우리는 애벌레 크롤링 데이터를 생성하기 위해 Astha Maltare 감사드립니다. 우리는 원고에 그의 의견을 주심에 닥터 니콜라스 Lanson 주니어 감사드립니다. 이 작품은 존스 홉킨스에​​서 루게릭병에 대한 로버트 패커드 센터 (UBP까지)와 루게릭병 협회 (UBP)에 의해 지원되었다, 그리고 정신 건강의 국립 연구소 (CDN)에서 R01MH083689.

A. 애벌레의 크롤링 분석 1. 애벌레 컬렉션 <ol><li> 파리 (10-15 10-15 남성 + 여성)의 8온스 병을 설정합니다. </li><li> 파리 그 다음 24 시간 동안 계란, 파리의 명확한 한병을 덮어두고. (새 병으로 성인 전송하고 필요에 따라 반복). </li><li> 3-4일위한 병 품어도, 세번째도 instar의 애벌레가 보일 때까지. </li><li> 애벌레와 병에 20 %의 자당 100 ML하고 20 분 동안 앉아 보자 - 50을 추가합니다. 애벌레는 정상에 떠 있습니다. </li><li> 팁과 25 ML serological 피펫을 사용하여 수집 유충 잘라, 그리고 메쉬 바구니로 곳이 네요. </li><li> 두 번 deionized H 2 O를 함께 메쉬 바구니에 애벌레 씻으십시오 애벌레는 이제 실험을위한 준비가되어 있습니다. </li></ol> 2. 약물과 애벌레를 치료하려면 <ol><li> 솔루션 5 % 자당 + 약물을 포함하는 5 ML의 비커에 유충의 원하는 숫자를 수송 브러쉬를 사용합니다. </li><li> 적어도 15 분 동안 애벌레 피드 구.</li><li> 메쉬 바구니로 약물 치료 애벌레를 붓고 린스. 그들은 이제 사용할 준비가 된 것입니다. </li></ol> 3. 전위의 분석 (총 거리를 측정하는 여행을하거나 신체 벽의 수축)을 <ol><li> 개별 유충의 수송을 브러쉬를 사용하여 <ol><li> 0.2 cm 2 그리드와 그래프 용지 위에 2퍼센트 아가로 오스를 (이전에 부어 및 강화에 허용)이 들어 15cm 배양 접시. <ol><li> 일분이 깨지는 그리드 라인의 숫자를 세어보세요. </li></ol></li><li> 묽은 효모 붙여넣기 솔루션을 포함하는 유리 해부 요리의 음. <ol><li> 해부 현미경 하에서 관찰하면서 일분 후에 연동 수축을 (후부 움직임 = 1 수축에 대한 전체 앞부분) 백작. </li></ol></li></ol></li><li> 애벌레의 원하는 숫자를 계산 때까지 반복합니다. </li></ol> B. 신속한 마이너스 geotaxis (RING) 프로토콜을 반복 이 분석은 원래 드되었습니다Gargano 외 5에 scribed. <ol><li> 표준 유리병이 포함된 식품 (또는 식품 + 약물)에 빛을 CO 2 anesthetization과 장소 하에서 새롭게 등장 성인 남자의 파리를 수집합니다. </li><li> 실온에서 파리를 유지 (벤치 위에. ~ 22 ° C) CO 2로부터 복구 (및 정상 상태 약물 수준의 축적 해당되는 경우)를 허용하기 위해 2-3 일동안은. </li><li> 준비된 폴리스티렌 유리병에 anesthetizing없이 25 파리에 대해 전송합니다. </li><li> 링 장치 (그림 1)에 파리와 튜브를 조립. </li><li> 파리는 15~20분위한 흐트러지지 환경에 적응하도록 허용합니다. </li><li> 이 시간 장소 디지털 카메라 중에 ~기구 앞에서 1m가 (플랫폼 튜브의 중간 높이에서 렌즈의 중심을 정렬하기 위해 필요한 경우에), 집중하며기구를 향해 카메라를 확대, 그리고 타이머를 설정 3.0 초. </li><li> 조심스럽게 dist로 왼손으로 링 장치를 확보하므로 같이하지 받아urb는 파리, 그리고 오른손으로 타이머를 누르고 있습니다. </li><li> 급격히 수돗물은 튜브의 바닥에 모든 파리들을 허물고 어려운 충분 있도록, 벤치의 표면에 세 번 장치를 누릅니다. </li><li> 동시에 세 번째 탭 완성과 함께, 삼초 카운트 다운 타이머를 시작합니다. </li><li> 삼초에서 사진을 찍었어요. </li><li> 1 분 타이머를 재설정하고 시작합니다. 이 시간 동안 장치에 카메라 초점을 재설정하고, 3 초 동안 타이머의 다른 채널을 설정합니다. </li><li> 일분 후, 반복 1.7-1.10 단계를 </li><li> 5-6 시련 총 후에, 컴퓨터에 이미지를 업로드하고 여십시오 좋아하는 이미지 뷰어를 사용하며, 평균 높이가 각 유리병에 대해 올랐다 득점. </li><li> 올라왔다는 평균 높이를 비교하여 서로 다른 그룹에 대한 통계 분석을 수행합니다. </li></ol> C.의 구애와 번식 분석 <ol><li> 아침 일찍, fli의 명확한 잘 생산 병초밖에 사용할 수 있습니다. </li><li> 하루 코스 (매 3-4시간)이 지남에 새롭게 등장 성적으로 순진한 남성과 여성을 수집합니다 : <ol><li> 매체와 튜브 또는 튜브에 개별적으로 남성을 놓습니다. </li><li> 유리병 / 튜브마다 함께 5-6 암컷를 놓습니다. </li><li> 5 일째 어두운 12 시간 조명 / 아래 25 ° C에서 수집된 파리를 분리. </li><li> 교배 바퀴 챔버에 한 명, 여자를 전송합니다. </li><li> 교배 바퀴 챔버로 남자 한을 전송합니다. </li><li> 다음 동작을위한 해부 현미경 하에서 쌍을 관찰 : <ol><li> 오리 엔테이션 (여성에 대한 남성 orients) </li><li> (남자가 여자를 도청) 태핑 </li><li> 날개의 노래 (수컷은 확장 하나의 날개를 진동) </li><li> (남성 찾고 있는데, 여성 성기) 빠는데 </li><li> 컬링 (남성 자체에 따른 복부를 곱슬머리) </li><li> (여성을 마운트하는 동안 활동을 컬링) 결합 시도 </li></ol></li></ol></li><li> 1 관찰0분 또는 성공적인 결합까지, 각 문제가 발생되는 시간을 지적 (지연), 구애 결합까지 동작 (구애 지수를 계산하는)뿐만 아니라 성공적으로 특정 동작을 수행 쌍 개수에 종사하는 총 시간 ( 주파수). 야생 타입 쌍의 100 %는 일반적으로 5 분 이내에 짝짓기 것입니다. </li><li> 계산 시간을 나누어 구애 지수 (CI)는 결합까지 총 시간으로 나눈 구애에 보냈다. 야생 형 쌍 이것은 보통 0.6-0.8 사이의 범위. </li></ol> D. 주재원 결과 분석을 크롤링 일반 야생 형 유충은 ~ 3cm / 분, 그리고 전시 ~ 잠시 후에 40-50 바디 벽의 수축을 돌아다니는 것입니다. Google은 최근 애벌레 기어 결함, 낮은 수명과 장애 6 등산 성인을 보여줍니다 FUS / TLS 관련 루게릭병의 Drosophila 모델을 개발했습니다. 우리는 표현 O에게 대상모터 뉴런 (OK-371 gal4 드라이버) 및 F 야생 유형 및 FUS / TLS의 돌연변이 형태는 애벌레 크롤링 분석을 수행. 아래 그림과 같이 야생 형 FUS 표현 약 6cm로 애벌레 크롤링 능력을 감소 반면 야생 형 유충은 최대 12cm까지 크롤 링합니다. FUS / TLS에서 루게릭병 유발 돌연변이의 R521C을 표현하는 동물은 약 ​​1cm / 분을 크롤 링, 그들의 크롤 링 운동에있어서 매우 심각한 장애 (그림 1)을 보여줍니다. 부정적 geotaxis 링 분석 영 야생 형 파리 성충은 3 번째 기간에 ~ 4~5센티미터 (시간이 주어진 동안 주어진 평균 높이를 정의하기 위해 다른 변종이나 활동 수준을 수용할 수있는 3 초에서 조절할 수의 평균 등산 높이가 있어야 / 치료)를 변형. 바닥에 남아있는 파리는 값 0을 할당됩니다. 그것은 그 후 높이를 측정하기위한 각 플라이의 위치를​​ 결정하기 어려워진다 있기 때문에 유리병 당 25 개 이상의 파리를 사용할 것을 권고하지 않습니다. NO의 desensitization는 우리가 고용했다는 떨어져 일분 간격의 6 연속 시련을 올려다 관찰되었습니다. 그것은 사용 튜브에 배치 새로운 파리 신선한 튜브에서와 동일한 범위까지 올라갈하지 않으므로 수집되는 데이터의 초기 집합 후에이 분석의 폴리스티렌 테스트 튜브를 다시 사용할 수없는 중요합니다. <img alt="그림 1" src="/files/ftp_upload/3795/3795fig1.jpg" /> 그림 1. 링 분석을위한 설정. 집중 장치를 향해 카메라를 확대; 디지털 카메라는 폴리스티렌 튜브 안에 파리를 포함하는기구 앞에서 ~ 1 미터에 위치 및 3.0 초로 타이머를 설정합니다. <img alt="그림 2" src="/files/ftp_upload/3795/3795fig2.jpg" /> 사용 애벌레 크롤링 분석에서 그림 2. 대표 데이터 ectopically UAS-FUS WT을 표현하고, 모터 신경 세포 드라이버 (OK371-gal4)의 통제하에 UAS-FUS의 R521C 버려요. <p클래스 = &quot;jove_content&quot;&gt;

<ol>
	<li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila+melanogaster+and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery.</a> Pharmacol. Rev. 63 (2), 411-436 (2011).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+Parkinson's+disease.">A Drosophila model of Parkinson's disease.</a> Nature Mar. 23 (6776), 394-398 (2000).</li><li>Auluck, P. K., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+prevention+of+Parkinson+disease+in+Drosophila.">Pharmacological prevention of Parkinson disease in Drosophila.</a> Nat. Med. 8 (11), 1185-1186 (2000).</li><li>Whitworth, A. J., Theodore, D. A., Greene, J. C., Benes, H., Wes, P. D., Pallanck, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutathione+Stransferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione Stransferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (22), 8024-8029 (2005).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40 (5), 386-395 (2005).</li><li>Lanson, N. A., Maltare, A., King, H., Smith, R., Kim, J. H., Taylor, J. P., Lloyd, T. E., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+FUS-related+neurodegeneration+reveals+genetic+interaction+between+FUS+and+TDP-43.">A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43.</a> Hum. Mol. Genet. 20 (13), 2510-2523 (2011).</li><li>Batlevi, Y., Martin, D. N., Pandey, U. B., Simon, C. R., Powers, C. M., Taylor, J. P., Baehrecke, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+light+chain+1+is+required+for+autophagy,+protein+clearance,+and+cell+death+in+Drosophila.">Dynein light chain 1 is required for autophagy, protein clearance, and cell death in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (2), 742-747 (2010).</li><li>Sang, T. K., Chang, H. Y., Lawless, G. M., Ratnaparkhi, A., Mee, L., Ackerson, L. C., Maidment, N. T., Krantz, D. E., Jackson, G. 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+Drosophila+model+of+mutant+human+parkin-induced+toxicity+demonstrates+selective+loss+of+dopaminergic+neurons+and+dependence+on+cellular+dopamine.">A Drosophila model of mutant human parkin-induced toxicity demonstrates selective loss of dopaminergic neurons and dependence on cellular dopamine.</a> J. Neurosci. 27 (5), 981-992 (2007).</li><li>Stacey, S. M., Muraro, N. I., Peco, E., Labb&#233;, A., Thomas, G. B., Baines, R. A., van Meyel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+glial+glutamate+transporter+Eaat1+is+regulated+by+fringe-mediated+notch+signaling+and+is+essential+for+larval+locomotion.">Drosophila glial glutamate transporter Eaat1 is regulated by fringe-mediated notch signaling and is essential for larval locomotion.</a> J. Neurosci. 30 (43), 14446-14457 (2010).</li><li>Repnikova, E., Koles, K., Nakamura, M., Pitts, J., Li, H., Ambavane, A., Zoran, M. J., Panin, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialyltransferase+regulates+nervous+system+function+in+Drosophila.">Sialyltransferase regulates nervous system function in Drosophila.</a> J. Neurosci. 30 (18), 6466-6476 (2010).</li><li>Repnikova, E., Koles, K., Nakamura, M., Pitts, J., Li, H., Ambavane, A., Zoran, M. J., Panin, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialyltransferase+regulates+nervous+system+function+in+Drosophila.">Sialyltransferase regulates nervous system function in Drosophila.</a> J. Neurosci. 30 (18), 6466-6476 (2010).</li><li>Nedelsky, N. B., Pennuto, M., Smith, R. B., Palazzolo, I., Moore, J., Nie, Z., Neale, G., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+functions+of+the+androgen+receptor+are+essential+to+pathogenesis+in+a+Drosophila+model+of+spinobulbar+muscular+atrophy.">Native functions of the androgen receptor are essential to pathogenesis in a Drosophila model of spinobulbar muscular atrophy.</a> Neuron. 67 (6), 936-952 (2010).</li><li>Lorenzo, D. N., Li, M. G., Mische, S. E., Armbrust, K. R., Ranum, L. P., Hays, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrin+mutations+that+cause+spinocerebellar+ataxia+type+5+impair+axonal+transport+and+induce+neurodegeneration+in+Drosophila.">Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Drosophila.</a> J. Cell Biol. 189 (1), 143-158 (2010).</li><li>Wang, J. W., Brent, J. R., Tomlinson, A., Shneider, N. A., 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=The+ALS-associated+proteins+FUS+and+TDP-43+function+together+to+affect+Drosophila+locomotion+and+life+span.">The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span.</a> J. Clin. Invest. , (2011).</li><li>Choi, J. K., Jeon, Y. C., Lee, D. W., Oh, J. M., Lee, H. P., Jeong, B. H., Carp, R. I., Koh, Y. H., Kim, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+GSS+syndrome+suggests+defects+in+active+zones+are+responsible+for+pathogenesis+of+GSS+syndrome.">A Drosophila model of GSS syndrome suggests defects in active zones are responsible for pathogenesis of GSS syndrome.</a> Hum. Mol. Genet. 19 (22), 4474-4489 (2010).</li><li>Ruan, H., 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=Social+interaction-mediated+lifespan+extension+of+Drosophila+Cu/Zn+superoxide+dismutase+mutants.">Social interaction-mediated lifespan extension of Drosophila Cu/Zn superoxide dismutase mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (21), (2008).</li><li>Slawson, J. B., Kim, E. Z., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+video+tracking+of+locomotion+in+adult+Drosophila+melanogaster.">High-resolution video tracking of locomotion in adult Drosophila melanogaster.</a> J. Vis. Exp. (24), (2009).</li><li>Becnel, J., Johnson, O., Luo, J., N&#228;ssel, D. R., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serotonin+5-HT7+Dro+receptor+is+expressed+in+the+brain+of+Drosophila,+and+is+essential+for+normal+courtship+and+mating.">The serotonin 5-HT7 Dro receptor is expressed in the brain of Drosophila, and is essential for normal courtship and mating.</a> PLoS One. 6 (6), e20800 (2011).</li><li>Johnson, O., Becnel, J., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+5-HT(2)+and+5-HT(1A)-like+receptors+differentially+modulate+aggressive+behaviors+in+Drosophila+melanoga-+ster.">Serotonin 5-HT(2) and 5-HT(1A)-like receptors differentially modulate aggressive behaviors in Drosophila melanoga- ster.</a> Neuroscience. 158 (2), 1292-1300 (2009).</li><li>Bastock, M., Manning, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Courtship+of+Drosophila+Melanogaster.">The Courtship of Drosophila Melanogaster.</a> Behaviour. , 85-111 (1955).</li><li>Greenspan, R. J., Ferveur, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Courtship+in+Drosophila.">Courtship in Drosophila.</a> Annu. Rev. Genet. 34, 205-232 (2000).</li><li>Villella, A., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenetics+of+courtship+and+mating+in+Drosophila.">Neurogenetics of courtship and mating in Drosophila.</a> Adv. Genet. 62, 67-184 (2008).</li></ol>

우리는 공개 할게 없다.

Drosophila 동작 긴밀 유전과 환경 요인에 의해 규제됩니다. 우리와 다른, 이전 행동과 Drosophila 5-19에서 모델로 인간 neurodegenerative 질병의 비행과 관련된 유전자를 검사하기 위해 데이터를 수집하려면 여기를 설명한 assays를 사용했습니다. 크롤링 분석 내용은 제 3 instar의 애벌레에주의 선택은 중요한 단계입니다. 약물로 치료를하면 그것이 좋은 용해도가있는 경우 최대?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 자당 </td><td> 피셔 과학 </td><td> S5-500 </td></tr><tr><td> 아가로 오스 </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> 6온스 Drosophila 병 </td><td> Genesee 과학 </td><td> 32-130 </td></tr><tr><td> 페인트 브러시 (# 1) </td><td> 테드 펠라 주식 회사 </td><td> 11,859 </td></tr><tr><td> 식품 구성 요소를 플라이 </td><td></td><td></td></tr><tr><td> Cornmeal </td><td> 피셔 과학 </td><td> NC9109741 </td></tr><tr><td> 한천 </td><td> Genesee 과학 </td><td> 66-104 </td></tr><tr><td> 당밀 </td><td> 피셔 과학 </td><td> NC9349176 </td></tr><tr><td> 프로피 온산 </td><td> Acros </td><td> 14930-0010 </td></tr><tr><td> Tegosept </td><td> 꼭대기 </td><td> 20-258 </td></tr><tr><td> 에탄올 </td><td> 어부과학적인 </td><td> BP2818-4 </td></tr><tr><td> 누룩 </td><td> Genesee 과학 </td><td> 62-107 </td></tr></tbody></table>

methods to assay drosophila behavior

Drosophila melanogaster, 과일 파리는 등 암, 심혈관 질환 및 각종 신경 질환 1 등 인간 질병의 광범위한 분자 메커니즘을 연구하는 데 사용되었습니다. 우리는 애벌레 운동, 성인 등반 능력 (링 분석), 그리고 Drosophila의 구애 행동을 결정하는 간단하고 강력한 행동 assays을 최적화했습니다. 이러한 행동 assays은 즉시 행동에 대한 유전 및 환경 요인의 역할을 연구를위한 광범위하게 적용됩니다. 애벌레 크롤링 능력을 안정적으로 또한 그들의 운동에 약물이나 인간 질병 유전자의 효과를 (유전자 변형 파리에서) 조사를 위해 Drosophila의 애벌레의 크롤링 능력에서 초기 단계의 변화를 결정하기 위해 사용될 수 있습니다. 그들이 달리 평가되어야 어디로 이들 파리가 성체로 생존하지 않는 한 유전자의 표현이나 폐지가 pupal 또는 성인 무대에서 보여 주죠가 발생하면 애벌레의 크롤링 분석 더 적용됩니다. 이것은 기본ssay은 Drosophila의 애벌레에 추가적인 행동 반응을 조사하기 위해 밝은 빛 또는 스트레스와 함께 사용할 수도 있습니다. 구애 널리 성적 행동의 유전적 기초를 조사하기 위해 사용되고, 또한뿐만 아니라 학습과 기억 등, 활동과 조정을 검토하는 데 사용할 수 있습니다. Drosophila의 구애는 시각, 청각, 그리고 chemosensory 신호 등 다양한 감각 자극의 교환을 포함 남성과 성공적인 결합에 culminating 잘 특징 모터 행동의 복잡한 시리즈로 이어질 여성 사이. 전통 성인 등반 assays (부정적 geotaxis)는 서로 다른 시련 2-4 사이에 상당한 편차가있는 지루하고, 집중적인 노동과 시간이 소요입니다. 급속한은 (RING) 분석 5 성인 운동력이있는 사람과 부정적인 geotaxis B를 정할, 재현성 민감한, 높은 처리량 접근 방식을 제공,보다 광범위하게 사용된 프로토콜을 통해 많은 장점을 가지고 부정적인 geotaxis 반복ehaviors. 링 분석에서는 여러 genotypes 또는 약물 치료는 높은 처리량 접근이 선별 실험에 대한 더 의무가 만들어 함께 동시에 동물의 큰 번호를 사용하여 테스트할 수 있습니다.

Watch this Scientific Journal Video about Methods to Assay Drosophila Behavior at JoVE.com

Methods to Assay Drosophila Behavior

Drosophila melanogaster은 한 세기 이상에서 많은 중요한 생물 학적 과정의 분자 및 세포 기초를 이해하기 위해 사용되었습니다 유전자와 behaviorally 다루기 쉬운 모델 시스템이다. Drosophila 잘 플라이 행동의 유전적 기초에 대한 통찰력을 얻기 위해 악용되었습니다.

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 ...

Research

JoVE Journal

Neuroscience

Methods to Assay Drosophila Behavior

42.5K 조회수.  Louisiana State University Health Sciences Center. Drosophila melanogaster은 한 세기 이상에서 많은 중요한 생물 학적 과정의 분자 및 세포 기초를 이해하기 위해 사용되었습니다 유전자와 behaviorally 다루기 쉬운 모델 시스템이다. Drosophila 잘 플라이 행동의 유전적 기초에 대한 통찰력을 얻기 위해 악용되었습니다.

비디오: Methods to Assay Drosophila Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

곤충의 단일 Sensillum 레코딩 Drosophila melanogaster과 아노 펠 레스 gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

연구

신경과학

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

의 이스케이프 문제의 Optogenetic의 자극<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

먹이 찾아 돌아다 행동에 대한 단일 비행 분석에<em&gt; 초파리</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

선형 아가로 오스 채널을 사용하여 공부하기<em&gt; 초파리</em&gt; 애벌레 크롤링 동작

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

사용 보상 추구 행동의 변화를 측정하는 간단한 방법<em&gt; 노랑 초파리</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila 그루밍 행동의 부량

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Drosophila 섬 분석 결과에서 운전 행동의 높은 처리량 분석

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

초파리 애벌레 동작 Optogenetic 후 각 신경의 자극에 대 한 응답 추적

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

초파리 멜라노가스터에서 학습 유발 시냅스 가소성의 생체 내 광학 칼슘 이미징

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

마우스의 통증 행동을 측정하기위한 기계적 충돌 회피 분석

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...