저자는 실험실에서 zebrafish를 확립하기 위해 초기 노력에 피터 카스트로와 매튜 Dinday 감사드립니다. 이 작품은 건강 유리카 기금 (# R01NS079214-01)의 국립 연구소에 의해 자금을 지원했다.

1. 계란 생산 및 수집 <ol><li> Zebrafish 축산는 이전 8 설명한 표준 절차를 따릅니다. 간단히, 성인 zebrafish는 장소에 디바이더와 탱크를 사육에 자리 잡고 있습니다. 방에 불이 다음 아침에 왔을 때, 디바이더는 탱크와 생선이 방해받지 결합 시간의 약 20-60 분 수 있습니다 사육에서 제거됩니다. </li><li> 번식 탱크에서 달걀은 스트레이너에 수집 달걀 물에 헹구어 있습니다. 달걀은 다음 달걀 물과 페트리 접시으로 전송됩니다. Unfertilized 달걀과 잔해가 전송 피펫을 사용하여 제거됩니다. </li><li> 보육에 수집 된 계란 (28-32 ° C)를 포함하는 장소 페트리 접시. 계란 이틀 후 수정을 (dpf) 주변, 부화 후, 전송 피펫과 융모막 및 기타 이물질을 제거합니다. </li><li> 원하는 일에 후 수정을 (3-8 dpf)는 실온에서 실험실 벤치에서 자유롭게 수영 유충과 장소를 포함하는 페트리 접시를 제거합니다. </li></ol><ol><li> micropipette의 풀러를 사용하여 1.2 mm OD 붕규산 유리는 바보 퍼티 램프 위에 누워하여 150mm 페트리 접시 또는 빈 피펫 상자에 두 개의 바늘과 가게에 모세관 당긴다. 바늘 사전에 당겨 수 있습니다. 다른 OD 유리 모세 혈관이 가능한 앰프 headstage의 종류에 따라 사용할 수 있습니다. </li><li> Nalgene 주사기 필터 (4 mm)과 Microfil 필라멘트 (워너 정밀 계측기) 첨부와 1 ML의 주사기를 사용하여 세포 녹화 솔루션 (2 M NaCl)과 Backload microelectrode. 남은 몇 또는 전혀 거품이 될 때까지 바늘 끝 방향으로 bolus를 흔들어하거나 누릅니다. </li></ol> 3. 한천의 고정 <ol><li> 계란 물에 1.2 % 낮은 녹는 점 한천의 새로운 솔루션을 준비합니다. ~ 37 ° C.에서 물 목욕 세트의 장소 한천 솔루션 </li><li> 냉장고에 기록 챔버 및 장소 (5-10 분)와 coverslip을 준비합니다. 녹음 챔버 matc해야합니다H는하는 전기 생리학의 장비에 사용됩니다. 우리는 워너 악기 (모델 RC-26GLP)에서 로우 프로파일 공개 다이아몬드 목욕 이미징 챔버를 사용합니다. </li><li> 파스퇴르 또는 전송 피펫, 깨끗한 페트리 접시 판에 달걀 물을 작은 물방울에 놓으 애벌레의 zebrafish을 사용합니다. 이 비말에, 마취 (0.02 % tricaine)과 전신 마비 에이전트 (1 MG / ML α-bungarotoxin)를 포함하는 솔루션의 비말을 추가합니다. </li><li> 운동의 손실 (5-10 분)에 대한 zebrafish를 모니터링합니다. </li><li> 냉동실에서 coverslip / 녹음 챔버를 제거합니다. stereomicroscope의 무대에 챔버를 놓습니다. 전송 피펫 사용 비말과 coverslip / 녹음 챔버로 전송 솔루션 (물고기)에 1.2 % 낮은 녹는 점 아가로 오스의 여러 ML을 섞는다. </li><li> 피펫 팁 또는 고급 무딘 바늘, 물고기의 등 부분은 아가로 오스 겔 표면에 노출 될 수 있도록 stereomicroscope 아래에 위치 애벌레을 사용합니다. 다음 평면 주걱 전송에게 한천 B를 사용하여 강화 (5-10 분)에 아가로 오스 허용전기 생리학의 유정에서 설정 기록 챔버에 고정. </li></ol> 3. 세포 필드 레코딩 <ol><li> 녹음 챔버에 zebrafish 기록 매체 2-5 ML을 추가합니다. 약물 변경 사항은 약 1 ML / 분의 속도로 레코딩 솔루션을 필요 perfuse 챔버 경우. 녹화 솔루션의 어떠한 산소가 필요하지 않습니다. </li><li> 3 차원 micromanipulator에 장착 증폭기 headstage에 microelectrode를 (약 1 μm 팁 직경 MΩ 2-7)를 넣습니다. micromanipulator은 움직임과 조정의 범위를 허용 할 수있는 적절한 위치에 있는지 확인합니다. 무대에서 내려 높은 현미경의 전망의 비행기, 그리고 위하고 약간 zebrafish의 머리 앞의 지점으로 낮은 정밀 조정을 사용하여에 microelectrode 팁을 가져와. </li><li> &quot;전류 클램프&quot;모드 제로 전극의 앰프를 갖추고 있습니다. </li><li> 그냥 s를 만지고 있도록 정밀 조정 할 낮은 전극 팁을 사용하여약간 forebrain 앞의 zebrafish의 urface. </li><li> 이 zebrafish의 피부를 구멍까지 미세 조정 단계를 사용하면 전극 팁을 부활 시켰습니다. forebrain에 몇 미크론를 해결 한 후 전극을 향상 할 수 있도록 허용합니다. </li><li> Axoscope 소프트웨어를 사용하여 전류 클램프 모드에서 전기 활동을 기록합니다. 데이터는 10 kHz에서의 샘플링 속도에서 획득됩니다. </li></ol>

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	<li>Clark, K. 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=Stressing+zebrafish+for+behavioral+genetics.">Stressing zebrafish for behavioral genetics.</a> Reviews in Neuroscience. 22 (1), 49 (2011).</li><li>Rinkwitz, S., 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=Zebrafish:+an+integrative+system+for+neurogenomics+and+neurosciences.">Zebrafish: an integrative system for neurogenomics and neurosciences.</a> Progress in Neurobiology. 93 (2), 231 (2011).</li><li>Penberthy, W. 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=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Frontiers in Bioscience. 7, d1439 (2002).</li><li>Letamendia, 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=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS One. 7, e36690 (2012).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nature Genetics. 26 (2), 216 (2000).</li><li>Haffter, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral+line.">Mutations affecting development of the zebrafish inner ear and lateral line.</a> Development. 123, 1 (1996).</li><li>Suster, M. L., 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=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Molecular Biology. 561, 41 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Baraban, S. C., 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=Pentylenetetrazole+induced+changes+in+zebrafish+behavior,+neural+activity+and+c-fos+expression.">Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.</a> Neuroscience. 131 (3), 759 (2005).</li><li>Baraban, S. C., 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=A+large-scale+mutagenesis+screen+to+identify+seizure-resistant+zebrafish.">A large-scale mutagenesis screen to identify seizure-resistant zebrafish.</a> Epilepsia. 48 (6), 1151 (2007).</li><li>Williams, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+radiotelemetry+to+evaluate+electrographic+seizures+in+rats+with+kainate-induced+epilepsy.">The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy.</a> Journal of Neuroscience Methods. 155 (1), 39 (2006).</li><li>Marsh, E. D., 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=Interictal+EEG+spikes+identify+the+region+of+electrographic+seizure+onset+in+some,+but+not+all,+pediatric+epilepsy+patients.">Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.</a> Epilepsia. 51 (4), 592 (2010).</li><li>Zhu, C., 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=Evaluation+and+application+of+modularly+assembled+zinc-finger+nucleases+in+zebrafish.">Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.</a> Development. 138 (20), 4555 (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

여기에 제시된 세포 녹화 방법은 뇌 활동의 매우 민감하고 빠른 분석이 가능합니다. 이 음반은 일반적으로 간질 환자 11 12 쥐 모델에서 비정상적인 전기 방전 (즉, 발작)의 존재를 평가하는 데 사용 electroencephalographic (EEG) 모니터링 유사합니다. 여기에 표시된대로 세포 녹음, 약리 조작과 함께 사용할 수 있습니다. 레코딩 이러한 유형의도 유전자 변형 zebrafish 잠재적 인 간질 phenotypes?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 아가로 오스 낮은 녹는 </td><td> 피셔 - 과학 </td><td> BP1360-100 </td><td> 1.2 %에서 배아 미디어에서 디졸브 </td></tr><tr><td> 기록 미디어 </td><td> 피셔 - 과학 </td><td> BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 </td><td> 1 ㎜ NaCl, 2.9 MM KCl, 10 MM의 HEPES, 1.2 MM MgCl 2, 10 MM 덱스 트로 오스, 2.1 MM CaCl 2 1 N NaOH로 약 7.3로 산도를 </td></tr><tr><td> Tricaine </td><td> 은빛 연구소 </td><td> MS-222 </td><td> 0.02 % </td></tr><tr><td> α-bungarotoxin </td><td> Bioscience Tocris </td><td> 2133 </td><td> 1 MG / ML </td></tr><tr><td> 모세관 유리 튜브 </td><td> 워너 악기 </td><td> G120TF -3 </td><td> MΩ이 -7의 저항에 당겨 </td></tr><tr><td> 패치 클램프 증폭기 </td><td> 워너 악기 </td><td> PC-505B </td><td> 우리는 전류 클램프 모드에서 워너 증폭기를 사용하여, 이득은 2K로 설정 2 MV / PA 및 베셀 필터에서 설정합니다. 비교 모델은 제조업체의 지침에 따라 사용할 수 있습니다. </td></tr><tr><td> 필터 / 앰프 </td><td> 백조 기술 </td><td> FLA-01 </td><td> 10-20에서 설정 게인; 1 2K로 설정 컷 - 오프 주파수, 노치 필터에 우리는 백조 사전 증폭기를 사용합니다. 비교 모델은 제조업체의 지침에 따라 사용할 수 있습니다. </td></tr><tr><td> 축삭 A / D 보드와 Axoscope 소프트웨어 </td><td> 분자 장치 </td><td> 축삭 Digidata 1320A, Axoscope 8.2 </td><td> 데이터는 간격이없는 수집 모드를 사용 Axoscope에 수집, 10 kHz에서에서 샘플링. 비교 모델 및 프로그램은 제조업체의 지침에 따라 사용할 수 있습니다. </td></tr><tr><td> 달걀 물을 </td><td&gt; 인스턴트 오션 </td><td></td><td> 3g 인스턴트 오션 바다 소금, 10 ML 탈 이온수 2 ML 0.1 % 메틸렌 블루 </td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

간질은 미국에서 3 백만 명에 이르는 사람들과 최대 50,000,000명 전 세계적으로 영향을 미칩니다. 자연 부당한 발작의 발생으로 정의, 간질은 뇌 또는 유전자 변이에 대한 모욕의 결과로 획득 할 수 있습니다. 동물 모델 압류에 대한 노력은 주로 활용 설치류의 모욕 (convul​​sant 약물, 자극 또는 뇌 손상)과 유전자 조작을 (안티센스 최저, 상동 재조합 또는 transgenesis) 획득했습니다. Zebrafish는 쥐 기반 간질 연구에 귀중한 대안을 제공 할 수 척추 모델 시스템 1~3아르. Zebrafish는 척추 동물의 유전자 또는 개발 연구에서 널리 사용되며, 포유 동물에 대한 유전 적 유사성의 높은 학위를 전시 및 알려진 인간 단일 유전자 간질 변이 ~ 85 %를 homologs을 표현한다. 때문에 자신의 작은 크기 (길이 4~6mm)로, zebrafish의 유충은 초기 개발 및 arra 동안 100 μl의 낮은 유체 볼륨에 유지 할 수 있습니다멀티 잘 접시에 yed. 시약은 배아는 약물 관리를 단순화하고 테스트 화합물 4 생체 검사에서 빠른 있도록 개발 된 솔루션에 직접 추가 할 수 있습니다. 합성 oligonucleotides (morpholinos), mutagenesis, 아연 손가락 nuclease와 유전자 변형 접근 방식은 빠른 속도로 5-7 zebrafish의 유전자 최저 또는 돌연변이를 생성하는 데 사용할 수 있습니다. 이 속성은 zebrafish 연구 한테 그런 간질과 같은 신경 질환의 연구에서 동물에 비해 전례없는 통계 전력 분석 이점을 여유. 간질 연구를위한 &quot;금 표준&quot;중앙 뇌 구조 (즉, 발작), 효율적으로 애벌레의 zebrafish의 뇌 활동을 기록하는 방법에 발생한 이상 전기 방전을 감시하고 분석하는 것입니다 때문에 여기에 설명되어 있습니다. 이 방법은 기존의 세포 녹화 기술의 적응이며, 그대로 zebrafish 애벌레의 뇌 활동의 안정적인 장기 모니터링 할 수 있습니다. 에스충분한 녹음은 유전자 변형 물고기에 기록 convul​​sant 마약과 자연 발작의 목욕 응용 프로그램에 의해 유도 급성 발작에 표시됩니다.

한천 - 임베디드 zebrafish 애벌레의 forebrain에 기록 electrographic 발작과 같은 방전의 예는 그림 1에 표시됩니다. 또는 1 밀리미터 picrotoxin (B에, 8 dpf),이 샘플에서 큰 진폭 멀티 스파이크 버스트 방전은 convul​​sant 약의 목욕 응용 프로그램을, 40 밀리미터 pilocarpine (6 dpf의)에 의해 evoked했다. 이 음반에 고정하고 한천 - 임베디드 zebrafish는 지속적으로 90 분에에 모니터링하고 있습니다. 물고?...

Watch this Scientific Journal Video about Forebrain Electrophysiological Recording in Larval Zebrafish at JoVE.com

Forebrain Electrophysiological Recording in Larval Zebrafish

애벌레의 zebrafish forebrain에서 세포 필드 잠재력을 기록하는 간단한 방법이 설명되어 있습니다. 방법은 강력한를 제공합니다<em&gt; 생체 내</em&gt; 읽기에서의 활동 압수 같은. 이 기술은 간질 관련 유전자 또는 convul​​sant 약의 투여에 의해 evoked 발작을 들고 유전자 변형 zebrafish의 애벌레와 함께 사용할 수 있습니다.

애벌레의 Zebrafish의 Forebrain Electrophysiological 기록

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

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JoVE Journal

Neuroscience

17.0K 조회수.  University of California, San Francisco. 애벌레의 zebrafish forebrain에서 세포 필드 잠재력을 기록하는 간단한 방법이 설명되어 있습니다. 방법은 강력한를 제공합니다 생체 내 읽기에서의 활동 압수 같은. 이 기술은 간질 관련 유전자 또는 convul​​sant 약의 투여에 의해 evoked 발작을 들고 유전자 변형 zebrafish의 애벌레와 함께 사용할 수 있습니다.

비디오: Forebrain Electrophysiological Recording in Larval Zebrafish

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

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신경과학

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

온전한 성인 Zebrafish의의 뇌에 전기 생리학 기록

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

에서 전기 생리학 기록<em&gt; 초파리</em&gt; Labellar는 Sensilla 맛

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

애벌레 Zebrafish의 척수 절개

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

동시 전기 생리 녹화 및 설치류의 뇌에서 억제 에이전트의 마이크로 주사

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

에서 전기 생리 학적 기록<em&gt; Drosophila</em&gt; 낮은 휘발성의 냄새에 반응하는 Trichoid Sensilla

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

애벌레 Zebrafish의 측면 라인 세포의 생체 내 칼슘 이미징

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

전기 생리학 필드 기록에 의하여 인터라멜라 해마 CA1에 있는 장기 시냅스 가소성 조사

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

자유롭게 수영 물고기에서 이동 식 Tetrodes에 의해 뉴런의 무선 전기 생리적 기록

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...