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>

<ol>
	<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. 262 (1), 189-214 (1976).</li><li>Katz, B., Miledi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+on+the+synaptic+delay+at+the+neuromuscular+junction.">The effect of temperature on the synaptic delay at the neuromuscular junction.</a> J Physiol. 181 (3), 656-670 (1965).</li><li>Macleod, G. T., Gan, J., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle-associated+proteins+and+quantal+release+at+single+active+zones+of+amphibian+(Bufo+marinus)+motor-nerve+terminals.">Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.</a> J Neurophysiol. 82 (3), 1133-1146 (1999).</li><li>Macleod, G. T., Farnell, L., Gibson, W. G., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+secretion+and+nerve-terminal+cable+properties+at+neuromuscular+junctions+in+an+amphibian+(Bufo+marinus).">Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).</a> J Neurophysiol. 81 (3), 1135-1146 (1999).</li><li>Zefirov, A., Benish, T., Fatkullin, N., Cheranov, S., Khazipov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+active+zones.">Localization of active zones.</a> Nature. 376 (6539), 393-394 (1995).</li><li>Macleod, G. T., Lavidis, N. A., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+dependence+of+quantal+secretion+from+visualized+sympathetic+nerve+varicosities+on+the+mouse+vas+deferens.">Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens.</a> J Physiol. 480 (Pt 1), 61-70 (1994).</li><li>Samigullin, D., Bill, C. A., Coleman, W. L., 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=Regulation+of+transmitter+release+by+synapsin+II+in+mouse+motor+terminals.">Regulation of transmitter release by synapsin II in mouse motor terminals.</a> J Physiol. 561 (Pt 1), 149-158 (2004).</li><li>Coleman, W. L., 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=Rab3a-mediated+vesicle+recruitment+regulates+short-term+plasticity+at+the+mouse+diaphragm+synapse.">Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse.</a> Mol Cell Neurosci. 41 (2), 286-296 (2009).</li><li>Atwood, H. L., Parnas, H., Parnas, I., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+currents+evoked+by+graded+intracellular+depolarization+of+crayfish+motor+axon+terminals.">Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals.</a> J Physiol. 383, 587-599 (1987).</li><li>Parnas, H., Dudel, J., Parnas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+release+and+its+facilitation+in+crayfish.+I.+Saturation+kinetics+of+release,+and+of+entry+and+removal+of+calcium.">Neurotransmitter release and its facilitation in crayfish. I. Saturation kinetics of release, and of entry and removal of calcium.</a> Pflugers Arch. 393 (1), 1-14 (1982).</li><li>Wojtowicz, J. M., Marin, L., 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=Activity-induced+changes+in+synaptic+release+sites+at+the+crayfish+neuromuscular+junction.">Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction.</a> J Neurosci. 14 (6), 3688-3703 (1994).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crayfish+neuromuscular+facilitation+activated+by+constant+presynaptic+action+potentials+and+depolarizing+pulses.">Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses.</a> J Physiol. 241 (1), 69-89 (1974).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+statistics+of+transmitter+release+during+facilitation.">Changes in the statistics of transmitter release during facilitation.</a> J Physiol. 229 (3), 787-810 (1973).</li><li>Worden, M. K., Bykhovskaia, M., 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=Facilitation+at+the+lobster+neuromuscular+junction:+a+stimulus-dependent+mobilization+model.">Facilitation at the lobster neuromuscular junction: a stimulus-dependent mobilization model.</a> J Neurophysiol. 78 (1), 417-428 (1997).</li><li>Bykhovskaia, M., Hackett, J. T., Worden, M. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+stability+of+Drosophila+larval+neuromuscular+preparations+in+haemolymph-like+physiological+solutions.">Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions.</a> J Comp Physiol A. 175 (2), 179-191 (1994).</li><li>Pawlu, C., DiAntonio, A., Heckmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postfusional+control+of+quantal+current+shape.">Postfusional control of quantal current shape.</a> Neuron. 42 (4), 607-618 (2004).</li><li>Akbergenova, Y., 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=Synapsin+maintains+the+reserve+vesicle+pool+and+spatial+segregation+of+the+recycling+pool+in+Drosophila+presynaptic+boutons.">Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons.</a> Brain Res. 1178, 52-64 (2007).</li><li>Akbergenova, Y., 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=Enhancement+of+the+endosomal+endocytic+pathway+increases+quantal+size.">Enhancement of the endosomal endocytic pathway increases quantal size.</a> Mol Cell Neurosci. 40 (2), 199-206 (2009).</li><li>Vasin, A., Volfson, D., Littleton, J. 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. 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+unit+populations+at+the+Drosophila+larval+neuromuscular+junction.">Quantal unit populations at the Drosophila larval neuromuscular junction.</a> J Neurophysiol. 82 (3), 1497-1511 (1999).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+reduced+calcium+on+quantal+unit+current+and+release+at+the+crayfish+neuromuscular+junction.">The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction.</a> Pflugers Arch. 391 (1), 35-40 (1981).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Ca2++inflow+to+quantal,+phasic+transmitter+release+from+nerve+terminals+of+frog+muscle.">Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.</a> Pflugers Arch. 422 (2), 129-142 (1992).</li><li>Marrero, H. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction.">FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.</a> Methods Mol Biol. 440, 349-369 (2008).</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=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Imlach, W., 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=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li><li>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=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li><li>Huntwork, S., Littleton, 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=A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li><li>Zhong, Y., 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=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, 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=Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

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

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Universidad de Cartagena UDEC

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JoVE Journal

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

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

모터 신경 세포의 이점 패치 클램프 레코딩과 Zebrafish의 대상 골격근

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

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 ...

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. ...

생물학

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 ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

해부 및 C. 엘레간스 신경근육학 융티온에서 레코딩

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

의 작성<em&gt; Drosophila</em에 대한&gt; 중부 뉴런<em&gt; 현장에서</em&gt; 패치 클램핑

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

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

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

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

배아 Zebrafish의 MAUTHNER 세포에서 패치 클램프 녹음

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

Drosophila 유충의 신경근 접합부의 투과 전자 현미경을 위한 시료 전처리 공정 최적화

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

초파리 신경근접합부(NMJ) 정량화: 시냅스 형태 및 기능을 평가하는 방법

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

모터 신경 세포의 이점 패치 클램프 레코딩과 Zebrafish의 대상 골격근

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

에서 시냅틱 후보의 Channelrhodopsin2 중재 자극 Drosophila 신경근육학 분기점

해부 및 C. 엘레간스 신경근육학 융티온에서 레코딩

의 작성

에 Electrophysiological 녹음 Drosophila 엠브료

배아 Zebrafish의 MAUTHNER 세포에서 패치 클램프 녹음

Drosophila 유충의 신경근 접합부의 투과 전자 현미경을 위한 시료 전처리 공정 최적화

초파리 신경근접합부(NMJ) 정량화: 시냅스 형태 및 기능을 평가하는 방법

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

모터 신경 세포의 이점 패치 클램프 레코딩과 Zebrafish의 대상 골격근

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

에서 시냅틱 후보의 Channelrhodopsin2 중재 ​​자극 Drosophila 신경근육학 분기점

해부 및 C. 엘레간스 신경근육학 융티온에서 레코딩

의 작성

에 Electrophysiological 녹음 Drosophila 엠브료

배아 Zebrafish의 MAUTHNER 세포에서 패치 클램프 녹음

Drosophila 유충의 신경근 접합부의 투과 전자 현미경을 위한 시료 전처리 공정 최적화

초파리 신경근접합부(NMJ) 정량화: 시냅스 형태 및 기능을 평가하는 방법

에서 시냅틱 후보의 Channelrhodopsin2 중재 자극 Drosophila 신경근육학 분기점