NIH 보조금 R01 수소 099557에 의해 지원

1. 제조의 기록 전극 microelectrode 끌어당기는 사람에 대 한 <ol><ol><li>사용 다음 유리 전극 당기 <li>프로토콜 (재료의 표 참조): <ol><li>1: 열 510 당겨-속도 30 시간 250; 2 호선: 열 490 풀-속도 30 250 시간. 
			참고: 단위; 당 0.5 ms에 해당 하는 시간 단위 다른 단위는 상대. 램프 테스트 수행 후 모든 필 라 멘 트에 대 한 열 값을 조정 한다.</li>
		</ol></li> <li>현미경 (35 x 배율)를 사용 하 여 가져온된 전극의 내경 ( 그림 1A) 7-10 µ m의 범위에 있는지 확인. 먼지 축적을 방지 하기 위해 단단히 닫힌된 용기에 모세를 저장.</li>
	</ol></li> <li>화재 연마 <ol><li>화재 폴란드어 모세 (1-2에 대 한 최대 열 값의 80-90%)는 마이크로-포지를 사용 하 여 (재료의 표 참조). 세련 된 전극의 최종 내부 직경은 5 µ m ( 그림 1A) 27 확인.</li>
	</ol></li> <li>굴곡 참고: 두 개의 굴절 높은 확대 목표 아래 근육 위에 전극 위치 하기 위해 만들어집니다.
	<li> 그림 1B에서 표시 하는 장치를 사용 하 여 <ol>. 조작자에 전극을 수정 하 고 필 라 멘 트, 아니라 그것을만 지는 팁 놓고</li>
		<li>집합 열 값의 최대 60-70%를 누르고 1-2에 대 한 마이크로-포지의 페달 (재료의 표 참조)는 필 라 멘 트가 열. L 자 모양의 바늘을 사용 하 여 부드럽게 풀 ( 그림 1B 확대) 전극의 팁 다운. 약 90 o ( 그림 1C)에 굴곡을 만들어.</li>
		<li>전극 토치의 불꽃에 집게를 사용 하 여 손으로 누른 첫 번째 벤드에서 7-10 밀리미터의 거리에 두 번째 벤드 약 120 o ( 그림 1C)의 각도 확인.</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" /> 그림 1. Micropipette 제조의 마지막 단계. (A) 전극 고 화재 연마 후 팁. (B.1) 팁 절곡에 대 한 설정. (B.2) 박스형된 영역 오른쪽에 확대 표시 됩니다. 전극과는 필 라 멘 트 전극 팁은 약간 와이어 위에 위치 하 고 감동 하지 있도록 고정 됩니다. (C.1) 기록 전극입니다. (C.2) 박스형된 영역 오른쪽에 확대 표시 됩니다. 첫 번째 벤드는 약 90 °의 각도 첫 번째 벤드와 전극의 끝 사이 거리가 약 1 m m. 규모 바 = 3 m m. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> 이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
 2. 추가 예비 단계 <ol><li>준비 (mM)에 heamolymph 같은 (HL3) 솔루션: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 트 레 할 로스, 115 자당, 5 HEPES, 및 1mm CaCl 2; pH 7.3-를 조정 7.4. 냉장고에 솔루션을 유지 하 고 매주 신선한 게.</li>
	<li>게 자극 pipets 같은 방법으로 녹음 유리 pipets, 그들을 구 부 필요가 없습니다 않습니다. 
	참고: 자극 전극의 준비는 28와 27에 자세히 설명 되어 있습니다. 마지막 지름 5-7 µ m의 범위에 있어야 화재 연마 후.</li>
	<li>삽입 주사기에 연결 된 microelectrode 홀더에서 자극 피 펫.</li>
	<li>작은 접시 (35 x 10 m m) 실리콘 고무 에폭시 코팅에서 제조 해 부 접시 (재료의 표 참조) 28에 설명 된 대로. 
	참고: 실리콘 고무 사용 하기 전에 완전히 경화 되어야 합니다.</li>
	<li>방황 3 탈피 애벌레를 선택 하 고 27 , 28 , , 29 30에 설명 된 대로 그것을 해 부. 
	참고: boutons를 시각화 하려면 비행 스트레인 CD8-GFP (재료의 표 참조) <ol><li>사용 집게 (재료의 표 참조), 선택 3 rd 탈피 애벌레는 유리병에서.</li>
		<li>핀 첫 번째 핀 후부 배치 애벌레와 다른 핀 앞쪽 (가까운 입 후크).</li>
		<li>추가 HL3 솔루션.</li>
		<li>애벌레의 등 쪽 측에 봄을가 위 (재료의 표 참조)에서 최고 핀 하단 하나를 사용 하 여 잘라 만들.</li>
		<li>애벌레 필렛, 애벌레의 왼쪽과 오른쪽에 2 추가 핀 배치를 핀.</li>
		<li>배 짱과 집게를 사용 하 여 tracheas 제거.</li>
		<li>봄이 위를 사용 하 여 복 부 신경 코드 밖에 신경을 잘라.</li>
	</ol></li></ol> 3. EJCs의 전기 레코딩 <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> 그림 2. 녹음 설정. 샘플 NMJ 실리콘 코팅된 페 트리 접시 (화살표) epifluorescence 기능, 높은 확대 목표 및 2 개의 micromanipulators 수직 화합물 현미경의 이동식 무대 위에 위치에 고정. 현미경은 진동 방지 테이블에 배치 됩니다. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<li>녹음 <ol><li>장소 현미경 단계 ( 그림 2)에 준비와 페 트리 접시 <ol>. 목욕에서 참조 전극 삽입.</li>
		<li>기록 전극 HL3 솔루션을 작성합니다. 10 배 목표 아래 (재료의 표 참조), 전극은 욕조에 담가 고 6-7 2, 3, 또는 4는 micromanipulator를 사용 하 여 복 부 세그먼트의 근육 이상 (재료의 표 참조) ( 그림 3 A.1, A.2).</li>
		<li>60 x 목표 전환 (재료의 표 참조). 피-형광 또는 DIC 광학을 사용 하 여 관심사의 지역에 초점. 시 냅 시스 bouton ( 그림 3 B.1-3) 위에 전극의 끝을 놓습니다.</li>
		<li>근육에 매우 부드럽게 전극을 누릅니다. 과도 한 압력은 NMJ를 손상 또는 자발적 시 냅 시스 활동에 증가 일으킬 수 있습니다. 전극의 팁은 안 막힌-그것은, 그것을 대체 하는 경우 확인</li>
		<li>앰프, A/D 보드, 그리고 컴퓨터에 전환.</li>
		<li>앰프에 전압 클램프 모드를 선택 하십시오.</li>
		<li>수집 소프트웨어를 시작 하 고 선택은 &#39; 간격-무료 &#39; 모드.</li>
		<li>컴퓨터 화면에 mEJCs의 모양을 관찰.</li>
		<li>0.2-의 범위에는 mEJCs의 진폭은 없는지 0.7 없음. 
		참고: 작은 EJCs 나타냅니다 기록 전극 결함이 있다 또는 그것이 제대로 위치 하지은.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> 그림 3. 시 냅 스 boutons의 시각화. Hemi-세그먼트 확대 (A.1)와 근육 6 및 7을 보여주는 확대 박스형된 영역 아래 10의 (A) A 명시 이미지 (A.2, 화살표 표시 기록 전극). 눈금 막대 = 50 µ m. (B) 시 냅 스 boutons는 유전으로 인코딩된 신경 마커 (CD8-GFP)와 초파리 라인에 시각 피 형광 이미징 (B.1) 또는 DIC 광학 (B.2)을 사용 하 여. 이미지는 객관적이 고 GFP 이미징에 대 한 필터 큐브 x 60 찍은 (재료의 표 참조). Synaptic boutons 화살표와 함께 표시 되 고 형광 및 DIC 이미지 오버레이 B.3에 표시 됩니다. 눈금 막대 = 10 µ m. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<ol start="2"> <li>자극 <ol><li>근처 복 부 세그먼트 2-4를 innervating 축 삭 자극 전극 배치 사용 시각적인 통제 micromanipulator.</li>
		<li>주사기의 피스톤을 당겨서 적용 부정적인 압력에 연결 전극 홀더, 그래서 축 삭 ( 그림 2) 전극의 내부 뽑아.</li>
		<li>는 자극에. 격리 단위에 노브를 설정 (재료의 표 참조) 제로 전류를 설정 하 여 다음 부드럽게 증가, EJCs 나타날 때까지 (또는 임계값에 도달할 때까지).</li>
		<li>현재 자극 suprathreshold 정권에서 자극 증가 약 두 번, EJCs의 관찰에 대 한 임계값에 비해 수행. 
		참고: 우리의 경험에서 이러한 자극 강도 실패 활동 전위와 활동 전위 발생을 피하기 위해 최적입니다. 예를 들어, EJCs 0.2의 자극 전류에 나타납니다 0.4의 전류를 사용 하는 엄마, 실험을 통해 엄마.</li>
	</ol></li> <li>물개 저항 <ol><li>앰프의 전극 저항 스위치를 설정 하 여 녹음 macropatch 전극의 인감 저항을 측정 &#34; 테스트 봉인 &#34; 위치. GΩ에 인감 저항의 값에 표시 됩니다 관찰 된 &#34; 현재 &#34; 창.</li>
		<li>인감 저항 0.5-2 M의 범위에 있는지 확인 &#8486;. 값이이 범위를 벗어난 표시 전극 제대로 광택 또는 하지 제대로 위치.</li>
		<li>인감 저항 녹음을 통해 모니터링, 실험을 통해 일정 하 게 유지 그것 다는 것을 확인.</li>
	</ol></li></ol> 4. 분석 분석용 소프트웨어 사용자 지정 <ol><li>분석 녹음 채용 (재료의 표 참조). 
	참고: 연구소 사내 소프트웨어 Quantan 31, EJCs 및 mEJCs focally 기록 ( 그림 4)의 검출에 대 한 사용자 지정을 사용 합니다. 이 소프트웨어 가우스 디지털 필터를 포함 하 고 겹치는 다중 quantal 이벤트 ( 그림 4A)에서 quantal 봉우리의 탐지를 허용 한다. 다른 접근 17에 설명 되어 있습니다.</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> 그림 4. Quantal 분석입니다. MEJCs (A)와 EJCs (B) Quantan 소프트웨어에 의해 감지. 이벤트 지역 녹색으로 표시 하 고 봉우리는 붉은 화살표에 의해 표시 된다. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchrony+of+quantal+events+in+evoked+multiquantal+responses+indicates+presynaptic+quantal+interaction.">Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction.</a> J Neurophysiol. 81 (5), 2234-2242 (1999).</li><li>Bykhovskaia, M., Polagaeva, E., Hackett, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechnisms+underlying+different+facilitation+forms+at+the+lobster+neuromuscular+synapse.">Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse.</a> Brain Res. 1019 (1-2), 10-21 (2004).</li><li>Cooper, R. L., Stewart, B. A., Wojtowicz, J. M., Wang, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+measurement+and+analysis+methods+compared+for+crayfish+and+Drosophila+neuromuscular+junctions,+and+rat+hippocampus.">Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus.</a> J Neurosci Methods. 61 (1-2), 67-78 (1995).</li><li>Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., Wu, C. 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T., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+Complexin+Accessory+Helix+with+Synaptobrevin+Regulates+Spontaneous+Fusion.">Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.</a> Biophys J. 111 (9), 1954-1964 (2016).</li><li>Wong, K., Karunanithi, S., Atwood, H. 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G., Lemos, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Loose-Patch-Clamp method. , (2007).</li><li>Wu, W. H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+recordings+of+high+and+low+output+NMJs+on+the+crayfish+leg+extensor+muscle.">Physiological recordings of high and low output NMJs on the crayfish leg extensor muscle.</a> J Vis Exp. (45), (2010).</li><li>Verstreken, P., Ohyama, T., Bellen, H. 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저자는 공개 없다.

초파리 는 공부 시 냅 스 전송에 유리한 모델 유기 체를 나타냅니다. 여러 녹음 구성 시 냅 스 전위, 시 냅 스 전류 두 전극 전압 클램프33,34, 및 초점 macropatch의 녹음의 세포내 녹음을 포함 한 애벌레 NMJ에 사용 되었습니다. 여기서 설명 하는 시 냅 스 전류 기록입니다. 후자의 방법을 시각화 된 boutons에 시 냅 시스 전송의 정확한 정량화를 허용합니?...

초파리 는 시 냅 스 전송 제어 하는 분자 메커니즘을 연구 하는 우수한 모델 시스템입니다. 초파리 에서 신경 근육 학 시스템 glutamatergic, 이며 따라서 초파리 신경 근육 학 교차로 (NMJ) glutamatergic 자료의 보존된 기능을 공부를 사용할 수 있습니다. 1 월과 1 월의 연구1, 이후 세 번째 탈피 애벌레는 갖는 자발적 시 냅 스 전송 흥분 성의 접합 잠재력 (EJPs) 또는 전류 (EJCs)을 모니터링 하 여 공부 하 광범위 하 게 사용 되었습니다. EJPs는 일반적으로 기록 침 날카로운 유리 마이크로 전극, 그들은 주어진된 근육 섬유에서 시 냅 스를 만드는 모든 boutons를 포함 하 여 전체 NMJ의 활동을 반영 하 고.
대조적으로, 출시의 사이트의 제한 된 수의 활동 신경 터미널 또는 시 냅 스 varicosities 근처 micropipette 팁을 배치 하 여 focally 기록할 수 있습니다. 이 기술은 원래 캐츠와 Miledi2에 의해 고용 되었다 및 초점 세포 외 녹음 개구리3,,45, 마우스6 를 포함 한 여러 NMJ 준비에서 성공적으로 고용 되어 , 7 , 8, 갑각류9,10,11,12,13,,1415,16및 초파리17,18,19,20,21,,2223. 이 이렇게는 최적화 된 레코딩 전극24,25macropatch 누가 Dudel에 의해 더 개발 되었다. Dudel의 구현에서이 기술을 밀접 하 게 일치 하는 느슨한 패치 클램프 방법26.
초파리 애벌레 NMJ 시 냅 스 boutons, 명확 하 게 정의 하 고 유전자 인코딩된 신경 형광 태그 ( 테이블의 자료참조)와 유전자 변형 라인은 쉽게 사용할 수 있습니다. 이러한 장점이 우리 EJCs와 mEJCs는 선택 된 시 냅 시스 bouton20,,2122에서 기록를 활성화. 여기, 우리는이 기술을 자세히 설명합니다.

<table width="1251">
	<tbody>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sutter P-97</td>
			<td style="width:288px;">Sutter instrument</td>
			<td style="width:180px;">P-97</td>
			<td style="width:495px;">Microelectrode puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narishige MF-830</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MF-830</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">WPI MF200</td>
			<td style="width:288px;">WPI</td>
			<td>MF200</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Glass capilaries</td>
			<td style="width:288px;">WPI</td>
			<td>B150-86-10</td>
			<td style="width:495px;">Glass capilaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtorch 1WG61</td>
			<td style="width:288px;">Grainer</td>
			<td style="width:180px;">1WG61</td>
			<td>Microtorch</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:288px;">Dow Corning</td>
			<td style="width:180px;">SYLGARD 184</td>
			<td>Silicone for dissection plates preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Dissection pins</td>
			<td style="width:288px;">Amazon</td>
			<td>B00J5PMPJA</td>
			<td>Pins for larvae positioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Tweezers</td>
			<td style="width:288px;">WPIINC</td>
			<td>500342</td>
			<td>Tweezers for placing pins, removing the guts and tracheas.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Scissors</td>
			<td style="width:288px;">WPIINC</td>
			<td>501778</td>
			<td>Scissors for cutting the cuticula of the larvae and nerves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus BX61WI</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">BX61WI</td>
			<td>Upright microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus Lumplan FL N 60x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">UPLFLN 60X</td>
			<td>Microscope objective 60X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus UPlan FL N 10x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">Uplanfl N 10X</td>
			<td>Microscope objective 10X</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Narishige Micromanipulator</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MHW-3</td>
			<td>Three-axis Water Hydraulic Micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">npi Electronic GmbH ELC-03XS</td>
			<td style="width:288px;">npi Electronic GmbH</td>
			<td style="width:180px;">ELC-03XS</td>
			<td>Electrophysiological amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Master 8</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Master 8</td>
			<td>Electrical stimulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Iso-Flex</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Iso-Flex</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">TMC antivibration table</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">63-9090</td>
			<td>Antivibration table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMC Faraday cage</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">81-333-90</td>
			<td>Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Digidata 1322A</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">Digidata 1322A</td>
			<td>Digidata</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Computer</td>
			<td style="width:288px;">Dell</td>
			<td style="width:180px;">Dell Dimension 5150</td>
			<td>Computer with Win XP OS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Electrode holder</td>
			<td style="width:288px;">WPI</td>
			<td>MEH3SW&#160;</td>
			<td style="width:495px;">Electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Optical filter</td>
			<td style="width:288px;">Omega optical</td>
			<td style="width:180px;">XF 115-2</td>
			<td style="width:495px;">Filter cube for Green Fluorescent Protein (GFP) detection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">pCLAMP 8</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">8.0.0.81</td>
			<td>Software for signal recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Quantan</td>
			<td style="width:288px;">In-house software</td>
			<td style="width:180px;">-</td>
			<td>Software for signal processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Canton-S (Wildtype)</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td>64349</td>
			<td>Control fly line</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">cpx SH1</td>
			<td style="width:288px;">Generous Gift of J.T. Littleton</td>
			<td style="width:180px;">-</td>
			<td>Complexin knock-out fly line with increased spontaneous exocytosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">CD8-GFP</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td style="width:180px;">5137</td>
			<td>Fly line with neuronal fluorescent (GFP) Tag</td>
		</tr>
	</tbody>
</table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

Drosophila 신경 근육 학 교차로 (NMJ) glutamatergic 시 냅 스 전송 연구에 우수한 모델 시스템입니다. 초파리 애벌레 NMJ에 시각화 된 boutons 시 냅 스 전류 초점 macropatch 녹음 기술을 설명합니다. 이 기술은 micropipettes, 높은 배율, 장거리 물 침수 목표, 미분 간섭 콘트라스트 (DIC) 광학와 형광 화합물 현미경 녹음의 맞춤된 제작을 해야 합니다. 첨부 합니다. 기록 전극 선택된 시 냅 시스 bouton DIC 광학, epi-형광, 또는 둘 다 시각 위에 배치 됩니다. 이 기법의 장점은 릴리스의 사이트의 제한 된 수의 시 냅 스 활동을 모니터링을 허용 한다입니다. 기록 전극은 여러 미크론의 직경 그리고 전극 가장자리 밖에 배치 된 릴리스 사이트 크게 영향을 주지 않습니다 기록된 전류. 기록 된 시 냅 스 전류 빠른 속도 고 쉽게 해결 될 수 있습니다. 이러한 장점은 향상 된 자연 또는 비동기 시냅틱 활동 돌연변이 플라이 라인의 연구에 대 한 특히 중요 하다.

초점 macropatch 녹음 선택 된 시 냅 스 boutons (그림 5)에서 모니터링 시 냅 시스 활동 가능 전극 (그림 5A, 사이트 1) 시 냅 시스 bouton 위에 가져갈 때 기록 된 mEJCs (그림 5C, 사이트 1)는 진폭 잡음 레벨을 크게 초과 하 고 (하위 밀리초 범위)에서 단계 상승 날카로운. 때 기록 전극은 이동 시 냅 시스 bouton 멀리 몇 ?...

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

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

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

시 냅 스 전류 초파리 3 탈피 애벌레 neuromuscular 접속점에 시각화 된 시 냅 스 boutons에서 focally 기록 될 수 있습니다. 이 기술은 단일 시 냅 시스 bouton의 활동을 모니터링 수 있습니다.

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

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Neuroscience

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

6.0K 조회수.  Wayne State University. 시 냅 스 전류 초파리 3 탈피 애벌레 neuromuscular 접속점에 시각화 된 시 냅 스 boutons에서 focally 기록 될 수 있습니다. 이 기술은 단일 시 냅 시스 bouton의 활동을 모니터링 수 있습니다.

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila 애벌레 Segmental Axons 이내 시냅틱 Vesicles의 생체내 시각화에

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

연구

신경과학

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

성상 세포 운송업자 전류의 디컨 볼 루션 분석 글루타민산 정리의 시간 과정을 도출

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

신경 근육 접합 : 측정 시냅스 크기, 공 초점 형광 현미경을 사용하여 시냅스 단백질 농도의 분열 및 변경

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

상기 표현형의 특성을 : 정량이 중요한 이유<em&gt; 초파리</em&gt; 애벌레 신경 근육 학 분기점

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

칼슘 염료를 개구부 신경 절편에 삽입하기 : 신경 개구부를 통해 개구리 신경 근육 계통에서 칼슘 과도 등록

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

높은 처리량 및 다중 파라 메트릭 정량화를위한 ​​두 알고리즘<em&gt; 초파리</em&gt; 신경 근육 접합 형태학

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...