이 작품은 LC 그리피스 건강 보조금 RO1GM - 33205와 MH - 067284 국립 연구소에 의해 그리고 NJ Hornstein에 브랜다 이스 대학 섬머 스쿨 학부 연구 장학 지원했다. 우즈 홀, MA에서이 기법에 대한 예비 실험은 2008 년 신경 시스템 및 행동 여름 코스 (R25 MH059472 NIMH 부여)의 일환으로 해양 생물학 연구소에서 수행되었다.

1 부 : 동물 관리 및 유전 십자가 <ol><li> UAS - ChR2 및 표준 비행 미디어 8을 포함하는 별도의 병의 OK371 - GAL4 플라이 라인 유지합니다. </li><li> OK371 - GAL4 플라이 라인과 UAS - ChR2 라인의 남성의 처녀를 수집합니다. </li><li> 1 ㎜ 모든 트랜스 망막 (ATR)과 혼합 표준 플라이 미디어가 들어있는 유리병에 남성과 여성 모두를 넣어. ATR 음식은 ~ 1 분간 전자렌지에 처음으로 용해 정기 비행 미디어로하여야한다. 일단 녹아, 1 분 30 초 동안 냉각 후 비행 미디어의 매 10 ML 100 %의 ETOH에서 ATR 100 MM 100 μl를 추가할 수 있습니다. 얼음 플레이스 튜브, 그때 4 어두운 영역에 저장 ° C.를 </li><li> 파리의 친구를하자 22-25에서 어두운 지역에서 계란 ° C.를하다 </li><li> 셋째 instar의 유충을 볼 때까지 3~4일 기다려, 그 시점에서 파리 성충 처분. </li></ol> 2 부 : 릭 설정 <ol><li> 블루 LED 어떤 10X 해부 범위 눈 조각 (우리의 경우, 칼 자이스 혈구 주식 회사, www.zeiss.com, 손우드, NY에서), 히트 싱크와 광원 (토르 연구소, www.thorlabs.com, 뉴턴, 뉴저지를 연결합니다 ). 게시하고 클램프와 자기 기지에 연결합니다. 광원에 의해 생성된 전기 노이즈를 줄이기 위해 전기 접지 방패로 히트 싱크를 커버. </li><li> 전기 생리학 장비의 공기 테이블에서 광원과 장소 자석 기지. </li><li> 회로를 제어 및 외부 전압 소스 (Powerlab 30분의 4, ADInstruments, www.adinstruments.com, 콜로라도 스프링스, CO)에 제어 회로를 연결하는 광원을 연결합니다. </li><li> 푸른 불빛을 활성화하기 위해 회로를 제어하기 1-5 V의 펄스를 제공합니다. </li><li> 마그네틱베이스와 광원 블루 라이트 빔은 애벌레 절개로 점유하는 영역에 초점을 맞춘까지를 조정합니다. </li></ol> 파트 3 : 해부 <ol><li> sylgard - 줄지어 접시의 바닥에 6 ~ 0.1 mm 곤충 핀. </li><li> 페트리 - 요리에 플라스틱으로 식품 미디어 및 장소에서 3 차 instar의 애벌레를 제거합니다. </li><li> 음식을 제거하는 호수와 애벌레를 씻어. </li><li> 플레이스 뽑아 핀 근처에 요리를 해부에 애벌레와 ½ 수준으로 살린 추가할 수 있습니다. </li><li> 당신은 동물의 등의 표면을 따라 실행이 은빛 튜브 (기관)을 볼 수 있도록 동양 애벌레. </li><li> tracheal 튜브 사이에있는 꼬리에 직접 큰 핀을 삽입합니다. 애벌레를 누른 상태의 머리에 두 번째 큰 핀을 장소, 길이 시체를 밖으로 스트레칭해야되고. </li><li> 꼬리 근처에 작은 절개를 확인하고, 신체의 길이를 그것을 계속합니다. 우연히 복부 신경 및 / 또는 신체 벽의 근육을 건드리지 않게으로 가위의 끝을 그렇게 사육되었는지 확인합니다. </li><li> 동물의 네 모퉁이에 플레이스 네 핀 이제 오픈 midsection의. 필릿 동물을 줄 수있는만큼 해부 접시에 그들을 설정합니다. 핀 준비 밖으로 가르쳤다. </li><li> 포셉 및 가위를 사용하여 동물의 내장, 기관 및 지방 기관을 제거하십시오. </li><li> 식염수에 준비를 씻어. </li><li> 이마 엽 (叶) (같은 그림에서 본)와 준비의 복부 신경절을 찾습니다. </li><li> 마이크로 가위를 사용하여, 신중하게 그냥 이마 엽 (叶) 뒤에있는 복부 신경절을 통해 했네요. </li></ol> 부 4 : 근육 녹음과 청색 광 자극 <ol><li> 전자 전극 풀러 (셔터 인 스트 루먼트)를 사용하여 10-20 μω 전극을 당기세요. </li><li> 3 M KCl로 뽑아 전극을 입력합니다. </li><li> 이곳은 전기 생리학 장비에 대한 해부 현미경으로 애벌레 준비 근처 micromanipulator와 전극 홀더 및 기동 전극 팁의 전극을 가득 채웠다. </li><li> 모든 신체 벽 세그먼트 (그림 A)의 근육 6 (M6)를 식별합니다. </li><li> 조심스럽게 M6에 전극을 삽입하고 신속한 hyperpolarization 살펴보십시오. 근육 쉬고 막 잠재력은 -30에서 -70 뮤직 비디오 MV 곳이되어야합니다. </li><li> 해부 준비가 광선에 중심 있도록 푸른 불빛을 조정합니다. </li><li> 회로를 제어하는​​ MS 20-100 전압 펄스를 제공합니다. 점진적으로 회로를 제어하는​​ 공급 전압을 증가시킵니다. 근육 세포 (그림 1B)에 흥분성의 접합을위한 잠재력보세요. </li></ol> 대표 결과 : 그림 1A는 녹화 설정 및 토막 준비의 구조를 보여줍니다. 그림 1B 짧은 광 펄스에 의해 evoked 전형 EJP를 보여줍니다. 그림 EJP 진폭은 신경을 분포시키다 M6 모두 두명의 모터 뉴런의 진폭을 표현입니다. 낮은 조명의 농도 하나의 모터 유닛 (데이터가 표시되지 않음)만을 활성화. <img src="/files/ftp_upload/1133/figure1.png" alt="그림 1" width="600" height="373" /> 그림 1 : 세포 레코딩 장비 및 청색 LED와) 일반 설계도. 뇌 (브라질)은 복부 신경절 (VG)의 리듬 활동을 억제하기 위해 제거됩니다. ChR2은 GAL4 - UAS 시스템을 사용 모터 뉴런에 표시됩니다. M6 근육에서 B) 세포 녹음. 40 MS 블루 라이트 펄스 (127에서 μW / mm 2) 안정 M6 큰 잠재력 시냅스 (별표)를 보여주고 있습니다.

<ol>
	<li>Keshishian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545 (1996).</li><li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+development:+insights+from+Drosophila.">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35 (2007).</li><li>Lagow, R. 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=Modification+of+a+hydrophobic+layer+by+a+point+mutation+in+syntaxin+1A+regulates+the+rate+of+synaptic+vesicle+fusion.">Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.</a> PLoS Biol. 5 (4), e72 (2007).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279 (2005).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940 (2003).</li><li>Schroll, 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=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr Biol. 16 (17), 1741 (2006).</li><li>Lin, D. M., Auld, V. J., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+neuronal+cell+ablation+in+the+Drosophila+embryo:+pathfinding+by+follower+growth+cones+in+the+absence+of+pioneers.">Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.</a> Neuron. 14 (4), 707 (1995).</li><li>Ralph Greenspan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The Theory and Practice of Drosophila Genetics, 2nd ed. , (2004).</li></ol>

우리는 공개의 관심 충돌이 없습니다.

중요 단계는 초기 절개와 근육 세포의 입력을 모두 포함하고 있습니다. 초기 절개하는 동안 신경이 절단되거나 근육이 손상된 경우 그것은 실험의 나머지 부분을 계속하기가 어렵습니다. 해부하는 동안, 하나 등 지느러미 절개하는 동안 이상 최대한 가위를 해부 각도 매우 신중해야합니다. 두 번째 중요한 단계 동안, 근육 세포를 입력할 하나는 과거 hyperpolarization ~ 30 뮤직 비디오에 대한 감시해?...

<table><tbody><tr><td>Sylgard</td><td>Ellsworth Adhesives</td><td>4019862</td><td>&lt;a href=&quot;http://www.ellsworth.com&quot; target=&quot;_blank&quot;&gt;www.ellsworth.com&lt;/a&gt;</td></tr><tr><td>Minutens Pins</td><td>Fine Science Tools</td><td>26002-10</td><td>&lt;a href=&quot;http://www.finescience.com&quot; target=&quot;_blank&quot;&gt;www.finescience.com&lt;/a&gt;</td></tr><tr><td>Dissecting Dish</td><td>Fisher Scientific</td><td></td><td>&lt;a href=&quot;http://www.fishersci.com&quot; target=&quot;_blank&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>Neuroprobe Intracellular Amplifier and Head Stage</td><td>A-M Systems</td><td>680100</td><td>&lt;a href=&quot;http://www.a-msystems.com&quot; target=&quot;_blank&quot;&gt;www.a-msystems.com&lt;/a&gt;</td></tr><tr><td>&lt;a href=&quot;http://www.adinstruments.com/products/hardware/corporate/product/ML866/&quot; target=&quot;_blank&quot;&gt;Powerlab 4/30 data acquisition system&lt;/a&gt;</td><td><a href="http://www.adinstruments.com" target="_blank"><img src="/img/new_window.png" /> ADInstruments</a></td><td></td><td>&lt;a href=&quot;http://www.adinstruments.com&quot; target=&quot;_blank&quot;&gt;www.adinstruments.com&lt;/a&gt;</td></tr><tr><td>Grass stimulator</td><td>Grass Technologies</td><td></td><td>&lt;a href=&quot;http://www.grasstechnologies.com&quot; target=&quot;_blank&quot;&gt;www.grasstechnologies.com&lt;/a&gt;</td></tr><tr><td>Desktop Computer</td><td>Dell</td><td></td><td>&lt;a href=&quot;http://www.dell.com&quot; target=&quot;_blank&quot;&gt;www.dell.com&lt;/a&gt;</td></tr><tr><td>Dissecting Scope</td><td>Leica Microsystems</td><td></td><td>&lt;a href=&quot;http://www.leica-microsystems.com&quot; target=&quot;_blank&quot;&gt;www.leica-microsystems.com&lt;/a&gt;</td></tr><tr><td>Light Source</td><td>Dolan-Jenner Industries</td><td>41446-062</td><td>&lt;a href=&quot;http://www.dolan-jenner.com&quot; target=&quot;_blank&quot;&gt;www.dolan-jenner.com&lt;/a&gt;</td></tr><tr><td>Fly Media</td><td></td><td></td><td></td></tr><tr><td>All-Trans-Retinal</td><td>Sigma-Aldrich</td><td>116-31-4</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com&quot; target=&quot;_blank&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>&lt;em&gt;OK-371 Gal4&lt;/em&gt; Flies</td><td>Bloomington Stock center</td><td></td><td></td></tr><tr><td>&lt;em&gt;UAS-ChR2&lt;/em&gt; Flies</td><td>Fiala lab, Griffith lab</td><td></td><td></td></tr><tr><td>LED controller circuit</td><td>Built in Griffith lab</td><td></td><td>&lt;a href=&quot;http://www.ledsupply.com&quot; target=&quot;_blank&quot;&gt;http://www.ledsupply.com&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.futureelectronics.com&quot; target=&quot;_blank&quot;&gt;http://www.futureelectronics.com&lt;/a&gt;&lt;br/&gt;Composed of: &lt;br/&gt;1. 200 mA Buck Puck&lt;br/&gt;2. Blue LED&lt;br/&gt;3. Insulated wire&lt;br/&gt;4. Circuit bread board&lt;br/&gt;</td></tr><tr><td>LED Heat Sink</td><td>Thorlabs Inc.</td><td></td><td>&lt;a href=&quot;http://www.thorlabs.com/&quot; target=&quot;_blank&quot;&gt;http://www.thorlabs.com/&lt;/a&gt;</td></tr><tr><td>Air Table</td><td>TMC</td><td></td><td>&lt;a href=&quot;http://www.techmfg.com/products/accessories/intro3.html&quot; target=&quot;_blank&quot;&gt;http://www.techmfg.com/products/accessories/intro3.html&lt;/a&gt;</td></tr><tr><td>Faraday Cage</td><td>Built in Griffith lab</td><td></td><td></td></tr><tr><td>Leica Leitz M Micro-Manipulator</td><td>Leica Microsystems</td><td>ACS01</td><td>&lt;a href=&quot;http://www.leica-microsystems.com&quot; target=&quot;_blank&quot;&gt;www.leica-microsystems.com&lt;/a&gt;</td></tr><tr><td>Electrode Holder</td><td>Axon Instruments</td><td></td><td>&lt;a href=&quot;http://www.axon.com&quot; target=&quot;_blank&quot;&gt;www.axon.com&lt;/a&gt;</td></tr><tr><td>Borosilicate Glass</td><td>FHC, Inc.</td><td></td><td>&lt;a href=&quot;http://www.fh-co.com/p14-15.pdf&quot; target=&quot;_blank&quot;&gt;www.fh-co.com/p14-15.pdf&lt;/a&gt;</td></tr><tr><td>Electrode Puller</td><td>Sutter Instrument Co.</td><td></td><td>&lt;a href=&quot;http://www.sutter.com&quot; target=&quot;_blank&quot;&gt;www.sutter.com&lt;/a&gt;</td></tr><tr><td>HL 3.1 Saline with 0.8mM Ca2+</td><td></td><td></td><td>Contents (mM):&lt;br/&gt; NaCl:70 KCl:5 CaCl&lt;sub&gt;2&lt;/sub&gt;: 0.8&lt;br/&gt; MgCl&lt;sub&gt;2&lt;/sub&gt;:4Sucrose:115&lt;br/&gt;NaHCO&lt;sub&gt;3&lt;/sub&gt;: 10 Trehalose: 5 HEPES</td></tr><tr><td>Micro-Dissection Tools</td><td>Fine Science Tools</td><td></td><td>&lt;a href=&quot;http://www.finescience.com&quot; target=&quot;_blank&quot;&gt;www.finescience.com&lt;/a&gt;</td></tr></tbody></table>

channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions

<em&gt; Drosophila</em&gt; 애벌레 신경근육학 준비는 시냅스 생리학을 공부에 유용한 도구로 입증되었습니다<sup&gt; 1,2,3</sup&gt;. 현재, 유일하게이 준비 흥분성의 junctional 잠재력 (EJPs)를 보여주고에 사용할 의미 흡입 전극의 사용을 포함한다. 연구 및 교육 실험실 모두에서, 학생들은 종종 어려움 자극 전극의 유형을 작전하고 조작 있습니다. 현재 작업에서 우리는 원격 흡입 전극을 사용하지 않고 애벌레 NMJ에 신경 잠재력을 자극하는 방법을 보여줍니다. channelrhodopsin2 (ChR2)을 표현함으로써<sup&gt; 4,5,6</sup&gt;에<em&gt; Drosophila</em를 사용하여&gt; 모터 뉴런<em&gt; GAL4 - UAS</em&gt; 시스템<sup&gt; 7</sup&gt;, 그리고 기본적인 전기 생리학의 장비에 약간 변화를주고, 우리는 푸른 빛의 펄스와 EJPs을 안정적으로 보여주고 수 있었다. 이 기술은 학생 릭 연습 시간과 자원이 제한됩니다 neurophysiology 교육 연구소에서 특정 사용이 될 수 있습니다.

Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

이 절차에 신경근육학 교차로 (NMJ)에서 광 펄스로 신경 잠재력을 보여주고에 푸른 빛을 활성화 algal 채널 및 셀 - 특정 유전자 표현 도구를 사용하여<em&gt; Drosophila</em&gt; 애벌레. 이 기술은 저렴하고 연구 및 교육 연구소에서 신경 생리학 연구를위한 흡입 전극 자극을 대체 사용하기 쉬운 것입니다.

에서 시냅틱 후보의 Channelrhodopsin2 중재 ​​자극 Drosophila 신경근육학 분기점

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

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Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

13.6K 조회수.  Brandeis. 이 절차에 신경근육학 교차로 (NMJ)에서 광 펄스로 신경 잠재력을 보여주고에 푸른 빛을 활성화 algal 채널 및 셀 - 특정 유전자 표현 도구를 사용하여 Drosophila 애벌레. 이 기술은 저렴하고 연구 및 교육 연구소에서 신경 생리학 연구를위한 흡입 전극 자극을 대체 사용하기 쉬운 것입니다.

비디오: Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila 애벌레 NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

연구

생물학

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

세미 그대로의 레이저 조명과 신경 활동의 Optogenetic 섭동<em&gt; 초파리</em모션&gt; 유충

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

신경과학

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

의 기록 세포 내 전압 응답에 대한 전기 생리 방법<em&gt; 초파리</em&gt; 빛 자극에 광수와의 interneurons<em&gt; 생체</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법<em&gt; Drosophila</em&gt; 광 수용체

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac.

Drosophila 테트로도톡신 저항 나트륨 채널을 사용 하 여 Monosynaptic 연결 검사

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

시 냅 스에서 사이클링 하는 FM 염료: 높은 칼륨 도발은, 전기 및 Channelrhodopsin 자극을 비교

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

청색 및 적색 이동 채널로도프신을 가진 열등한 콜리큘러스 뉴런의 장거리 채널로돕신 보조 회로 매핑

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, ...

Synaptic Modulation in Drosophila After Exposure to Prolonged Light

Source: Sugie, A., et al. <a href="https://app.jove.com/v/55176">Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light.</a> J. Vis. Exp. (120)
This video demonstrates the rearing of Drosophila melanogaster under specific light conditions to study the role of Bruchpilot proteins in neurotransmitter release and the resulting modulation of synaptic activity.

Synaptic Modulation in Drosophila After Exposure to Longlonged Light (장시간 빛에 노출된 후 초파리의 시냅스 조절)

1. Light Exposure Conditions

	From the larva stage until eclosion, keep the flies in a 12 h light/12 h dark cycle (LD) at 25 &#176;C (Figure 1A).
	...

에서 시냅틱 후보의 Channelrhodopsin2 중재 자극 Drosophila 신경근육학 분기점

요약

더 많은 비디오 탐색

이 비디오의 챕터

Drosophila 애벌레 NMJ Immunohistochemistry

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

세미 그대로의 레이저 조명과 신경 활동의 Optogenetic 섭동

의 기록 세포 내 전압 응답에 대한 전기 생리 방법

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila 테트로도톡신 저항 나트륨 채널을 사용 하 여 Monosynaptic 연결 검사

시 냅 스에서 사이클링 하는 FM 염료: 높은 칼륨 도발은, 전기 및 Channelrhodopsin 자극을 비교

청색 및 적색 이동 채널로도프신을 가진 열등한 콜리큘러스 뉴런의 장거리 채널로돕신 보조 회로 매핑

Synaptic Modulation in Drosophila After Exposure to Longlonged Light (장시간 빛에 노출된 후 초파리의 시냅스 조절)

에서 시냅틱 후보의 Channelrhodopsin2 중재 ​​자극 Drosophila 신경근육학 분기점

요약

더 많은 비디오 탐색

이 비디오의 챕터

Drosophila 애벌레 NMJ Immunohistochemistry

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

세미 그대로의 레이저 조명과 신경 활동의 Optogenetic 섭동

의 기록 세포 내 전압 응답에 대한 전기 생리 방법

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila 테트로도톡신 저항 나트륨 채널을 사용 하 여 Monosynaptic 연결 검사

시 냅 스에서 사이클링 하는 FM 염료: 높은 칼륨 도발은, 전기 및 Channelrhodopsin 자극을 비교

청색 및 적색 이동 채널로도프신을 가진 열등한 콜리큘러스 뉴런의 장거리 채널로돕신 보조 회로 매핑

Synaptic Modulation in Drosophila After Exposure to Longlonged Light (장시간 빛에 노출된 후 초파리의 시냅스 조절)

에서 시냅틱 후보의 Channelrhodopsin2 중재 자극 Drosophila 신경근육학 분기점