토크 보상의 원래 디자인은 칼 괴쯔와 유래. 이 프레 젠 테이션의 특정 설치 대출에 대한 광범위하고 원래 마틴 Heisenberg와 하드 늑대에 의해 개발되었다. 난 그들의 지속적인 지원, 격려와 전문 지식에 대한이 두 사람 특히 빚을졌습니다.

미디어 플라이 파리 식품의 구성은 (. 국영 외, 1996) 학습을위한 중요 : <ul><li> 워터 1,000 ML </li><li> Cornmeal 180g </li><li> 콩 10g </li><li> 효모 18.5 g </li><li> 한천 7.5 g </li><li> 몰레 40g </li><li> 시럽 (사탕무) 40g </li><li> Nipagin 2.5 g </li></ul> 모든 병을는 신선하고 살아있는 효모 붙여넣기의 명인 파리와 pupae에 대한 추가적인 표면을 제공하기 위해 필터 종이와 함께 제공됩니다. 번식 및 준비 플리 다음 절차는 정확하게 적절한 밀도로 성장 동물을 무대로 이어지는, 매일 수행됩니다. 전날의 마지막 절차부터 모든 새로 ecclosed 파리는 사육 및 실험 수집하고 있습니다. 남아있는 생활 pupae없이 가장 오래된 튜브가 삭제됩니다. 네 일 옛 파리는 하루 계란 증착을위한 신선한 유리병에 추가됩니다. 여성 파리의 밀도는 약 20 각 유리병에 대해, 유리병의 크기에 따라 조정되어야하며 응력이 사용됩니다. 이상적인 밀도 즉시 매체는 모든 애벌레가 첫 번째 파리의 ecclose 전에 pupated 가지고 같은 애벌레 단계와 낮아 동안 녹일 정도로 높은 하나입니다. 전날부터 달걀 누워 파리 제거와 삭제됩니다. 준비 플라이 25 위에서 설명한대로 파리는 표준 cornmeal / 당밀 매체에 유지됩니다 ° C와 12hr light/12hr 어두운 정권 60 %의 습도. 간단히 추위 - 마취로 24 - 48h 오래된 파리 immobilizing 후, 파리는 (직경 0.05mm 삼각형 모양의 구리 갈고리로 머리와 흉부와 (과 SuperGlue 자외선 유리 접착제, 505127A, 페이서 기술, 쿠카몽가, CA. 미국) 붙어 아르 실험 전) 하루. 동물 다음 자당의 몇 가지 곡물을 포함하는 작은 습한 실에서 야간 개별적으로 유지됩니다. 기구 설정의 핵심 장치는 토크 보상 (토크 미터) (Götz, 1964)입니다. 그것은 의도 비행 기동에 의한 수직의 신체 축을 중심으로 파리의 각운 동량을 측정합니다. 위에서 설명한대로 연결에 붙어 비행은, homogeneously 뒤에서 조명하는 원통형 파노라마 (경기장, 직경 58mm)의 중심에 정지 비행을 수행하는 클램프를 통해 토크 미터에 첨부되어 있습니다. 광원은 100W, 12V 텅스텐 - 요오드 전구입니다. 경기장의 녹색과 파란색의 조명은 빛이 단색 광범위한 대역 코닥 레튼 젤라틴 필터 (각각 # 47 # 99)를 통해 전달됩니다. 필터는 0.1s 이내에 빠른 솔레노이드에 의해 교환하실 수 있습니다. 또한, 경기장은 푸른 녹색 필터 (로스코 &quot;surfblue&quot;번호 5433) 또는 전혀 필터를 통해 전달하여 &quot;일광&quot;로 켜집니다. 본 연구에 사용된 로스코 푸른 녹색 필터의 전송 스펙트럼은 BG18 필터 (쇼트, 마인츠)의에 해당되고 코닥 파란색과 녹색 필터 (Brembs 및 헴펄 드 이바라 2006 년 사이의 중간을 구성, 리우 외 ., 1999). 경기장은 컴퓨터 제어 전기 모터를 사용하여 비행 주위에 회전 수 있습니다. 이러한 &quot;비행 시뮬레이터&quot;상황에서 경기장의 각속도는 비례하지만, (계수 K를 결합 =- 11 ° / s의 • 10 - 10Nm)를 파리의 편주 토크에 대한 감독. 이것은 파노라마를 안정화하고 각도 방향을 제어하기 위해 파리를 수 있습니다. 이 가상 &quot;비행 방향&quot;(즉, 투기장 위치)은 원형 전위 차계 (Novotechnik, A4102a306)를 통해 지속적으로 기록됩니다. 디지털 변환기 카드에 아날로그 (PCL812, Advantech 주)는 나중에 분석을위한 흔적 (주파수 20Hz를 샘플링) 상점 컴퓨터에 경기장의 위치와 침로에서 빗나가다 토크 신호를 피드. 처벌은 파리의 머리와 흉부에 뒤에 이상의 감독 조정 적외선 레이저 (825 nm의 150 MW)에서 열을 적용하여 얻을 수 있습니다. 레이저는 펄스 (~ 4Hz에서 약 200ms 펄스 폭)와 강도는 파리의 생존을 보장하기 위해 줄어 듭니다. 실험 패턴 학습 전통적인 패턴 학습 실험 (딜와 Heisenberg, 1995; 딜 외, 1993, 1995;. 리우 외, 2006;. 리우 외, 1998;. 리우 외, 1999;. 늑대와 Heisenberg, 1991) , 교대 방향 네 가지 검정, T - 모양의 패턴 (즉, 두 직립 두 거꾸로)이 균일하게 경기장 벽 (가로는 ψ = 40 °, 높이 θ = 40 °, 바 너비가 = 14 °로 패턴을 본에 간격 아르 파리의 위치에서). 컴퓨터 프로그램 4 가상 90 °의 quadrants에 경기장의 360 °를 분할의 센터는 패턴으로 표시됩니다. 파리는 편주 토크 (비행 시뮬레이터 상황)와 패턴의 각도 위치를 제어합니다. 훈련 기간 동안, 열 처벌은 전두엽 영상 분야에서 패턴 방향 중 하나의 모양과 인접 이루어집니다. 각 패턴의 강화항상 그룹 내에 같도록합니다. 테스트가 진행되는 동안, 더위가 영구적으로 꺼져 있고 파리의 패턴 환경 설정 기록됩니다. 컬러 학습 컬러 학습 이전에 설명된대로 수행됩니다 (Brembs 및 Heisenberg, 2000; Brembs 및 헴펄 드 이바라, 2006; Brembs과 위너, 2006, 늑대와 Heisenberg, 1997). 아레나 네개가 가상 90 °의 quadrants로 나누어의 중심은 네 동일한 수직 줄무늬 (폭 ψ = 14 °, 높이 θ = 40 °)으로 표시됩니다. 파리가 위의 T - 모양의 패턴에 대한 설명은 편주 토크와 함께 네 개의 동일한 줄무늬의 각도 위치를 제어합니다. 가상 사분 테두리 중 하나가 날아 앞에 지점을 통과 때마다 전체 경기장의 조명의 색상이 변경됩니다. 훈련 기간 동안, 열 처벌은 두 색상 중 하나를 파견 이루어집니다. 테스트 동안, 더위가 영구적으로 꺼져 있고 파리의 색상 환경 설정 기록됩니다. 물론, 색상은 복합 시설 (Brembs 및 Heisenberg, 2001)에 대한 패턴과 결합 수 있습니다. 편주 토크 학습 편주 토크 학습 (; Heisenberg와 늑대, 1993 Brembs 및 Heisenberg, 2000) 이전에 설명된대로 수행됩니다. 파리의 자연 편주 토크 범위는 왼쪽이나 오른쪽으로 돌리고 중 하나에 해당하는 약 &quot;왼쪽&quot;과 &quot;오른쪽&quot;도메인으로 나뉘어져 있습니다. 경기장 벽에는 패턴이 없습니다. 훈련 기간 동안, 열은 파리의 편주 토크 하나의 도메인에 언제든지 적용과 토크가 다른에 전달하면 꺼져 있습니다. 테스트 단계에서 열은 영구적으로 꺼져 있으며, 편주 토크 도메인의 비행의 선택이 기록됩니다. 종합 학습 (Brembs 및 Heisenberg, 2000) 이전에 설명한대로 종합 학습, 편주 토크 학습의 확장 기능입니다. 기본적으로, 편주 토크 학습 및 색상 학습 동등한 operant (편주 토크)와 predictors (색상) 클래식과 함께 실험에 결합됩니다. 훈련 동안 비행은 비행의 편주 토크는 형벌과 관련된 도메인에 전달 때마다 가열합니다. 파리뿐만 아니라 온도뿐만 아니라 아레나 착색이 (녹색에서 청색 또는 반대로) 변경, 편주 토크 도메인을 스위치 때마다. 따라서, 편주 토크 도메인 및 색상은 열을 동등 predictors 역할을합니다. 테스트 단계에서 열은 영구적으로 꺼져 있으며, 편주 토크 도메인 / 색상은 파리의 선택이 기록됩니다. 토론 이 실험 설정은 고급 유전자 모델 생물로 실험 환경을 통해 최상의 컨트롤을 결합합니다. 이 프레 젠 테이션에 설명되어있는 절차를 사용하여 행동 특성의 다양한 분자와 신경 생물학 토대가 포함하게 조사하며 이에 제한되지 않음 수 자발적인 행동 생성 메커니즘, operant 및 고전 시설, 패턴 인식, 컬러 비전이나 코스 관리 .

<ol>
	<li>Guo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila;+Dependence+on+age,+practice+and+diet.">Conditioned visual flight orientation in Drosophila; Dependence on age, practice and diet.</a> Learn. Mem.. 3, 49-59 (1996).</li><li>Goetz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optomotorische+Untersuchung+des+visuellen+Systems+einiger+Augenmutanten+der+Fruchtfliege+Drosophila.">Optomotorische Untersuchung des visuellen Systems einiger Augenmutanten der Fruchtfliege Drosophila.</a> Kybernetik. 2, 77-92 (1964).</li><li>Liu, L., Wolf, R., Ernst, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+in+Drosophila+visual+learning+requires+the+mushroom+bodies.">Context generalization in Drosophila visual learning requires the mushroom bodies.</a> Nature. 400, 753-756 (1999).</li><li>Brembs, B., Hempel de Ibarra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+parameters+support+generalization+and+discrimination+learning+in+Drosophila+at+the+flight+simulator.">Different parameters support generalization and discrimination learning in Drosophila at the flight simulator.</a> Learn. Mem. 13, 629-637 (2006).</li><li>Liu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+memory+traces+for+two+visual+features+in+the+Drosophila+brain.">Distinct memory traces for two visual features in the Drosophila brain.</a> Nature. 439, 551-556 (2006).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+organization+of+operant+behavior+as+revealed+in+Drosophila+flight+orientation.">Basic organization of operant behavior as revealed in Drosophila flight orientation.</a> J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 169, 699-705 (1991).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila+melanogaster+abolished+by+benzaldehyde.">Conditioned visual flight orientation in Drosophila melanogaster abolished by benzaldehyde.</a> Pharmacol Biochem Behav. 61, 349-355 (1998).</li><li>Dill, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+memory+without+shape+recognition.">Visual pattern memory without shape recognition.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 349, 143-152 (1995).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+recognition+in+Drosophila+involves+retinotopic+matching.">Visual pattern recognition in Drosophila involves retinotopic matching.</a> Nature. 365, 751-753 (1993).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+analysis+of+Drosophila+landmark+learning+in+the+flight+simulator.">Behavioral analysis of Drosophila landmark learning in the flight simulator.</a> Learn. Mem. 2, 152-160 (1995).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Space+from+Visual+Motion:+Turn+Integration+in+Tethered+Flying+Drosophila.">Visual Space from Visual Motion: Turn Integration in Tethered Flying Drosophila.</a> Learn. Mem. 4, 318-327 (1997).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+operant+and+the+classical+in+conditioned+orientation+in+Drosophila+melanogaster+at+the+flight+simulator.">The operant and the classical in conditioned orientation in Drosophila melanogaster at the flight simulator.</a> Learn. Mem. 7, 104-115 (2000).</li><li>Brembs, B., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+and+occasion+setting+in+Drosophila+visual+learning.">Context generalization and occasion setting in Drosophila visual learning.</a> Learn. Mem. 13, 618-628 (2006).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+with+compound+stimuli+in+Drosophila+melanogaster+in+the+flight+simulator.">Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator.</a> J Exp Biol. 204, 2849-2859 (2001).</li><li>Heisenberg, M., Wolf, 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+sensory-motor+link+in+motion-dependent+flight+control+of+flies.">The sensory-motor link in motion-dependent flight control of flies.</a> Rev. Oculomot. Res. 5, 265-283 (1993).</li></ol>

The authors have nothing to disclose.

이 실험 설정은 고급 유전자 모델 생물로 실험 환경을 통해 최상의 컨트롤을 결합합니다. 이 프레 젠 테이션에 설명되어있는 절차를 사용하여 행동 특성의 다양한 분자와 신경 생물학 토대가 포함하게 조사하며 이에 제한되지 않음 수 자발적인 행동 생성 메커니즘, operant 및 고전 시설, 패턴 인식, 컬러 비전이나 코스 관리 .

operant learning of drosophila at the torque meter

토크 미터에서 실험을 위해, 파리가 25 표준 플라이 매체에 보관됩니다 ° C와 12hr light/12hr 어두운 정권 60 %의 습도. 표준 번식 정권은 적절한 애벌레 밀도와 연령 일치하는 동료를 보장합니다. 콜드 - anesthetized 파리는 삼각형 모양의 후크 실험 전날에 머리와 흉부와 함께 붙어 있습니다. 클램프를 통해 토크 미터에 부착된, 파리의 의도 비행 연습은 그 수직 바디 축 주위의 각운 동량으로 측정됩니다. 비행기가 정지 비행을 수행하는 원통형 파노라마의 중앙에 배치됩니다. 디지털 변환기 카드에 아날로그는 나중에 분석을위한 추적을 저장하는 컴퓨터에 편주 토크 신호를 피드. 컴퓨터가 이러한 자극 및 편주 토크 추적 사이의 피드백 루프를 폐쇄하여 파리의 통제 가지고 수있는 자극의 다양한 제어합니다. 처벌은 조정 적외선 레이저에서 열을 적용하여 얻을 수 있습니다.

Watch this Scientific Journal Video about Operant Learning of Drosophila at the Torque Meter at JoVE.com

Operant Learning of Drosophila at the Torque Meter

토크 미터와 닿는 Drosophila의 편주 토크를 측정하는 것은 실험 동물의 자극 상황의 신경학 절묘한 컨트롤을 수 있습니다. 함께 과일 파리에서 사용할 수있는 고유한 유전자 도구를 사용하여,이 패러다임은 신경 생물학 연구의 광범위한 사용됩니다.

토크 미터에서 Drosophila의 Operant 학습

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

operant-learning-of-drosophila-at-the-torque-meter

Research

JoVE Journal

Biology

13.4K 조회수.  Free University of Berlin. 토크 미터와 닿는 Drosophila의 편주 토크를 측정하는 것은 실험 동물의 자극 상황의 신경학 절묘한 컨트롤을 수 있습니다. 함께 과일 파리에서 사용할 수있는 고유한 유전자 도구를 사용하여,이 패러다임은 신경 생물학 연구의 광범위한 사용됩니다.

비디오: Operant Learning of Drosophila at the Torque Meter

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

칼슘 지표로 로딩 Drosophila 신경 터미널

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

연구

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 ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

의 형광 라벨링 Drosophila 심장 구조

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

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 ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

의 양적 비교 CIS</em트렌스 제닉에서> - 규제 요소 (CRE) 활동 Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

에서 상동 재조합 구문을 Recombineering<em&gt; 초파리</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

해부와의 설치<em&gt; 초파리</em&gt; 번데기 아이 디스크

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...