시작하기 전에 준비 : <ul><li> 완더링 yhird instar Drosophila의 애벌레 </li><li> HL3.1 (hemolymph 같은 수정된) 솔루션 </li><li> Sylgard (투명 실리콘 고무) 해부 접시는 브렌트와 맥케 이브 (2008) 3 설명한 방법을 사용하여 소형 (35 × 10 ㎜) 플라스틱 배양 접시에 준비했습니다. </li><li> 해부 핀 짧은 컷 </li><li> 전극 pipettes을 자극 </li><li> 샤프 기록 pipettes </li></ul> HL3 솔루션 : <ol><li> 70 NaCl, 5 KCl, 4 MgCl 2, 10 NaHCO 3, 5 trehalose, 115 자당, 5 HEPES, pH를 7.2 : 해부 및 electrophysiological 실험 동안, 유충은 (MM)를 포함하고 HL3.1 솔루션 2 열중하고 있습니다. </li><li> CaCl 2의 농도는 세포 칼슘 2 +의 필요한 농도를 제공하는 HL3.1 솔루션에 추가됩니다. <ul><li> 애벌레 해부는 HL3.1를 사용하여 + 농도 (근육 수축을 피하기 위해) + 0.25 MM CaCl 2, 얼음에 보관 낮은 칼슘 2 수행됩니다. </li><li> Electrophysiological 실험은 일반적으로 HL3.1에 기록됩니다 + 1 ㎜ CaCl 2, 그러나 이것은 0.4에서 조정할 수 있습니다 - 1 MM 칼슘 2 +로 필요합니다. </li></ul></li><li> HL3.1 솔루션은 필터를 사용하기 전에 소독, 4에 저장된 ˚ C. </li></ol> pipettes을 자극하고 기록 준비 : <ol><li> 열 = 390, 필라멘트 = 4, 속도 = 35 = 200 지연과 = 0 당겨 :. 녹화 및 자극 pipettes은 다음과 같은 설정을 사용하여 셔터 P - 2000 레이저 기반 micropipette의 풀러와 함께 행진 </li><li> 자극 전극은 절단 모터 axons의 넓이보다 약간 크게되었습니다 넓은 끝을 가진 얇은 벽으로 둘러싸인 borosilicate 유리 모세관의 준비가되어 있습니다. 모양 엔드는 신경 손상을 최소화하기 위해 부드러운 마무리 (Narashiga 피펫 폴리셔 사용), 줄 firepolished 있습니다. 자극 전극 목욕 솔루션 (HL3.1 + 1 ㎜ 칼슘 2 +)로 채워진다. </li><li> 기록 전극은 날카로운 피펫 (MΩ 30-60)를 형성 냈고, 3M KCl으로 가득 1.2mm borosilicate 유리에서 준비가되어 있습니다. </li></ol> 1 부 : Drosophila의 애벌레의 해부 <ol><li> 제 instar 방황 애벌레는 이전에 다음 ID로 4 설명된대로 몸을 벽의 근육을 폭로 해부하고 있습니다. </li><li> 해부는 HL3.1에서 수행됩니다 + 0.25 MM 칼슘 2 + 작은 실리콘 접시에 (35 X 10mm). HL3.1 솔루션은 유충을 마취시키다하기 위해 해부를위한 얼음처럼 차가운해야하며, 해부 전과 도중 얼음에 보관됩니다. </li><li> 애벌레는 HL3.1 해부 솔루션 + 0.25 MM에게 칼슘 2 추가 및 적고 짧은 절개 핀을 (~ 2mm 길이)를 사용하여 전기 생리학 실험에 대한 수정 브렌트와 맥케 이브 (2008)에 의해 설명된 방법을 사용 해부 아르 실험 기간 동안 현미경의 객관적이고 전극에 충돌 가능성. </li><li> 해부 다음, 애벌레의 모터 axons이 절단됩니다. 이것은 부드럽게 CNS를 개최하고 복부 신경절에 후부 근육 신경을 분포시키다 주변 신경이 근육 (그림 1)을 손상하지 않고 컷 수 있도록 약간을 높이는 통해 이루어집니다. 두뇌 뺍니다. </li><li> 해부 애벌레 준비는 HL3.1로 두 번 씻은 있습니다 + 1 ㎜ 칼슘 2 +합니다. </li></ol><img src="/files/ftp_upload/1109/fig1.png" alt="그림 1" /> 그림 1. 해부 애벌레의 모터 axons 절단에 관련된 단계를 보여주는 도식. CNS는 집게와 함께 해제되고 모터 axons은 뇌의 바닥에 그만입니다. 2 부 : 애벌레 근육 세포에서 세포내 기록. <ol><li> 전기 생리학 현미경과 잠수함 HL3.1과 준비에 대한 해부 접시 + 1.5 MM 칼슘 2 +를 삽입하고 용액과 접촉되도록 욕조 전극을 수정. </li><li> 모든 실험은 세 번째 복부 세그먼트 (A3) 이내에 근육 6 수행됩니다. 근육 신경을 분포시키다 주변 신경은 흡입 전극 5를 사용하고 자극하고 있습니다. </li><li> 세포내 전극이 자리에있을 때 진동을 피하기 위해 먼저 접시에 자극 전극을 위치. 근육 6 innervating 모터 신경에 가까운 주변에 자극 전극을 놓고 컷 신경이 유리 피펫 안에 때까지 부드럽게 흡입을 적용합니다. 흡입이 적용되면 신경을 스트레칭하지 않도록주의하십시오. 약간 그래서 그것이 그렇게 그것이 절단 신경 섬유 (그림 2)에 당기하지 않는 근육과 위치를 만지지 않아 피펫을 올립니다. <img src="/files/ftp_upload/1109/fig2.png" alt="그림 2" width="600" height="296" /> 그림 2. Drosophila의 유충에서 한 복부 세그먼트의 근육과 innervating 모터 뉴런의 구조. 근육 6 위치에 자극 피펫 및 절단 신경 및 기록 전극의 위치는 그림입니다. </li><li> 인트라휴대폰 녹음 3 M KCl로 가득 날카로운 microelectrodes로 만들어집니다. 세 번째 복부 세그먼트 (A3)에 근육 6 중​​심 위의 기록 전극을 위치하고 목욕 솔루션 제로 읽기 때문에 오프셋 입력을 조정합니다. 천천히 전극을 절감하고 그것이 근육 표면을 접촉까지 높은 광학 배율 아래에있는 근육에 접근한다. 근육이 침투되었는지 확인하기 위해 오실로 스코프를보세요. 쉬고 막 가능성은 적어도 -60 MV해야하거나 동물을 사용해서는 안됩니다. 산발적 미니어처 endplate의 잠재력은 이제 볼 수 있습니다. 기록 시작하기 전에 일분 안정화하기 위해 세포를 남겨주세요. </li><li> 멤브레인 전위의 변화는 액슨 HS - 2A 헤드 무대와 Axoclamp 2B 증폭기로 감지하고 Clampex V 8.2.0.235 (액슨 인 스트 루먼트)로 기록됩니다. Axoclamp의 2B는 pCLAMP 소프트웨어와 Digidata 1322A 인터페이스와 함께 작동을 실행하는 컴퓨터에 인터페이스입니다. </li><li> 세포 mEJP 반응을 측정하기 위해 어떤 자극없이 3 분 녹음되었습니다. 성분은 미니 분석 소프트웨어 (Synaptosoft, V 6.0.3)와 결정 mEJP 진폭과 주파수를 사용하여 분석했다. </li></ol> 신경 자극 : <ol><li> 모터 신경의 절단 부분이 전체 근육의 지속적인 반응을 보여주고을 모두 모터 axons에 대한 충분한 강도의 제곱 전압 펄스 (0.3 MS 기간)의 일련의 자극이다. </li><li> 자극은 기간 및 간격 시간에 따라 연속적으로 펄스를 제공하는 프로그램입니다 마스터 - 8 펄스 발생기와 함께 생성됩니다. </li><li> NMJ에서 시냅스 복구를 위해 자극 사이에 가는데 허용합니다. </li><li> 자극의 강도를 결정할 때, 가장 낮은 수준으로 시작하여 여러 실험을 통해 천천히 강도를 증가시킵니다. evoked 응답 결과 최소 자극 강도는 실험에 사용해야합니다. </li><li> 적절한 자극 강도에 도달하면 하나의 복합 EJP과 자극에 의​​해 evoked 한 근육 수축이 있어야합니다. </li><li> 각 근육 평균 amplitudes에서 적어도 10 evoked 잠재력을 기록합니다. </li></ol> 파트 3 : 대표 결과 <img src="/files/ftp_upload/1109/fig3.png" alt="그림 3" width="600" height="358" /> 그림 3. evoked EJP의 근육을 보여주는 6 대표 세포 녹화 segmental 신경의 전기 자극, 그리고 산발적 미니어처 endplate의 잠재력, 또는 mEJP의에 응답합니다. 같은 캔턴 S와 같은 건강한 야생 형 애벌레의 근육 6 EJP amplitudes는 일반적으로 40 뮤직 비디오와 1-3 MV 사이 mEJP amplitudes 수 있습니다.

<ol>
	<li>Atwood, H. L., Govind, C. K., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+ultrastructure+of+synaptic+terminals+on+ventral+longitudinal+abdominal+muscles+in+Drosophila+larvae.">Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae.</a> J. Neurobiol. 24 (8), 1008-1024 (1993).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li><li>Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., Ramaswami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+of+dynamin+within+individual+Drosophila+synaptic+boutons+relative+to+compartment-specific+markers.">Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.</a> J Neurosci. 16, 5443-5456 (1996).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dissection+of+Drosophila+larvae.">Protocol for dissection of Drosophila larvae.</a> J Vis Exp. , (2008).</li><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster.">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> J Physiol (Lond). 262, 189-214 (1976).</li><li>Katz, L. C., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+and+the+construction+of+cortical+circuits.">Synaptic activity and the construction of cortical circuits.</a> Science. 274, 1133-1138 (1996).</li></ol>

방법은 NMJ에서 시냅스 기능의 변화를 감지하기 위해 상대적으로 신속하고 광범위한 방법을 제공합니다 여기에서 설명한. 생체내 그대로 동​​물을 사용하여 electrophysiological 레코딩을 수행하고, 유전이나 약리 조작을 수행할 수있는 능력, Drosophila neurotransmission의 생리 및 유전적 측면을 조사하는 데 이상적인 동물 모델 확인하십시오. 근육 세포가 매우 큰 때문에, 일부는 두...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Small Petri dishes (35 x 10 mm)</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>1008</td>
<td>&nbsp;</td>
<tr>
<td>SYLGARD 182 Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
<td>3097366-1004</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>475002-9902</td>
<td>&nbsp;</td>
<tr>
<td>Light for microscope</td>
<td>&nbsp;</td>
<td>Schott</td>
<td>KLI500</td>
<td>&nbsp;</td>
<tr>
<td>Dissection pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>pClamp 9 software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>PCLAMP 9 STANDARD</td>
<td>&nbsp;</td>
<tr>
<td>Dissection scissors: 3mm Vannas Spring Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-0</td>
<td>&nbsp;</td>
<tr>
<td>Dumont SS Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Thin-walled borosilicate glass capillaries, with filament (1.0 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillaries, with filament (1.2 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B120F-4</td>
<td>&nbsp;</td>
<tr>
<td>Sutter P-2000 Laser Based Micropipette Puller </td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Model P-2000</td>
<td>&nbsp;</td>
<tr>
<td>Pipette polisher</td>
<td>&nbsp;</td>
<td>Narishiga</td>
<td>MF-83</td>
<td>&nbsp;</td>
<tr>
<td>Axon HS-2A head stage</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>Model HS-2A</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Axoclamp 2B amplifier</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>AXOCLAMP 2B</td>
<td>&nbsp;</td>
<tr>
<td>Clampex Software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>v 8.2.0.235</td>
<td>&nbsp;</td>
<tr>
<td>Mini analysis software. v 6.0.3</td>
<td>&nbsp;</td>
<td>Synaptosoft</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Brownlee Precision Amplifier</td>
<td>&nbsp;</td>
<td>Brownlee</td>
<td>Model 410</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Baker</td>
<td>4058-01</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>p-9333 </td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>s6297-1kg</td>
<td>&nbsp;</td>
<tr>
<td>Trelahose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>TO167</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>bp220-212 </td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>h-3375</td>
<td>&nbsp;</td>
<tr>
<td>MgCl-6H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>m2670-1kg </td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>c79-500</td>
<td>&nbsp;</td>
<tr>
<td>Master-8 Pulse Generator</td>
<td>&nbsp;</td>
<td>A.M.P.I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibration table for electrophysiology set up</td>
<td>&nbsp;</td>
<td>Technical manufacturing corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrophysiological methods for recording synaptic potentials from the nmj of drosophila larvae

이 비디오에서는, 우리는 Drosophila의 유충의 신경근육학 교차점 (NMJ)에서 신경 전달 기록에 대한 electrophysiological 방법을 설명합니다. 애벌레 신경근육학 시스템은 시냅스 생리학과 neurotransmission 연구를위한 모델 버렸네이며, 유전학을 정의하고 실험 조작에 액세스할 수있는 가치있는 연구 도구입니다. 애벌레는 몸을 벽 근육, 중추 신경계 및 말초 신경을 노출 해부하실 수 있습니다. Drosophila과 innervation 패턴의 근육이 잘 특징과 근육 세포 기록에 대한 액세스하기 쉽습니다. A7 - 개인 근육은 8 복부 세그먼트, 세그먼트에서 A2를 반복하는 패턴으로 배열된 30 근육과 각 내에 위치와 방향에 의해 확인할 수 있습니다. 해부 drosophila의 애벌레가 얇고 개인 근육과 운동 신경의 axons의 번들하는 것은 transillumination하여 시각 수 있습니다<sup&gt; 1</sup&gt;. 유전자 변형 구조는 시각적인 신원 확인을 위해 또는 특정 조직에 유전자 조작을위한 제품을 대상 세포를 분류하는 데 사용할 수 있습니다. 애벌레에서 흥분성의 연결 잠재력 (EJPs)은 버렸네에서 motoneurons에서 글루 탐 산염의 기공을 갖는 릴리스에 대응 생성됩니다. 해부 애벌레에서 EJP는 세포내 전극과 근육에 기록하실 수 있습니다. 액션 잠재력은 인위적으로, 복부 신경절에 후부 절단 부드러운 흡입으로 유리 피펫에 그려 및 전극과 자극했습니다 모터 뉴런에 evoked 수 있습니다. 이러한 모터 뉴런은 자극 별개의 소성 한계를 가지고 있고, 그들이 동시에 불을 때, 그들은 근육에 응답을 생성합니다. NMJ 버렸네를 통해 전송되는 신호는 모터 뉴런 신경을 분포시키다 그 근육에 기록하실 수 있습니다. EJPs과 미니어처 흥분성의 접합 잠재력 (mEJPs)가 막 잠재력의 변화로 볼 수 있습니다. Electrophysiological 답변 수정 최소한의 hemolymph 같은 솔루션에서 실온에서 기록됩니다<sup&gt; 2</sup&gt; (HL3) 5 MM의 MG가 포함되어<sup&gt; 2 +</sup&gt; 그리고 1.5 MM 칼슘<sup&gt; 2 +</sup&gt;. evoked EJPs의 진폭의 변화는 시냅스 기능과 구조의 차이를 나타낼 수 있습니다. 디지털 녹음은 EJP 진폭, mEJP 주파수와 진폭, 그리고 quantal 콘텐츠에 대한 분석입니다.

Watch this Scientific Journal Video about Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae at JoVE.com

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

여기 Drosophila의 유충의 신경근육학 교차점에서 시냅스 전송을 측정 electrophysiological 방법을 설명합니다. Evoked 릴리스는 모터 신경의 axons을 자극하여 인공적으로 시작되며, NMJ을 통해 전송은 근육에 evoked postsynaptic 반응에 의해 측정할 수 있습니다.

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. ...

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Research

JoVE Journal

Biology

20.9K 조회수.  Columbia University College of Physicians and Surgeons. 여기 Drosophila의 유충의 신경근육학 교차점에서 시냅스 전송을 측정 electrophysiological 방법을 설명합니다. Evoked 릴리스는 모터 신경의 axons을 자극하여 인공적으로 시작되며, NMJ을 통해 전송은 근육에 evoked postsynaptic 반응에 의해 측정할 수 있습니다.

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

셋째 Instar Drosophila의 유충에서 Imaginal 디스크의 해부

연구

생물학

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Drosophila Larval NMJ Dissection

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 35 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. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila 애벌레 NMJ의 해부

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

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

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

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 ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

수확 및 준비 Drosophila</emElectrophysiological 녹화 및 기타 수속을위한> 태아

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

에 Electrophysiological 녹음 Drosophila 엠브료

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

크레이 피쉬 오프너 근육의 NMJ에서 역사보기 및 생리학 시범

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Whitefly에서 Endosymbionts의 추출을위한 방법<em&gt; 베미 시아 타바시</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

셋째 Instar Drosophila의 유충에서 Imaginal 디스크의 해부

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

Drosophila 애벌레 NMJ의 해부

Drosophila 애벌레 NMJ Immunohistochemistry

에서 시냅틱 후보의 Channelrhodopsin2 중재 자극 Drosophila 신경근육학 분기점

수확 및 준비 Drosophila 태아

에 Electrophysiological 녹음 Drosophila 엠브료

크레이 피쉬 오프너 근육의 NMJ에서 역사보기 및 생리학 시범

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Whitefly에서 Endosymbionts의 추출을위한 방법

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

셋째 Instar Drosophila의 유충에서 Imaginal 디스크의 해부

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

Drosophila 애벌레 NMJ의 해부

Drosophila 애벌레 NMJ Immunohistochemistry

에서 시냅틱 후보의 Channelrhodopsin2 중재 ​​자극 Drosophila 신경근육학 분기점

수확 및 준비 Drosophila 태아

에 Electrophysiological 녹음 Drosophila 엠브료

크레이 피쉬 오프너 근육의 NMJ에서 역사보기 및 생리학 시범

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Whitefly에서 Endosymbionts의 추출을위한 방법

에서 시냅틱 후보의 Channelrhodopsin2 중재 자극 Drosophila 신경근육학 분기점