunostaining

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

연구

신경과학

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

유 전적으로 정의 된 신경 회로의 추적 바이러스

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

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 ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

배아 마우스의 뇌에 추적 조건부 유전 Transsynaptic

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

레이저 유도 신경 세포의 추적에서 뇌 외식

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

3 차원 영상 및 인간 Intraepidermal 신경 섬유 내 미토 콘 드리 아의 분석

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

CLARITY를 사용하여 마우스 척수에서 세로토닌 섬유 이미징 / CUBIC 기술

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

생체 외에서 쥐에 있는 저 승 구심 성 섬유의 Electrophysiological 속성의 특성

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

마우스에 인할지 모터 신경의 퇴행 성 신경 해부학 추적

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

자유 부동 면역 형광 분석에 의하여 절단 말초 신경 섬유에 있는 알파 Synuclein 응집체를 표적으로 하는

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...