Name: use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule
Uploaded: 2015-11-25T15:00:00.000+00:00
Duration: 13 min 30 s
Description: Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

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The authors have nothing to disclose.

함께 특정한 이온 전극 램지 분석의 사용은, 절연 곤충 Malpighian (신장)에서 유체 세뇨관 분비 속도 및 이온 플럭스를 측정 할 수있다. 스물 이상의 세관은 체외 microperfused 세뇨관에서 개별의 분석법에 비해 더 높은 처리량을 가능하게 한 번에 분석 될 수있다. 또한, 이온 전극을 특정 제 2 장치로의 유체의 소량의 전사에 도입 될 수있는 오류를 제한 시츄 분비 유체 내 이온 농도?...

<html> 신장 상피 이온 수송은 유기체의 iono- 및 삼투의 기초가. 초파리 melanogaster의 Malpighian (신장) 세관 상피 이온 수송의 분자 메커니즘을 연구 할 수있는 최고의 기회를 제공합니다. 이것은 생리 학적 연구에의 신장 세뇨관의 접근성과 짝을 초파리의 강력한 유전학의 조합 때문이다. 기술 1을 개척 연구자의 ​​이름을 따서 명명 램지 분석, 격리 Malpighian 세관에서 유체 분비 속도를 측정하고, 다우와 동료 2에 의해 1994 년에 초파리 년에 설립되었습니다. 이것은 유체 분비 조절 셀 고유 신호 전달 경로를 정의하는, 초파리 등을 GAL4-UAS 시스템 3,4- 유전 도구를 사용하여 추가 연구를위한 기반을 마련. 예는 많은 다른 6,7 사이에 펩티드 호르몬 (5)에 응답하여 칼슘 신호를 포함한다. 비행 세뇨관의 주요 부분에서 염화칼륨이 풍부한 액체의 분비를 통해 발생에 ve_content &quot;&gt; 유전자 기술 및 고전 생리 학적 연구의 조합 뇨 생성을 도시하고있다.이 주로 양이온 평행 transepithelial 분비 통해 이루어 K는 +뿐만 아니라 나트륨 +는 주체 세포, 및 CL를 통해 -. 성상 세포 8-12 내지 분비 별도로 transepithelial K + 및 나트륨 + 플럭스를 측정하는 능력은 유체 분비의 측정보다 반송기구의 상세한 특성화를 허용 홀로. 예를 들어, 자극되지 초파리 세뇨관에서, 나 +가 / K + -ATPase 억제제 인 ouabain 유체 분비 2에 영향을주지 주체 세포 내로의 흡수량은 유기 음이온 수송 체 억제제 타우로 콜산 (13)에 의해 억제 된 경우에도. 그러나, 린톤 및 오도​​넬은 ouabain과는 탈분극 것을 보여 주었다기저 막 잠재력, 그리고 나 + 플럭스 (9)을 증가시킨다. 대표 결과에 도시 된 바와 같이, 우리는 이러한 발견을 복제하고, 14 병용 감소 K + 플럭스가 있음을 보여 주었다; 증가 나 + 플럭스 및 K 감소 + 플럭스 분비없는 순 변화의 결과로 유체 분비에 반대하는 효과를 가지고있다. . 따라서, &quot;ouabain과 모순&quot;, 즉 두 가지 해결책이 존재하며, 초파리 세뇨관 유체 분비에 영향을주지 ouabain과 초기 관찰 : 제 자극 세뇨관에서 유체 분비 ouabain과 효과는 명백하지 않다 인한 유기 음이온 수송 (13)에 의해 그 흡수; 둘째, 자극되지 세관에서, ouabain과는 (대표 결과와 심판을 참조하십시오. 9) 유체 분비없는 순 변화의 결과로, transepithelial 나 +와 K + 플럭스에 영향을 반대하고있다. 따라서, 나 +의 주요 역할 / K + -ATP비자극 세뇨관에서 ASE는 나트륨 +는 기저 외측 막에 걸쳐 전송 프로세스 -coupled위한 유리한 농도 기울기를 생성하는 세포 나 + 농도를 낮출 수있다. 실제로, 별도로 나 +와 K + 플럭스를 측정하여, 우리는 세관이 비행 나트륨 - 칼륨 - 2 - 염화 송체 (NKCC)를 부족하지 ouabain과 첨가 한 후 더 이상의 감소, transepithelial K + 플럭스 감소했으며, transepithelial의 변화가 있음을 보여 주었다 나 + 14 플럭스. 이러한 연구 결과는 NKCC를 통해 세포를 입력 나 +가 나 + / K + -ATPase를 통해 재활용 우리의 결론을지지했다. 다른 예에서, Ianowski는 외. mM의 10 mM 내지 6 욕 K + 농도를 낮추면 유체 분비없는 순 변화와 Rhodnius의 prolixus에서 세뇨관에서 transepithelial의 K + 플럭스 증가 transepithelial 나 + 플럭스 감소 관찰 <sup&gt; 15. 애벌레 세관에서 나 + 플럭스 및 K + 플럭스에 차등 효과는 염분 (17) 양육에 대한 응답으로 다양한 소금 다이어트 (16)와 두 모기 종에 대한 응답으로 초파리 세관에서 관찰되었다. 램지 분석 준비 transepithelial 이온 플럭스의 측정에서 가장 큰 도전은 분비 된 유체 내 이온 농도의 판정이다. 이 문제는 불꽃 photometery (18), 방사성 이온 (19)의 사용, 전자 프로브 파장 분산 분광기 (20)를 포함하여 다양한 솔루션을 만났다되었습니다. 이러한 기술은 이온 농도의 측정을위한 도구로 분비되는 액체 방울의 전송을 필요로한다. 비자극 초파리 세뇨관 분비 유체의 부피가 작기 때문에, 일반적으로 ~ 0.5 NL / 분, 이것은 기술적 도전을 제기하고 분비 된 유체의 일부가 오류가있는 경우에도 소개이전에 잃었다. 대조적으로, 특정 이온 전극의 사용은 인 시츄 (이온 농도가 계산 될 수있는) 이온 활성의 측정을 허용한다. 현재 프로토콜은 K + 이온 운반체 (21)로 발리 노마 이신 (Valinomycin) 사용 Rhodnius의 세뇨관 걸쳐 transepithelial의 K + 플럭스를 측정하도록 Maddrell 동료가 사용하는 각색 및 또한 4- 급의 -butylcalix [4] 아렌 - 테트라 아세트산의 사용을 설명했다 테트라 에틸 에스테르 계 나은 등 Messerli 특징으로 특이 이온 특정 전극을 +. 등. (22). 이온 특정 전극 성인 9,23과 애벌레 (16) 초파리에서 램지 분석에 Malpighian 세관에 의해 분비되는 유체의 이온 농도를 측정하는 데 사용 된, 뉴질랜드 알파인 웨타 (Hemideina 마오리어) (24) 모기 (17). 여기서는 상세히 램지의 사용 등을 서술유체 분비 초파리 melanogaster의 발 Malpighian 세뇨관의 속도뿐만 아니라 분비 유체 내 K + 및 나트륨 +의 농도를 결정하는 이온 고유의 전극을 사용하므로 transepithelial 이온 플럭스의 계산을 측정하기 말한다. 분석의 개요는도 1에 제공된다. <img alt="그림 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Malpighian 세관의 그림 1. 도식과 이온 농도를 측정하기 위해 이온 특정 전극의 사용과 램지 분석. 이 그림은 램지 분석에 대한 설정을 보여줍니다. (A) 각 플라이는 네 세뇨관, 전방 세뇨관의 쌍 및 후방 세뇨관의 쌍에 의해 둘러싸인 체액 복강 그 플로트있다. 각 쌍에서 두 세관은 중장 hindgu과의 교차점에 뇨를 비운다 요관에서 합류티. 세관은 블라인드 종료됩니다. 소변 (빨간색으로 표시) 유체 분비 주요 세그먼트에 의해 발생하고, 장에 요관으로 밖으로 흐른다. 절개 후 세관 쌍 요관을 직감에 병행하여 해리된다. (B)은 세뇨관의 쌍은 분석 접시의 웰 내에서 입욕 식염수 방울로 전달된다. 여기에 언급 된 두 세뇨관의 일 &quot;앵커 세관은,&quot;금속 핀 감싸 불활성이다. 다른 세관은 분비의 가느 다란 관이다. 초기 세그먼트 (유체 분비하지 않는)과 분비 세관의 주요 부분은 목욕 식염수의 액적 내에 남아있다. 생체 내에서 발생하는 것에 이온과 목욕 식염수에서 주 세그먼트의 세관 루멘에 물 이동, 그리고, 요관으로 이동합니다. 낮은 세그먼트 (파란색) 목욕 식염수 때문에 불활성 밖에 있습니다. 요관이 절단되기 때문에, 유체는 분비 요관의 절단 단부로부터 액적으로 나온다. 티그 분비는 계속 유체 액적 시간 경과 확대 분비, 그 직경은 접안 마이크로 미터를 사용하여 측정된다. 미네랄 오일 층은 분비 된 유체의 증발을 방지한다. 참조 및 이온 특정 전극이 분비 유체의 이온 농도를 측정한다. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td><td>May be purchased from multiple distributors</td></tr><tr><td>Petri dish, polystyrene, 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Specific brand is not important</td></tr><tr><td>Petri dish, polystyrene, 35 mm x 10 mm</td><td>Corning Life Sciences</td><td>Fisher 08-757-100A</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Blades #1</td><td>Fine Science Tools</td><td>10011-00</td><td>Specific brand is not important; use appropriate sharps precautions</td></tr><tr><td>Needle, 30 G x 1/2</td><td>Becton Dickinson</td><td>305106</td><td>Use appropriate sharps precautions</td></tr><tr><td>Minutien pins, black anodized, 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Stereomicroscope with ocular micrometer</td><td>Nikon</td><td>SMZ800</td><td>Specific brand is not important; this is given as an example</td></tr><tr><td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td><td>Hobby shop</td><td></td><td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td></tr><tr><td>Glass cutting tools (glass cutter, glass cutting pliers)</td><td>Hobby shop</td><td></td><td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td></tr><tr><td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0042</td><td></td></tr><tr><td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0044</td><td></td></tr><tr><td>Nitric acid, 70%</td><td>Sigma</td><td>438073</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td></tr><tr><td>Cimarec 7 in x 7 in hotplate</td><td>Fisher</td><td>11675911Q</td><td>Specific brand is not important; caution when heated</td></tr><tr><td>Selectophore dichlorodimethylsilane</td><td>Sigma</td><td>40136-1ML</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Two-step vertical pipet puller</td><td>Narishige</td><td>PC-10</td><td>Other pipet pullers can be used; this is given as an example</td></tr><tr><td>Glass petri dish, 150 mm diameter x 15 mm height</td><td>Fisher</td><td>08-748E</td><td>Specific brand is not important; only one dish needed</td></tr><tr><td>World Precision Instruments E210 1 mm micropipette storage jar</td><td>Fisher</td><td>50-821-852</td><td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td></tr><tr><td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td><td>Fisher</td><td>S161-500</td><td>Indicating silica useful for determining whether silica gel retains desiccating ability</td></tr><tr><td>World Precision Instruments MicroFil, 34G</td><td>Fisher</td><td>50-821-914</td><td>May be available from other distributors.</td></tr><tr><td>1 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309659</td><td>May be available from other distributors.</td></tr><tr><td>3 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309657</td><td>May be available from other distributors.</td></tr><tr><td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td><td>Cole-Parmer</td><td>UX-30600-02</td><td>Specific brand is not important</td></tr><tr><td>Female Luer Locking Connector</td><td>4 Medical Solutions</td><td>ADC 9873-10</td><td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td></tr><tr><td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td><td>Fisher</td><td>14-179-110</td><td>Specific brand is not important</td></tr><tr><td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td><td>Fisher</td><td>14171208</td><td>Specific brand is not important</td></tr><tr><td>Modeling clay</td><td></td><td></td><td>Specific brand is not important</td></tr><tr><td>Selectophore potassium ionophore I, cocktail B</td><td>Sigma</td><td>99373</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Selectophore sodium ionophore X</td><td>Sigma</td><td>71747</td><td>Sodium ionophore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td></tr><tr><td>Selectophore 2-nitrophenyl octyl ether</td><td>Sigma</td><td>73732</td><td></td></tr><tr><td>Selectophore sodium tetraphenylborate</td><td>Sigma</td><td>72018</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Schneider&#039;s Drosophila medium</td><td>Life Technologies</td><td>21720024</td><td></td></tr><tr><td>High impedance electrometer</td><td>World Precision Instruments</td><td>FD223a</td><td></td></tr><tr><td>Microelectrode holder 1 mm with 45&amp;deg; body, vented, with handle</td><td>Warner Instruments</td><td>64-1051</td><td></td></tr><tr><td>Microelectrode holder 1 mm with straight body, vented</td><td>Warner Instruments</td><td>64-1007</td><td></td></tr><tr><td>Silver wire</td><td>Warner Instruments</td><td>64-1318</td><td></td></tr><tr><td>Micromanipulators, pair</td><td>Leitz</td><td></td><td>Various brands/models will work; this is an example</td></tr><tr><td>Faraday cage</td><td>Technical Manufacturing Corporation</td><td>81-334-03</td><td>This is an example; any Faraday cage will work</td></tr><tr><td>Single gooseneck fiberoptic light</td><td>Nikon</td><td></td><td>Specific brand is not important</td></tr><tr><td>mineral oil</td><td>Fisher</td><td>BP-2629</td><td>Specific brand is not important</td></tr><tr><td>forceps, Dumont #5 with Biologie tip</td><td>Fine Science Tool</td><td>11295-10</td><td>May be available from other distributors.</td></tr></tbody></table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

신장 상피 이온 수송의 변조는 생물체가 외부 환경 변화에 직면 이온과 삼투압 항상성을 유지할 수 있습니다. 초파리 melanogaster의 Malpighian (신장) 가느 다란 관으로 인해이 생물 및 생리 학적 연구에의 신장 세뇨관의 접근성의 강력한 유전학, 상피 이온 수송의 분자 메커니즘을 연구 할 수있는 최고의 기회를 제공합니다. 여기서는 유체 분비 나트륨 및 칼륨 농도를 측정하는 이온 고유의 전극을 이용하여, 절연 플라이 신 세뇨관에서 유체 분비 속도를 측정하기 램지 분석의 사용을 기술한다. 이 분석은 이온 농도를 측정하는 별도의 장치에 분비 된 유체를 전송할 필요없이, 20 시간에 세관의 transepithelial 유체와 이온 플럭스 ~ 연구를 허용한다. 유전자는 별개의 세뇨관 수송 공정에서 특정 유전자의 역할을 평가하기 위해 분석 될 수있다. 또한, Bathing 식염수 첨가 화학적 특성, 또는 약물 또는 호르몬의 효과를 조사하기 위해 수정 될 수있다. 요약하면,이 기술은 이러한 반송기구의 상피 이온 초파리 세뇨관 운송뿐만 아니라, 규정의 기본 메커니즘의 분자 특성화를 허용한다.

도 7 및도 8은 유전 학적 및 약리학 적으로 별개의 K + 및 나트륨 + 플럭스만으로는 유체 분비 속도를 측정함으로써 캡쳐되지 않은 정보를 구분할 수있는 K + 및 나트륨 + 농도를 측정하기 위하여 이온 - 특정 전극 램지 분석의 사용을 보여준다.도를 나트륨 + 플럭스 (도 7b)이 변경되지 않은 상태 NKCC에서 동형 널 돌연변이...

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

이 프로토콜은 초파리에서 격리 Malpighian (신장) 세관에서 유체 분비 속도를 측정 할 수있는 램지 분석의 사용을 설명합니다. 또한, 이온 전극의 특정 사용 transepithelial 이온 플럭스의 계산을 가능하게 분비 유체 나트륨 및 칼륨 농도를 측정하기 위해, 설명한다.

램지 분석의 사용은 유체 분비 및 이온 플럭스 요금을 측정하는<em&gt; 초파리</em&gt; Malpighian하는 Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

use-ramsay-assay-to-measure-fluid-secretion-ion-flux-rates-drosophila

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Biology

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

11.8K 조회수.  University of Texas Southwestern Medical Center.  이 프로토콜은 초파리에서 격리 Malpighian (신장) 세관에서 유체 분비 속도를 측정 할 수있는 램지 분석의 사용을 설명합니다. 또한, 이온 전극의 특정 사용 transepithelial 이온 플럭스의 계산을 가능하게 분비 유체 나트륨 및 칼륨 농도를 측정하기 위해, 설명한다. 

비디오: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

확인된 뉴런과 먹이 행동에서 칼슘 신호의 동시 녹화 Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

연구

신경과학

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

분석의 방법 Drosophila 행동

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

초파리 멜라노가스터의 혈액-뇌 장벽 무결성에 대한 분석

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

발생학

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

의 생리적 반응을 측정<em&gt; 초파리</em&gt; 라이브 칼슘 이미징을 사용하여 지질 페로몬에 감각 뉴런

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

면역학 및 감염병학

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

모세관 공급 분석은 식품 섭취를 측정<em&gt; 노랑 초파리</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

드로소필라 애벌레를 사용하여 열유전학 신경 화면에서 다량 영양소 섭취의 정량화

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

에서 애벌레 활동의 측정<em&gt; 초파리</em&gt; 활동 모니터

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

생물학

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

형광 유전자 인코딩 ph 지시자 초파리 melanogaster 말 관 Epithelia에 광학 정량화의 세포내 pH

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

성인 모기에 있는 배설 시스템의 Neuropeptides 및 그밖 규제기관의 활동을 공부하기

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ

Source: Rossano, A. J. and Romero, M. F. <a href="https://www.jove.com/video/55698/" target="_blank">Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator</a>. J. Vis. Exp. (2017).
This article describes the dissection of the anterior Malpighian tubules (MT) from a female Drosophila fly and an example protocol from Rossano et al. (2017) in which the MTs are prepared for live imaging in a perfusion chamber.

성충 파리의 Malpighian Tubule Dissection: Isolating an Ossmoregulatory and Excretory Organ(성충 파리의 Malpighian Tubule Dissection: Isolating an Osmoregulatory and Excretory Organ)

램지 분석의 사용은 유체 분비 및 이온 플럭스 요금을 측정하는

요약

더 많은 비디오 탐색

이 비디오의 챕터

확인된 뉴런과 먹이 행동에서 칼슘 신호의 동시 녹화 Drosophila melanogaster

분석의 방법 Drosophila 행동

초파리 멜라노가스터의 혈액-뇌 장벽 무결성에 대한 분석

의 생리적 반응을 측정

모세관 공급 분석은 식품 섭취를 측정

드로소필라 애벌레를 사용하여 열유전학 신경 화면에서 다량 영양소 섭취의 정량화

에서 애벌레 활동의 측정

형광 유전자 인코딩 ph 지시자 초파리 melanogaster 말 관 Epithelia에 광학 정량화의 세포내 pH

성인 모기에 있는 배설 시스템의 Neuropeptides 및 그밖 규제기관의 활동을 공부하기

성충 파리의 Malpighian Tubule Dissection: Isolating an Ossmoregulatory and Excretory Organ(성충 파리의 Malpighian Tubule Dissection: Isolating an Osmoregulatory and Excretory Organ)