We thank Rachelle Kanippayoor for her help in identifying the new wild-type strain as being of the melanogaster species. R.W.F, O.F. and A.F.S were responsible for research design; R.W.F, M.N. and O.F. performed the experiments. R.W.F, M.N., O.F. and A.F.S. analyzed the data; R.W.F., I.S.M. and A.F.S. wrote the manuscript. 
This work was supported by PSC-CUNY research awards, jointly funded by The Professional Staff Congress and The City University of New York to A.F.S.; by internal funding from Western University to A.F.S. and I.S.M.; by a training support from the National Alliance for Hispanic Health&#8217;s Alliance/Merck Ciencia (Science) Hispanic Scholars Program and a University Fellowship from the Yale Graduate School of Arts and Sciences to R.W.F.

1. 장비 및 사내 만든 시약 (다른 사람을위한 재료의 목록을 참조하십시오) <ol><li> 플라이 작업을 수행 할 수있는 초파리 차가운 마취 장치를 준비합니다. <ol><li> 작은 플라스틱 상자 (10.2 cm, 폭 12.7 cm 길이), 피펫 팁 상자의 일반적으로 상단 커버 (3.8 cm 깊이 10.2 cm, 폭 12.7 cm 길이)을 충당하기 위해 다공성 폴리에틸렌 시트를 잘라. </li><li> 짓 눌린 얼음 상자 채우기, 다공성 폴리에틸렌 시트 커버. </li></ol></li><li> T-미로 장치를 준비; 이전에 설명 된 세부 28-31 장치 적응. </li><li> (32) 기술로 플라이 흡인기를 준비합니다. </li><li> 및 테스트 튜브의 스트레스없이 파리를 대체하는 phototaxis 응답 장치를 준비합니다 <ol><li> 29,33에 의해 설명 된 바와 같이, 역류 장치를 사용합니다. </li></ol></li></ol> 참고 : 28에 의해 기술 된 바와 같이 T 미로 또한, 다른 목적을 위해 이용 될 수있다. <ol start="5"><li> 동음 확인ogeneous 조명 조건 : 흰색 벤치 커버로 덮여 벤치에 실험을 수행하고, 화이트 보드 앞에. 참고 :이 실험을 위해 사용 된 플라이 균주는 초파리는 야생 유형을 melanogaster의했다 : 광저우-S, 오레곤과 사마르 칸트는 우리의 실험실 주식 34에서 날아; 엘우드 파리는 롱 아일랜드, 뉴욕, 미국에, 헌팅턴의 엘우드 동네에서 2011 년 가을에 수집되었다. </li></ol> 2. 실험 전에 파리를 준비 실험을 수행하기 전에 1-2일 : <ol><li> 12 시간 빛 / 어둠주기에 25 ° C에서 표준 옥수수 가루 한천 - 당밀 효모 매체에 파리를 유지한다. </li><li> 실험을 수행하는 1 ~ 2 일 전에 차가운 마취하에 응답자 파리를 수집 <ol><li> 그들은 깔때기를 사용하여, 50 ㎖ 팔콘 튜브에 제기되고있다있는 병에서 파리를 전송합니다. </li><li> 로, 얼음 수준에서 파리와 팔콘 튜브를 놓습니다절연 얼음 양동이. </li><li> -4 C가 진정 ° 분쇄 얼음으로 냉각 마취 장치를 놓습니다. </li><li> 파리 냉각 혼수를 입력 할 수 5 분을 기다립니다. </li><li> 추위 마취 장치의 다공성 폴리에틸렌 시트에 파리를 전송합니다. </li><li> 추위 마취 장치에 응답자의 파리를 수집합니다. RT에서, 실체 현미경, 여성 파리 3-7 일 이전 남성을 분리하고 (40)의 그룹이 파리를 유지한다. </li></ol></li><li> 실험을 수행하기 1-2 일 전에 이미 터 파리를 수집합니다. <ol><li> 부드럽게 광저우-S 혼합 성이 자신의 번호를 제어, 입 흡입기를 사용하여, 자신의 병에서 파리 수집합니다. </li><li> 그들이 방출되는 바와 같이, 60 ~ 100의 그룹이 파리를 유지한다. </li><li> 데이터 포인트는 필요에 따라 방출의 많은 샘플을 준비합니다. </li></ol></li><li> 추위 마취의 혼란 효과를 최소화하기 위해, 대응 및 이미 터의 수집 후 회복 중 적어도 밤 허용행동에 입 흡입기 모음. </li><li> 적어도 일일 전에 실험, 신선한 공기로 대체 할 수있는 가방에 오래된 플라스틱 냄새가 공기 수 있도록, 테스트 튜브의 가방을 엽니 다. </li></ol> 실험을 수행하기 전에 2 시간 : <ol start="6"><li> 심지어 빛, 실험이 수행 된 실내 온도가 약 23 ~ 25 ° C에 있는지 확인하고, 상기 습도 30 %. </li><li> 이 파리는 굶어되지 않도록 신선한 식품 유리 병에 대응하고 방출 과일 파리를 전송합니다. </li><li> 초파리 실험이 수행 될에 벤치에 2 시간 동안 환경에 적응하자. </li></ol> 3. 사회 회피 실험 수행 <ol><li> Zeitgeber 시간 ZT5 ZT9과 오후 사이에서 3-4 시간의 범위로, 하루 중 동시에 각 실험을 수행한다. </li><li> 타격 패드 T-미로를 놓고 t 그래서 나사 클립을 조그는 엘리베이터 안정적​​이다. </li><li> 신선한 테스트 유리 병에 응답자의 파리를 전송합니다. T-미로의 상단 부분에 응답자의 파리를 포함하는이 병을 스냅. </li><li> T-미로 장치를 경사와 초파리가 엘리베이터에 빠지지 않도록 두근 두근 매트를 누릅니다. </li><li> 파리는 T-미로의 상단과 하단 부분 사이 있도록 아래로 엘리베이터의 상단 부분을 이동하지만, 선택 포인트 이상의 엘리베이터를 유지하기 위해주의 - 탈출 파리를 방지하기 위해. </li><li> DSO (이전에 파리에 의해 점유 된 DSO없는 튜브를 생성하는 제 5 절 참조) 병을 얻습니다. <ol><li> 응답자 파리는이 새로운 환경에 적응하는 동안, 테스트 유리 병에 터 파리를 배치합니다. </li><li> 초파리 탈출하지 않도록 유리 병을 닫습니다면의 조각을 놓습니다. </li><li> 15 초 동안 소용돌이를 5 초 동안 소용돌이에서 유리 병을 제거 : 기계적으로 다음과 같이 미니 소용돌이에이 병을 선동. 55 초 총 3 번 반복합니다. </li><li> 렘식품 유리 병에 전송하여 - - 신선한 유리 병에서 이미 터 파리를 비켜 빠르게 T-미로 장치의 두 측면 중 하나에이 DSO 채워진 유리 병을 배치합니다. </li><li> 각각의 새로운 실행에 DSO 채워진 유리 병에 대한 배치의 측면을 교대한다. </li></ol></li><li> T-미로의 반대편에 신선한 테스트 유리 병을 놓습니다. </li><li> 초파리는 신선한 테스트 유리 병 및 DSO 테스트 유리 병 사이에 선택할 수 있도록 완전히 엘리베이터를 가지고, 그리고 타이머를 시작합니다. </li><li> 다른 설명이없는 파리, 1 분 동안 신선한 유리 병 및 DSO 유리 병 사이에서 선택할 수 있습니다. </li><li> 1 분 후, 신선한 유리 병, DSO 유리 병, 엘리베이터에 갇혀 사람들의 파리를 분리하기 위해 엘리베이터를 이동합니다. </li><li> 각각의 유리 병에 파리의 수를 계산하고 엘리베이터에. </li><li> 각각의 유전자형 / 조건에 대해이 작업을 반복합니다. </li></ol> 4. 사회 회피 데이터 분석 <ol><li> 각 genot 각 유리 병에 과일 파리의 수를 계산YPE 및 스프레드 시트에 데이터를 전송. </li><li> 스프레드 시트 프로그램을 사용하여, 파리의 총 개수로 나누어 다음 DSO없는 유리 병에 파리 번호에서 DSO 바이알 파리의 수를 감산하고, 각 유전자형에 대한 성능 지수 (PI)를 계산한다. </li><li> 결과의 통계적인 분석을위한 분석 소프트웨어를 이용하여 PI들 &#39;비교 수단. <ol><li> 이상적으로, 각 실험 2-3 내부 반복, 3 독립적 인 반복을 준비, 다른 병으로, 다른 일을 수행 또는 (30) 십자가. </li><li> 데이터가 정규 분포를 따르는 것처럼, 가우스 분포의 통계 시험을 사용한다. </li><li> 하나의 효과를 테스트 할 때 (즉, 밀도 - 그림 2), 컨트롤에 각각의 결과를 비교하기 위해 (열을 기준으로 열) 다중 비교에 대한 시험 후, 또는 간단한 t-test로, 일원 분산 분석을 사용합니다. </li><li> 그룹의 실험이 수행 될 때, 조건 및 성별 (빈 유리 병을 모두 테스트 강조D 않은 스트레스 파리; 성별 효과 - 그림 1), 양방향 ANOVA를 사용하여. </li></ol></li></ol> 5. 이전에 강세 파리 점령 튜브를 생성 - 대신 3.5-3.6의 수행 <ol><li> 대조군으로 강조되지 않은 파리에 의해 점유 된 유리 병을 준비하는 phototaxis을 사용합니다. </li><li> 반대 위치에 신선한 빈 유리 병으로, 테스트 유리 병에 파리를 전송하고, 역류 장치에 끼워 넣습니다. </li><li> 직접 15 W 형광 차가운 백색 빛의 앞에 말단 유리 병에 수평으로 장치를 마련하고, 검은 천으로 커버. </li><li> 파리 부드럽게 신선한 유리 병으로 이동하자. 이 작업은 몇 분 정도 걸릴 수 있습니다. 파리의 대부분이 말단 유리 병에 빛을 향해 걷고되면, 1 분 타이머를 시작합니다. </li><li> 부드럽게, 빛의 위치를​​ 온, 검은 천으로 제거 검은 천으로 복구하고, 파리 테스트 유리 병을 떠날 수 있습니다. </li><li> 그 때, 나는3.2-3.5에서와 같이 선택 실험을 nitiate. 3.6 및 3.7를 건너 뜁니다. </li><li> 다른 측면에서 테스트 바이알을 비웠다 파리를 분리하는 장치의 슬라이더를 이동. </li><li> 바로 선택 실험, 3.7에서와 T-미로에서 물러 테스트 유리 병을 사용합니다. </li></ol>

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S., 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=A+single+population+of+olfactory+sensory+neurons+mediates+an+innate+avoidance+behaviour+in+Drosophila.">A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila.</a> Nature. 431, 854-859 (2004).</li><li>Bonini, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tribute+to+Seymour+Benzer+1921-2007.">A Tribute to Seymour Benzer 1921-2007.</a> 180, 1265-1273 (2008).</li><li>Castermans, D., 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=The+neurobeachin+gene+is+disrupted+by+a+translocation+in+a+patient+with+idiopathic+autism.">The neurobeachin gene is disrupted by a translocation in a patient with idiopathic autism.</a> Journal of Medical Genetics. 40, 352-356 (2003).</li><li>Medrihan, 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=Neurobeachin,+a+protein+implicated+in+membrane+protein+traffic+and+autism,+is+required+for+the+formation+and+functioning+of+central+synapses.">Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses.</a> J Physiol. 587, 5095-5106 (2009).</li><li>Simon, A. F., 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=A+simple+assay+to+study+social+behavior+in+Drosophila:+measurement+of+social+space+within+a+group.">A simple assay to study social behavior in Drosophila: measurement of social space within a group.</a> Genes Brain Behav. 11, 243-252 (2012).</li><li>Venkatesh, T., 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=.">.</a> Cold Spring Harbor Meeting: From Molecules to Circuit Behavior. , (2013).</li><li>Hamilton, P. J., 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=De+novo+mutation+in+the+dopamine+transporter+gene+associates+dopamine+dysfunction+with+autism+spectrum+disorder.">De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.</a> Mol Psychiatry. 18, 1315-1323 (2013).</li><li>Gadow, K. D., 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=Association+of+dopamine+gene+variants,+emotion+dysregulation+and+ADHD+in+autism+spectrum+disorder.">Association of dopamine gene variants, emotion dysregulation and ADHD in autism spectrum disorder.</a> Research in Developmental Disabilities. 35, 1658-1665 (2014).</li><li>Fernandez, R. W., Akinleye, A. A., Nurilov, M., Rouzyi, Z., Simon, A. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical-conditioning+and+retention+in+normal+and+mutant+Drosophila+melanogaster.">Classical-conditioning and retention in normal and mutant Drosophila melanogaster.</a> J Comp Physiol [A]. 157, 263-277 (1985).</li><li>Krashes, M. J., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Aversive+Olfactory+Conditioning.">Drosophila Aversive Olfactory Conditioning.</a> Cold Spring Harbor Protocols. 2011, (2011).</li><li>Ejima, A., Griffith, L. C., Zhang, B., Freeman, M. R., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+30.">Ch. 30.</a> Drosophila Neurobiology, A Laboratory Manual. , 475-481 (2010).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+mutants+of+Drosophila+melanogaster+isolated+by+countercurrent+distribution.">Behavioral mutants of Drosophila melanogaster isolated by countercurrent distribution.</a> PNAS. 58, 1112-1119 (1967).</li><li>Simon, A. F., Shih, C., Mack, A., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+control+of+longevity+in+Drosophila+melanogaster.">Steroid control of longevity in Drosophila melanogaster.</a> Science. 299, 1407-1410 (2003).</li><li>Vaux, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+methods:+Know+when+your+numbers+are+significant.">Research methods: Know when your numbers are significant.</a> Nature. 492, 180-181 (2012).</li><li>Stowers, L., Logan, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+olfactory+signaling.">Sexual dimorphism in olfactory signaling.</a> Curr. Opin. Neurobiol. 20, 770-775 (2010).</li><li>Simon, A. F., Liang, D. T., Krantz, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+decline+in+behavioral+performance+of+Drosophila+melanogaster+with+age.">Differential decline in behavioral performance of Drosophila melanogaster with age.</a> Mechanisms of Ageing and Development. , 127-647 (2006).</li></ol>

The authors declare that they have no competing financial interests.

이 프로토콜은 사회적 회피 분석에 대한 자세한 절차를 설명합니다. 광저우-S는 이전에 기계적으로되어 강조했다 날아간 유리 병을 방지하며, 응답자의 성별과 수는 사회적 회피 성능 것이 영향을주지 않습니다. 그러나 응답자의 유전 적 배경이 큰 영향을 미친다. 성공적으로이 실험을 수행하기위한 몇 가지 중요한 단계를 다음과 같습니다 : 1) 항상 실험 전에 파리 2 시간을 ...

이 방법의 목적은 쉽게 구애와 침략에서 독립적 인 초파리에서 간단한 사회적 행동의 새로운 측면을 정량화하는 것입니다. 사회적 상호 작용은 적절한 발전과 개인의 건강 사회에서뿐만 아니라 전체 사회 집단의 기능에 중요하다. 이러한 상호 작용의 높은 복잡도가 큰 샘플 크기 및 사회적 행동의 유전 적 신경 염기 여전히 제대로 이해되는 바와 같이 동작의 단순화를 허용하는 시스템을 필요로한다. 초파리는 유전자를 식별하는 데 사용될 수있는 강력한 유전 적 모델이고 사회적 상호 작용의 신경 기지. 사실, D. melanogaster의 복잡한 사회적 행동의 레퍼토리를 가지고 있으며, 사회화의 일부 직접 측정은 이미 1-7을 수행하고있다. 그러나, 이러한 노력의 대부분은 상대적으로 복잡한 aggres 사회적 행동에 집중되어왔다시브 상호 작용 3,6, 구애 3,8-12의 다양한 측면, 어떻게 사회적 경험은 학습, 또는 일주기 리듬 13-17 다른 행동에 영향을 미친다. 또한, 이들 분석법은 많은, 파리의 기의 복잡한 상호 작용 패턴을 분석하고 결과 데이터를 분석하는 풍부한 비디오 추적 및 컴퓨터 소프트웨어를 사용에 의존한다. 이러한 분석은 매우 중요하며, 이러한 그룹 7 플라이 플라이 상호 작용의 동적 중요한 새로운 통찰로 이어집니다. 한 가지 제한은, 그러나, 크게는 지역 사회에 이러한 분석의 어려움, 그리고 다른 사람의 인식의 기초가되는 메커니즘의 제한된 지식입니다. 즉, 다른 한 개인에 의해 인식으로 그 신호의 방출에 기초하여 여전히 저조한 18 이해된다. D. : 반면에, 또한 간단한 동작, 개인이 스트레스를 파리에 의해 방출 신호에서 벗어나 사회적 회피, 전시 파리 MELAN스트레스 후각 또는 DSO (19) ogaster. 높은 처리량 분석에서,이 동작은 다른 파리, 또는 사회적 회피 (19)에 의해 방출 된 신호의 응력을 회피 정량화 할 수있다. 파리 T 미로 장치에 넣고 DSO 함유 바이알을 피하기 위해 선택 주어진다. 이 분석을 이용하여, CO 2 2 19 해부 하였다 CO에 응답하도록 필요한 신경 회로 DSO의 성분, 및 일부가 도시되었다. 여기에 제시된 사회적 회피 분석은 복잡한 행동 (20)를 해부 연구자의 ​​세대를 활성화 모어 Benzer의 연구실에서 개발 된 간단한 행동 분석 개념적으로 유사하다. 사회적 회피 분석 경제적 사회적 행동의 더 광범위한 연구를 허용 T 미로 분석을 이용하여 수행 될 수있다. 예를 들어,이 분석을 사용하여 우리는 최근에 보여 자폐증에 대해 서로 다른 유전 적 위험 사회 B의 대조 효과를 가지고ehavior 분석. 21, 22 neurobeachin - - 자폐증에 대한 후보 유전자에 대한 돌연변이 존재하는 결함은 모두 사회적 공간에서 (다른 곳에서 23 설명), 사회적 회피 (24). 이상 도파민 시그널링은 또한 인간 25,26 자폐증의 병인에서 역할을하는 것으로 제안된다. neurobeachin 얻어진 결과와 대조적으로, 우리는 증가 또는 도파민 세포 초파리 소낭 모노 아민 전송기 (VMAT)의 수준을 감소함으로써 사회적 공간 직접 VMAT (27)의이 수준과 상관되었지만 사회적 회피 성능이 영향을받지 않는 것을 발견했다. neurobeachin 및 VMAT 얻은 대조 결과를 따라서 다양한 비 사교적 행동 양식 및 다른 사람에게 응답을 변조하는 다른 기본 신경 회로들을 식별 가능성을 강조한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereo Zoom Microscope&#160;</td>
			<td>Nikon&#160;&#160;</td>
			<td>SMZ-645</td>
			<td>Any other standard scope for fly handling would work</td>
		</tr>
		<tr>
			<td>Small paint brushes&#160;</td>
			<td></td>
			<td></td>
			<td>for pushing flies</td>
		</tr>
		<tr>
			<td>Porous Polyethylene, 12&#34; x 12&#34; Sheet</td>
			<td>Flystuff - Genesee</td>
			<td>46-100</td>
			<td>http://www.flystuff.com/ProductInfo.php?productID=46-100 
			Porous Plastic sheet for the cold anesthesia box</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Alarm Timer/Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sharpie pens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini vortex</td>
			<td>Fisher</td>
			<td>14-955-151&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail 
			For mechanical agitation of the flies - any vortex would work.</td>
		</tr>
		<tr>
			<td>Corning Life Sciences DL No.:352017, Falcon test tube; round bottom; disposable; no closure, 14mL;&#160; 17 x 100mm</td>
			<td>Fisher</td>
			<td>14-959-8 &#160;&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalogId=29104&#38;productId=2771811&#38;distype=0&#38;highlightProductsItemsFlag=Y&#38;fromSearch=1&#38;searchType=PROD&#38;hasPromo=0 
			These snap in place in the in-house made T-maze and counter-current apparatus (see text)</td>
		</tr>
		<tr>
			<td>cotton balls</td>
			<td></td>
			<td></td>
			<td>to close the vials after the experiment.</td>
		</tr>
		<tr>
			<td>trifold board and white bench cover</td>
			<td></td>
			<td></td>
			<td>to provide a white background, and a homogeneous light.</td>
		</tr>
		<tr>
			<td>white bench cover</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pounding pad</td>
			<td></td>
			<td></td>
			<td>any mouse pad works.</td>
		</tr>
		<tr>
			<td>large black cloth</td>
			<td></td>
			<td></td>
			<td>to cover the counter-current apparatus in phototaxis response.</td>
		</tr>
		<tr>
			<td>cool-white light&#160;</td>
			<td>Home Depot</td>
			<td>1000516563</td>
			<td>http://www.homedepot.ca/product/illume-26-fluorescent-plug-in-linear/911423 
			any similar linear light with fluorescent light bulb cool-white at 13-15W would work</td>
		</tr>
	</tbody>
</table>

straightforward assay for quantification of social avoidance in drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

사회적 회피 분석은 다른 플라이에서 방출 응력 신호를 인식 초파리 melanogaster의 능력 (DSO)을 정량화하고, 따라서 사회적 상호 작용의 일 양상을 평가 강력한 시험이다. 분석은 일반적으로 두 가지 옵션 사이에서 선택과 파리를 제시 T-미로로 알려진 다양한 행동 분석에 사용 된 장치를 사용하여 수행됩니다 - 왼쪽 또는 오른쪽으로 19,28-31. 이 경우, 파리 DSO를 방지하는 계산 효율 및 ?...

Watch this Scientific Journal Video about Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster at JoVE.com

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

사회 회피의 정량화에 대한 간단한 분석<em&gt; 초파리</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

straightforward-assay-for-quantification-social-avoidance-drosophila

Research

JoVE Journal

Neuroscience

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

11.1K 조회수.  Yale University. Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

비디오: Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

를 사용하여 화합물 Bioactivity위한 이점 Nanoinjection 및 화면에 대한 전기 생리학 분석 Drosophila melanogaster 거대 섬유 시스템

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

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 ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

의 자발적인 운동 활성의 결정<em&gt; 초파리</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

사회적 공간에 영향을 미치는 조건<em&gt; 초파리</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

에서 냄새 유도 행동의 고해상도 정량화<em&gt; 초파리</em사용&gt;<em&gt; Flywalk</em&gt; 패러다임

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

의 Mechanosensation에 대한 행동 분석 클론의 MARCM은 기반<em&gt; 초파리</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

높은 처리량 분석은 개인의 알을 낳는 환경 설정을 검사합니다<em&gt; 노랑 초파리</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

대한 꼴 경로 길이 프로토콜<em&gt; 노랑 초파리</em&gt; 애벌레

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

살아있는 세포에 Sod1 돌연변이 단백질 집계 형성의 높은 콘텐츠 정량화에 대 한 분석 결과 개발

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...